**Part 2**

**Myocardial Infarction** 

110 Advances in Electrocardiograms – Clinical Applications

Yeh HI, Lai YJ, Lee SH, Chen ST, Ko YS, Chen SA, Severs NJ, Tsai CH.(2006). Remodeling of

Zhou SH, He XZ, Liu QM, Du WH, Li XP, Zhou T, Tang JQ, Li RS.(2008). Study on the

coronary heart disease. *Cardiol J,* Vol.15, No.1, pp. 50-56, ISSN 1897-5593

pacing. *Basic Res Cardiol,* Vol.101, No.4, pp. 269-280, ISSN 0300-8428

myocardial sleeve and gap junctions in canine superior vena cava after rapid

spatial distribution pattern of Cx40 gap junctions in the atria of patients with

**8** 

*Italy* 

**ECG in Acute Myocardial Infarction** 

Acute myocardial infarction can be defined from a number of different perspectives related to clinical, electrocardiographic, biochemical and pathological characteristic. The electrocardiogram (ECG) is the most important diagnostic tool in the diagnosis of ST-segment elevation myocardial infarction (STEMI), and therefore it should be accomplished immediately at hospital admission. In fact, it represents an important step not only for STEMI diagnosis, but also and more importantly for the therapeutic plan. The present article pertains to electrocadiographic findings in patients affected by persistent STEMI. Moreover, it takes into account the clinical utility of ECG in the diagnosis and therapeutic decisions of evolving STEMI, as well as the prognostic implications of the ECG evolutions in the reperfusion era.

**2. Evolving ECG changes occurring in the early phase of ST-elevation** 

Typically, the ECG in the evolving STEMI shows five abnormalities, which develop in turn: hyperacute T waves, ST-segment elevation, abnormal Q waves, T-waves inversion,

The T-waves represent the period of ventricular repolarization on the surface ECG. During the first minutes of coronary arterial occlusion (Dressler et al., 1947), the earliest ECG changes are represented by an increase in the amplitude of the T-wave, the so-called "Hyperacute T-waves" (Figure 1B,C). The morphologic characteristic of hyperacute T-wave are typical of ischemic event: they are asymmetric with a broad base and generally associated with reciprocal ST segment depression. In the evolving STEMI the hyperacute Twaves turn into giant R wave (Figure 1E). Hyperacute T-waves represent the electrocardiographic expression of ischemia before the beginning of necrosis; for this reason they are considered as the most significant phase during which the reperfusion therapy may achieve the greatest benefit in term of myocardial salvage (Lee et al., 1995). Prominent Twaves, however, are also associated with other diagnoses, including hyperkalemia, early repolarization end left ventricular hypertrophy (Somers et al., 2002). Thus in the differential diagnosis, the clinicians must consider additional features related to patient, including age,

**1. Introduction** 

**myocardial infarction** 

**2.1 Hyperacute T waves** 

normalization of the ST-segment (Figure 1).

comorbidity and current medical status.

**in the Reperfusion Era** 

*University of Padova,* 

Massimo Napodano and Catia Paganelli

## **ECG in Acute Myocardial Infarction in the Reperfusion Era**

Massimo Napodano and Catia Paganelli *University of Padova, Italy* 

### **1. Introduction**

Acute myocardial infarction can be defined from a number of different perspectives related to clinical, electrocardiographic, biochemical and pathological characteristic. The electrocardiogram (ECG) is the most important diagnostic tool in the diagnosis of ST-segment elevation myocardial infarction (STEMI), and therefore it should be accomplished immediately at hospital admission. In fact, it represents an important step not only for STEMI diagnosis, but also and more importantly for the therapeutic plan. The present article pertains to electrocadiographic findings in patients affected by persistent STEMI. Moreover, it takes into account the clinical utility of ECG in the diagnosis and therapeutic decisions of evolving STEMI, as well as the prognostic implications of the ECG evolutions in the reperfusion era.

### **2. Evolving ECG changes occurring in the early phase of ST-elevation myocardial infarction**

Typically, the ECG in the evolving STEMI shows five abnormalities, which develop in turn: hyperacute T waves, ST-segment elevation, abnormal Q waves, T-waves inversion, normalization of the ST-segment (Figure 1).

### **2.1 Hyperacute T waves**

The T-waves represent the period of ventricular repolarization on the surface ECG. During the first minutes of coronary arterial occlusion (Dressler et al., 1947), the earliest ECG changes are represented by an increase in the amplitude of the T-wave, the so-called "Hyperacute T-waves" (Figure 1B,C). The morphologic characteristic of hyperacute T-wave are typical of ischemic event: they are asymmetric with a broad base and generally associated with reciprocal ST segment depression. In the evolving STEMI the hyperacute Twaves turn into giant R wave (Figure 1E). Hyperacute T-waves represent the electrocardiographic expression of ischemia before the beginning of necrosis; for this reason they are considered as the most significant phase during which the reperfusion therapy may achieve the greatest benefit in term of myocardial salvage (Lee et al., 1995). Prominent Twaves, however, are also associated with other diagnoses, including hyperkalemia, early repolarization end left ventricular hypertrophy (Somers et al., 2002). Thus in the differential diagnosis, the clinicians must consider additional features related to patient, including age, comorbidity and current medical status.

ECG in Acute Myocardial Infarction in the Reperfusion Era 115

dependent on gender, age, and ECG lead. Thus, the current thresholds recommended by the American Heart Association Electrocardiography and Arrhythmias, the Amrican College of

V3 should be 0.25 mV (2.5 mm).

Women of all ages The threshold value for abnormal J-point elevation should be

through V9 should be 0.05 mV (0.5 mm).

(1 mm) in all other leads.

mm) in all other leads.

Table 1. **Threshold values for ST-segment elevation according to age, gender, and ECG leads.** Adapted from AHA/ACCF/HRS (2009) Recommendations for standardization and interpretation of the electrocardiogram . *J Am Coll Cardiol*, Vol. 53, No. 11, pp. 1003-10011,

However, ST-segment elevation can also attributed to other causes, different from acute myocardial infarction: a normal variant, frequently referred as *early repolarization,* commonly characterized by J-point elevation and rapidly upsloping or normal ST-segment; ventricular dyskinesis, often characterized by a small ST elevation; pericarditis, in which usually the ST elevation can be detected in more than one discrete region, as the inflammation involves a large portion of the epicardial surface, and reciprocal ST-depression is absent; elevated serum potassium; acute myocarditis; cardiac tumors or intra-thoracic mass. An additional ECG criteria in diagnosis of evolving STEMI is represented by the morphology of STsegment elevation. In fact, two patterns of ST-segment morphology can be distinguish, according to the direction of the ST slope: a concave morphology and a convex morphology (Figure 2A,B). The concave morphology (Figure 2A) is hardly consistent with STEMI diagnosis, and rather related to other conditions, such as benign early repolarization, acute pericarditis. On the other hand, the convex morphology is usually associated with STEMI (Brady et al., 2001) (Figure 2B). The assessment of ST-segment elevation during STEMI is also useful to evaluate the extension of the myocardial at risk, and then the prognosis. In fact the number of leads with ST segment elevation and the sum of the total ST deviation have been related to the extension of area of myocardium at risk, defined as the extent of jeopardize ischemic myocardium, and consequently to the extent of necrotic area if

The threshold value for abnormal J-point elevation should be 0.2 mV (2 mm) in leads V2 and V3 and 0.1 mV (1 mm) in all

The threshold value for abnormal J-point elevation in V2 and

0.15 mV (1.5 mm) in leads V2 and V3 and greater than 0.1 mV

The threshold for abnormal J-point elevation V3R and V4R should be 0.05 mV (0.5 mm), except for males less than 30 years of age, for whom 0.1 mV (1 mm) is more appropriate.

The threshold value for abnormal J-point elevation in V7

The threshold value for abnormal J-point depression should be – 0.05 mV (-0.5 mm) in leads V2 and V3 and – 0.1 mV (- 1

Cardiology vary according to age, gender, and ECG lead (Table 1).

other leads.

Men 40 years old of age

Men less than 40 years of

Men and women of all

Men and women of all

Men and women of all

ISSN 0735-1097/09/

reperfusion is not undertaken (Aldrich et al., 1988).

and older

age

ages

ages

ages

**A Normal**: Normal ST-segment and T-wave; **B Early, Hyper-acute T wave**: Development of Prominent T Wave; **C Hyper-acute T Wave**: Prominent T-wave with early ST-segment elevation; **D ST-segment elevation**: Progressive ST-segment elevation with persistent prominent T-wave; **E Giant R Wave**: ST segment elevation continues with development of giant R-wave. **F ST-segment Elevation:** ST-segment elevation with oblique morphology.

Fig. 1. Evolving ECG changes occurring in the early phase of ST-elevation myocardial infarction

### **2.2 ST-segment elevation**

The ST-segment, defined as the segment beginning at the J point and ending at the apex of the T-wave, represents the electrocardiographic period between ventricular depolarization (QRS) and repolarization (T-wave) (Figure 1A). The ST-segment changes on the standard ECG that are associated with infarction are due to flow of current across the boundary between the ischemic and nonischemic zones. ST-segment elevation generally occurs with reciprocal ST depression in ECG leads in which the axis is opposite in direction from those with ST elevation (Figure 1D). The best criteria to classify abnormally elevated ST-segment are resumed in the Minnesota code 9-2 and are defined as ST-segment elevation of 1 mm in at least 1 peripheral lead, or 2 mm elevation in at least 1 precordial lead. These criteria have 94% of specificity for STEMI with a sensitivity of 56% in STEMI diagnosis (Menown et al., 2000). The threshold values results from recognition that some elevation of the junction of the QRS complex and the ST-segment (J-point) is a normal finding. Indeed, these are

**A Normal**: Normal ST-segment and T-wave; **B Early, Hyper-acute T wave**: Development of Prominent T Wave; **C Hyper-acute T Wave**: Prominent T-wave with early ST-segment elevation; **D ST-segment elevation**: Progressive ST-segment elevation with persistent prominent T-wave; **E Giant R Wave**: ST segment elevation continues with development of giant R-wave. **F ST-segment Elevation:** ST-segment

The ST-segment, defined as the segment beginning at the J point and ending at the apex of the T-wave, represents the electrocardiographic period between ventricular depolarization (QRS) and repolarization (T-wave) (Figure 1A). The ST-segment changes on the standard ECG that are associated with infarction are due to flow of current across the boundary between the ischemic and nonischemic zones. ST-segment elevation generally occurs with reciprocal ST depression in ECG leads in which the axis is opposite in direction from those with ST elevation (Figure 1D). The best criteria to classify abnormally elevated ST-segment are resumed in the Minnesota code 9-2 and are defined as ST-segment elevation of 1 mm in at least 1 peripheral lead, or 2 mm elevation in at least 1 precordial lead. These criteria have 94% of specificity for STEMI with a sensitivity of 56% in STEMI diagnosis (Menown et al., 2000). The threshold values results from recognition that some elevation of the junction of the QRS complex and the ST-segment (J-point) is a normal finding. Indeed, these are

Fig. 1. Evolving ECG changes occurring in the early phase of ST-elevation myocardial

elevation with oblique morphology.

**2.2 ST-segment elevation** 

infarction

dependent on gender, age, and ECG lead. Thus, the current thresholds recommended by the American Heart Association Electrocardiography and Arrhythmias, the Amrican College of Cardiology vary according to age, gender, and ECG lead (Table 1).


Table 1. **Threshold values for ST-segment elevation according to age, gender, and ECG leads.** Adapted from AHA/ACCF/HRS (2009) Recommendations for standardization and interpretation of the electrocardiogram . *J Am Coll Cardiol*, Vol. 53, No. 11, pp. 1003-10011, ISSN 0735-1097/09/

However, ST-segment elevation can also attributed to other causes, different from acute myocardial infarction: a normal variant, frequently referred as *early repolarization,* commonly characterized by J-point elevation and rapidly upsloping or normal ST-segment; ventricular dyskinesis, often characterized by a small ST elevation; pericarditis, in which usually the ST elevation can be detected in more than one discrete region, as the inflammation involves a large portion of the epicardial surface, and reciprocal ST-depression is absent; elevated serum potassium; acute myocarditis; cardiac tumors or intra-thoracic mass. An additional ECG criteria in diagnosis of evolving STEMI is represented by the morphology of STsegment elevation. In fact, two patterns of ST-segment morphology can be distinguish, according to the direction of the ST slope: a concave morphology and a convex morphology (Figure 2A,B). The concave morphology (Figure 2A) is hardly consistent with STEMI diagnosis, and rather related to other conditions, such as benign early repolarization, acute pericarditis. On the other hand, the convex morphology is usually associated with STEMI (Brady et al., 2001) (Figure 2B). The assessment of ST-segment elevation during STEMI is also useful to evaluate the extension of the myocardial at risk, and then the prognosis. In fact the number of leads with ST segment elevation and the sum of the total ST deviation have been related to the extension of area of myocardium at risk, defined as the extent of jeopardize ischemic myocardium, and consequently to the extent of necrotic area if reperfusion is not undertaken (Aldrich et al., 1988).

ECG in Acute Myocardial Infarction in the Reperfusion Era 117

and away from the positive pole of lead III, which will show depression of the ST segment. When the occlusion is located more distally, that is, below both the first septal and first diagonal branches, the basal portion of the left ventricle will not be involved, and the STsegment vector will be oriented more inferiorly. Thus, the ST segment will not be elevated in leads V1, aVR, or aVL, and the ST segment will not be depressed in leads II, III, or aVF. Indeed, because of the inferior orientation of the ST-segment vector, elevation of the ST segment in leads II, III, and aVF may occur. In addition, ST-segment elevation may be more prominent in leads V3 through V6 and less prominent in V2than in the more proximal occlusions (Engelen et al., 1999). Inferior wall infarction that results in ST-segment elevation in only leads II, III, and aVF may be the result of occlusion of either the right coronary artery or the left circumflex coronary artery, depending on which provides the posterior descending branch, that is, which is the dominant vessel. When the right coronary artery is occluded, the spatial vector of the ST segment will usually be directed more to the right than when the left circumflex is occluded. This will result in greater ST-segment elevation in lead III than in lead II and will often be associated with ST-segment depression in leads I and aVL, leads in which the positive poles are oriented to the left and superiorly. However, recently these criteria resulted less accurate in patients with electrocardiographic small inferior myocardial infarction (Verouden et al., 2009). Indeed, when the RCA is occluded in its proximal portion, ischemia/infarction of the right ventricle may occur, which causes the spatial vector of the ST-segment shift to be directed to the right and anteriorly, as well as inferiorly. This will result in ST-segment elevation in leads placed on the right anterior chest, in positions referred to as V3R and V4R, and often in lead V1 (Correale et al., 1999). Lead V4R is the most commonly used right-sided chest lead. It is of great value in diagnosing right ventricular involvement in the setting of an inferior wall infarction and in making the distinction between right coronary artery and left circumflex coronary artery occlusion and between proximal and distal right coronary artery occlusion. It is important to recognize that the ST elevation in the right-sided chest leads associated with right ventricular infarction persists for a much shorter period of time than the ST elevation connoting inferior wall infarction that occurs in the extremity leads. For this reason, leads V3R and V4R should be recorded as rapidly as possible after the onset of chest pain. STsegment depression in leads V1, V2, and V3 that occurs in association with an inferior wall infarction may be caused by occlusion of either the right coronary or the left circumflex artery. This ECG pattern has been termed posterior or posterolateral ischemia since the early reports based on anatomic and pathological studies of ex vivo. However, recent in vivo imaging studies, including magnetic resonance imaging, have demonstrated that the region referred to as the posterior wall was lateral rather than posterior since the oblique position of the heart within the thorax: correlating the ECG patterns of healed myocardial infarctions to their anatomic location as determined by magnetic resonance imaging, the most frequent cause of abnormally tall and broad R waves in leads V1 and V2 was involvement of the lateral and not the posterior wall of the left ventricle (Bayes de Luna et al., 2006a). On these basis it has been proposed that the term posterior be replaced by the designation lateral (Cerqueira et al., 2002). Therefore, the terms posterior ischemia and posterior infarction be replaced by the terms lateral, inferolateral, or basal-lateral depending on the associated changes in II, III, aVF, V1, V5, and V6. Such terminology has been endorsed by the International Society for Holter and Noninvasive Electrocardiography (Bayes de Luna A et al., 2006b). It is not possible to determine whether the right coronary artery or left circumflex

**A Concave Morphology:** the concave morphology is characterized by downward ST slope; the ST slope remains below the virtual line drawn from the J-point to the apex of T-wave. **B Convex Morphology:** the convex morphology is characterized by upward ST-slope; the ST slope remains above the virtual line drawn from the J-point to the apex of T-wave

Fig. 2. Patterns of ST-segment elevation at ECG

Moreover , the analysis of the electrocardiographic leads revealing ST-segment elevation as well as of those showing ST depression, permits an almost accurate identification of the occluded coronary artery and also the proximal or distal location of the occlusion within that artery (Wagner et al., 2009). Anterior wall ischemia/infarction is invariably due to occlusion of the left anterior descending coronary artery and results in the spatial vector of the ST segment being directed to the left and laterally. This will be expressed as ST elevation in some or all of leads V1 through V6. The location of the occlusion within the left anterior descending coronary artery, that is, whether proximal or distal, is suggested by the chest leads in which the ST-segment elevation occurs and the presence of ST-segment elevation or depression in other leads. Occlusion of the proximal left anterior descending coronary artery above the first septal and first diagonal branches results in involvement of the basal portion of the left ventricle, as well as the anterior and lateral walls and the interventricular septum. This will result in the ST-segment spatial vector being directed superiorly and to the left and will be associated with ST-segment elevation in leads V1 through V4, I, aVL, and often aVR. It will also be associated with reciprocal ST-segment depression in the leads whose positive poles are positioned inferiorly, that is, leads II, III, aVF, and often V5 (Birnbaum et al., 1993). When the occlusion is located between the first septal and first diagonal branches, the basal interventricular septum will be spared, and the ST segment in lead V1 will not be elevated. In that situation, the ST-segment vector will be directed toward aVL, which will be elevated,

*Apex Apex*

**A Concave Morphology <sup>B</sup> Convex Morphology** 

**A Concave Morphology:** the concave morphology is characterized by downward ST slope; the ST slope remains below the virtual line drawn from the J-point to the apex of T-wave. **B Convex Morphology:** the convex morphology is characterized by upward ST-slope; the ST slope remains above the virtual

Moreover , the analysis of the electrocardiographic leads revealing ST-segment elevation as well as of those showing ST depression, permits an almost accurate identification of the occluded coronary artery and also the proximal or distal location of the occlusion within that artery (Wagner et al., 2009). Anterior wall ischemia/infarction is invariably due to occlusion of the left anterior descending coronary artery and results in the spatial vector of the ST segment being directed to the left and laterally. This will be expressed as ST elevation in some or all of leads V1 through V6. The location of the occlusion within the left anterior descending coronary artery, that is, whether proximal or distal, is suggested by the chest leads in which the ST-segment elevation occurs and the presence of ST-segment elevation or depression in other leads. Occlusion of the proximal left anterior descending coronary artery above the first septal and first diagonal branches results in involvement of the basal portion of the left ventricle, as well as the anterior and lateral walls and the interventricular septum. This will result in the ST-segment spatial vector being directed superiorly and to the left and will be associated with ST-segment elevation in leads V1 through V4, I, aVL, and often aVR. It will also be associated with reciprocal ST-segment depression in the leads whose positive poles are positioned inferiorly, that is, leads II, III, aVF, and often V5 (Birnbaum et al., 1993). When the occlusion is located between the first septal and first diagonal branches, the basal interventricular septum will be spared, and the ST segment in lead V1 will not be elevated. In that situation, the ST-segment vector will be directed toward aVL, which will be elevated,

line drawn from the J-point to the apex of T-wave Fig. 2. Patterns of ST-segment elevation at ECG and away from the positive pole of lead III, which will show depression of the ST segment. When the occlusion is located more distally, that is, below both the first septal and first diagonal branches, the basal portion of the left ventricle will not be involved, and the STsegment vector will be oriented more inferiorly. Thus, the ST segment will not be elevated in leads V1, aVR, or aVL, and the ST segment will not be depressed in leads II, III, or aVF. Indeed, because of the inferior orientation of the ST-segment vector, elevation of the ST segment in leads II, III, and aVF may occur. In addition, ST-segment elevation may be more prominent in leads V3 through V6 and less prominent in V2than in the more proximal occlusions (Engelen et al., 1999). Inferior wall infarction that results in ST-segment elevation in only leads II, III, and aVF may be the result of occlusion of either the right coronary artery or the left circumflex coronary artery, depending on which provides the posterior descending branch, that is, which is the dominant vessel. When the right coronary artery is occluded, the spatial vector of the ST segment will usually be directed more to the right than when the left circumflex is occluded. This will result in greater ST-segment elevation in lead III than in lead II and will often be associated with ST-segment depression in leads I and aVL, leads in which the positive poles are oriented to the left and superiorly. However, recently these criteria resulted less accurate in patients with electrocardiographic small inferior myocardial infarction (Verouden et al., 2009). Indeed, when the RCA is occluded in its proximal portion, ischemia/infarction of the right ventricle may occur, which causes the spatial vector of the ST-segment shift to be directed to the right and anteriorly, as well as inferiorly. This will result in ST-segment elevation in leads placed on the right anterior chest, in positions referred to as V3R and V4R, and often in lead V1 (Correale et al., 1999). Lead V4R is the most commonly used right-sided chest lead. It is of great value in diagnosing right ventricular involvement in the setting of an inferior wall infarction and in making the distinction between right coronary artery and left circumflex coronary artery occlusion and between proximal and distal right coronary artery occlusion. It is important to recognize that the ST elevation in the right-sided chest leads associated with right ventricular infarction persists for a much shorter period of time than the ST elevation connoting inferior wall infarction that occurs in the extremity leads. For this reason, leads V3R and V4R should be recorded as rapidly as possible after the onset of chest pain. STsegment depression in leads V1, V2, and V3 that occurs in association with an inferior wall infarction may be caused by occlusion of either the right coronary or the left circumflex artery. This ECG pattern has been termed posterior or posterolateral ischemia since the early reports based on anatomic and pathological studies of ex vivo. However, recent in vivo imaging studies, including magnetic resonance imaging, have demonstrated that the region referred to as the posterior wall was lateral rather than posterior since the oblique position of the heart within the thorax: correlating the ECG patterns of healed myocardial infarctions to their anatomic location as determined by magnetic resonance imaging, the most frequent cause of abnormally tall and broad R waves in leads V1 and V2 was involvement of the lateral and not the posterior wall of the left ventricle (Bayes de Luna et al., 2006a). On these basis it has been proposed that the term posterior be replaced by the designation lateral (Cerqueira et al., 2002). Therefore, the terms posterior ischemia and posterior infarction be replaced by the terms lateral, inferolateral, or basal-lateral depending on the associated changes in II, III, aVF, V1, V5, and V6. Such terminology has been endorsed by the International Society for Holter and Noninvasive Electrocardiography (Bayes de Luna A et al., 2006b). It is not possible to determine whether the right coronary artery or left circumflex

ECG in Acute Myocardial Infarction in the Reperfusion Era 119

presentation of acute coronary occlusion, denying patients potentially beneficial reperfusion therapy. It is important to note that, in the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries (GUSTO-I) trial patients who did not develop Q-wave after fibrinolysis for STEMI had a lower mortality rate when compared to those who did develop Q wave at 30 days post infarction and 1 year post infarction (Bargelata et al., 1997). Thus, the absence of Q-wave after reperfusion therapy is a powerful

In healthy patients, T-wave are normally upright in the left-sided leads (I, II, V3-V6). Within hours to days, an evolving STEMI will typically demonstrate T-wave inversion (Goldberger, 1991).The inverted T-wave appear generally in the same leads showing ST-segment elevation (Oliva et al., 1993). The morphology of inverted T-wave tends to be symmetric (Goldschlager & Goldman,1989). In the course of an evolving STEMI, T-wave inversion occurs when ischemia involves the epicardium. T-wave inversion is hypothesized by Mandel et al. to occur because of delayed depolarization in ischemic tissue (Mandel et al., 1968). In normal hearts, the epicardium is the first to depolarize, whereas the endocardium is the last. Delayed repolarization of the epicardium during ischemia reverses the direction of the ventricular repolarization current. With repolarization moving in the direction of endocardium to epicardium, the repolarization vector also reverses, causing a downward deflection of the T-wave (Smith & Whitwam, 2005). T-wave inversion occurs in approximately 3/4 of all patients with a completed myocardial necrosis (Goldschlager & Goldman,1989). Presence of T-wave inversion in precordial leads, of at least 2 mm, has a positive predictive value of 86% for left anterior descending artery stenosis (Haines et al., 1983). Indeed, a deepening T-wave soon after fibrinolysis may then determine successful reperfusion. However, normalization of T-waves may also predict a lower morbidity months after STEMI. One study by Tamura et al. found that patients with T-wave normalization within 6 months of infarction had higher left ventricular ejection fraction than those who did not, indicating that patients with normalization of inverted T waves had improved myocardial recovery (Tamura et al., 1999). The morphology of the T-wave inversion may help to differentiate between these other causes of T-wave inversion. Pacemaker T wave, in other words T wave inversion related to permanent ventricular pacemakers, tend to be broader than the narrower infarction T waves. A prolonged QTc distinguishes long QT syndrome. In mitral valve prolapse, T wave may be flattened or even inverted in inferior or lateral leads (Goldberger, 1991). In stroke, T waves tend to be very

marker of non-transmural necrosis and then of favorable prognosis.

wide and the QT interval prolonged (Cropp & Manning, 1960).

In not-reperfused STEMI, after a peak elevation approximately 1 hour after the onset of chest pain, the ST segment reaches a plateau at about 12 hours (Essen et al., 1979), and a complete resolution within 2 weeks in 95% of patients with inferior STEMI and 40% of patients with anterior STEMI (Mills et al., 1975). Even if the resolution of the ST-segment elevation may rarely occur from spontaneous reperfusion (Parikh & Shah, 1997), nowadays the normalization of the ST-segment can be observed in the majority of patients as result of successful reperfusion therapy. In fact, after successful fibrinolysis or mechanical reopening of infarct-related artery, abrupt changes occur in ECG as result of recovery in depolarization

**2.5 Normalization of the ST segment** 

**2.4 T-wave inversion** 

vessel is occluded when changes of inferior wall ischemia/infarction are accompanied by depression of the ST-segment in leads V1, V2, and V3; however, the absence of such changes is more suggestive of right coronary than left circumflex artery occlusion. When the left circumflex is occluded, the spatial vector of the ST-segment in the frontal plane is more likely to be directed to the left. For this reason, the ST-segment may be elevated to a greater extent in lead II than in lead III and may be isoelectric or elevated in leads I and aVL (Bairey et al., 1987). Conversely, when a dominant right coronary artery is occluded proximally, left posterolateral and right ventricular wall involvement will be present, and the posteriorly directed ST-segment vector associated with this involvement may cancel the ST-segment elevation in lead V1 anticipated by right ventricular involvement and vice versa. The American College of Cardiology (ACC)/American Heart Association (AHA) guidelines for the management for patients with acute myocardial infarction (ACC/AHA, 2009) note the presence of electrocardiographic ST-segment elevation of greater than 0.1 mV in two anatomically contiguous leads; they suggest that such a finding is a Class I indication for urgent reperfusion therapy in the patient presumed to have STEMI. However, in few patients the presence of a left bundle branch block make the ECG less specific for the diagnosis of STEMI, because LBBB resembles STEMI changes. In this setting, the presence of suggestive symptoms and/or the certainty of the new-onset of conduction disorders may be helpful in diagnosis. Nevertheless, when these are not conclusive for diagnosis, the presence of some ECG criteria, pertaining the ST shift in relation to QRS vectors, may still indicate the diagnosis. To this regard, the ECG should be interpreted using the "rule of appropriate discordance", described by Sgarabossa and colleagues (Sgarbossa, 1996, 1998). They identified three independent electrocardiographic criteria suggesting for STEMI diagnosis in presence of LBBB: ST-segment elevation of at least 1mm that is concordant with the QRS complex; ST-segment depression of at least 1mm in leads V2 and V3; and ST-segment elevation of at least 5 mm that is discordant with the QRS complex. The Sgarbossa criteria provide a simple and practical diagnostic approach to identify STEMI in presence of LBBB, contributing to better address risk stratification and to optimize the risk-benefit ratio of reperfusion therapy in this challenging and high-risk population. In fact, the presence of LBBB in patients with acute myocardial infarction is usually related to large necrosis and consequently to high risk of complications and death. In fact, the new onset LBBB is related to the occlusion of the proximal left anterior descending artery and a large amount of jeopardized myocardium (Opolski et al., 1986). On the other hand, a pre-existing left bundle branch block is a powerful marker of depressed left ventricular systolic function, and any additional loss of myocardium is likely to result in large infarction and cardiogenic shock (Hamby et al., 1983)

### **2.3 Abnormal Q-wave**

Q-wave are commonly present in normal ECG. Abnormal Q-wave suggesting myocardial necrosis have grater negative deflection and longer duration. Pathologic Q-wave typically appear within the first 9 hours of infarction, with a wide interval, ranging from few minutes to 24 hours (Perera, 2004; Goldberger, 1991). In particular in the evolution of non-reperfused myocardial infarction, Q-wave usually appear within 9 hours from coronary occlusion (Bär et al., 1996). However, it is not infrequent to observe Q-wave early after symptom onset. Abnormal Q-wave may be related to ischemia of the conduction system (Raitt at al., 1995; Smith & Whitwam, 2006). Thus, Q-wave should not be used exclusively as a marker of late presentation of acute coronary occlusion, denying patients potentially beneficial reperfusion therapy. It is important to note that, in the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries (GUSTO-I) trial patients who did not develop Q-wave after fibrinolysis for STEMI had a lower mortality rate when compared to those who did develop Q wave at 30 days post infarction and 1 year post infarction (Bargelata et al., 1997). Thus, the absence of Q-wave after reperfusion therapy is a powerful marker of non-transmural necrosis and then of favorable prognosis.

### **2.4 T-wave inversion**

118 Advances in Electrocardiograms – Clinical Applications

vessel is occluded when changes of inferior wall ischemia/infarction are accompanied by depression of the ST-segment in leads V1, V2, and V3; however, the absence of such changes is more suggestive of right coronary than left circumflex artery occlusion. When the left circumflex is occluded, the spatial vector of the ST-segment in the frontal plane is more likely to be directed to the left. For this reason, the ST-segment may be elevated to a greater extent in lead II than in lead III and may be isoelectric or elevated in leads I and aVL (Bairey et al., 1987). Conversely, when a dominant right coronary artery is occluded proximally, left posterolateral and right ventricular wall involvement will be present, and the posteriorly directed ST-segment vector associated with this involvement may cancel the ST-segment elevation in lead V1 anticipated by right ventricular involvement and vice versa. The American College of Cardiology (ACC)/American Heart Association (AHA) guidelines for the management for patients with acute myocardial infarction (ACC/AHA, 2009) note the presence of electrocardiographic ST-segment elevation of greater than 0.1 mV in two anatomically contiguous leads; they suggest that such a finding is a Class I indication for urgent reperfusion therapy in the patient presumed to have STEMI. However, in few patients the presence of a left bundle branch block make the ECG less specific for the diagnosis of STEMI, because LBBB resembles STEMI changes. In this setting, the presence of suggestive symptoms and/or the certainty of the new-onset of conduction disorders may be helpful in diagnosis. Nevertheless, when these are not conclusive for diagnosis, the presence of some ECG criteria, pertaining the ST shift in relation to QRS vectors, may still indicate the diagnosis. To this regard, the ECG should be interpreted using the "rule of appropriate discordance", described by Sgarabossa and colleagues (Sgarbossa, 1996, 1998). They identified three independent electrocardiographic criteria suggesting for STEMI diagnosis in presence of LBBB: ST-segment elevation of at least 1mm that is concordant with the QRS complex; ST-segment depression of at least 1mm in leads V2 and V3; and ST-segment elevation of at least 5 mm that is discordant with the QRS complex. The Sgarbossa criteria provide a simple and practical diagnostic approach to identify STEMI in presence of LBBB, contributing to better address risk stratification and to optimize the risk-benefit ratio of reperfusion therapy in this challenging and high-risk population. In fact, the presence of LBBB in patients with acute myocardial infarction is usually related to large necrosis and consequently to high risk of complications and death. In fact, the new onset LBBB is related to the occlusion of the proximal left anterior descending artery and a large amount of jeopardized myocardium (Opolski et al., 1986). On the other hand, a pre-existing left bundle branch block is a powerful marker of depressed left ventricular systolic function, and any additional loss of myocardium is likely to result in large infarction and cardiogenic shock

Q-wave are commonly present in normal ECG. Abnormal Q-wave suggesting myocardial necrosis have grater negative deflection and longer duration. Pathologic Q-wave typically appear within the first 9 hours of infarction, with a wide interval, ranging from few minutes to 24 hours (Perera, 2004; Goldberger, 1991). In particular in the evolution of non-reperfused myocardial infarction, Q-wave usually appear within 9 hours from coronary occlusion (Bär et al., 1996). However, it is not infrequent to observe Q-wave early after symptom onset. Abnormal Q-wave may be related to ischemia of the conduction system (Raitt at al., 1995; Smith & Whitwam, 2006). Thus, Q-wave should not be used exclusively as a marker of late

(Hamby et al., 1983)

**2.3 Abnormal Q-wave** 

In healthy patients, T-wave are normally upright in the left-sided leads (I, II, V3-V6). Within hours to days, an evolving STEMI will typically demonstrate T-wave inversion (Goldberger, 1991).The inverted T-wave appear generally in the same leads showing ST-segment elevation (Oliva et al., 1993). The morphology of inverted T-wave tends to be symmetric (Goldschlager & Goldman,1989). In the course of an evolving STEMI, T-wave inversion occurs when ischemia involves the epicardium. T-wave inversion is hypothesized by Mandel et al. to occur because of delayed depolarization in ischemic tissue (Mandel et al., 1968). In normal hearts, the epicardium is the first to depolarize, whereas the endocardium is the last. Delayed repolarization of the epicardium during ischemia reverses the direction of the ventricular repolarization current. With repolarization moving in the direction of endocardium to epicardium, the repolarization vector also reverses, causing a downward deflection of the T-wave (Smith & Whitwam, 2005). T-wave inversion occurs in approximately 3/4 of all patients with a completed myocardial necrosis (Goldschlager & Goldman,1989). Presence of T-wave inversion in precordial leads, of at least 2 mm, has a positive predictive value of 86% for left anterior descending artery stenosis (Haines et al., 1983). Indeed, a deepening T-wave soon after fibrinolysis may then determine successful reperfusion. However, normalization of T-waves may also predict a lower morbidity months after STEMI. One study by Tamura et al. found that patients with T-wave normalization within 6 months of infarction had higher left ventricular ejection fraction than those who did not, indicating that patients with normalization of inverted T waves had improved myocardial recovery (Tamura et al., 1999). The morphology of the T-wave inversion may help to differentiate between these other causes of T-wave inversion. Pacemaker T wave, in other words T wave inversion related to permanent ventricular pacemakers, tend to be broader than the narrower infarction T waves. A prolonged QTc distinguishes long QT syndrome. In mitral valve prolapse, T wave may be flattened or even inverted in inferior or lateral leads (Goldberger, 1991). In stroke, T waves tend to be very wide and the QT interval prolonged (Cropp & Manning, 1960).

### **2.5 Normalization of the ST segment**

In not-reperfused STEMI, after a peak elevation approximately 1 hour after the onset of chest pain, the ST segment reaches a plateau at about 12 hours (Essen et al., 1979), and a complete resolution within 2 weeks in 95% of patients with inferior STEMI and 40% of patients with anterior STEMI (Mills et al., 1975). Even if the resolution of the ST-segment elevation may rarely occur from spontaneous reperfusion (Parikh & Shah, 1997), nowadays the normalization of the ST-segment can be observed in the majority of patients as result of successful reperfusion therapy. In fact, after successful fibrinolysis or mechanical reopening of infarct-related artery, abrupt changes occur in ECG as result of recovery in depolarization

ECG in Acute Myocardial Infarction in the Reperfusion Era 121

2010). Generally factors which preclude "waiting for PCI" include young age, anterior MI, and early (<3 hrs of pain) presentation. Factors which make delayed P-PCI the preferred strategy include contraindications to fibrinolysis, cardiogenic shock, advanced age, inferior MI, and delayed presentation (Antman et al., 2004). Whichever reperfusion strategy is chosen, it is important to maximize the effectiveness of that therapy by applying not only prompt, but also appropriate anti-platelet and anti-thrombin adjuncts. The recommendations for these therapies differ with the reperfusion method chosen. Appropriate protocol development demands maximization of the effectiveness of anti-

Ventricular tachycardia (VT), Ventricular Fibrillation (VF) and complete atrio-ventricular (BAVC) block, may be the first manifestation of ischemia and requires immediate correction, since these arrhythmias may cause sudden cardiac death. VF and VT have been reported up to 20% of patients with STEMI (Henkel et al., 2006). Often arrhythmias are the manifestation of a serious underlying disorder, such as persisting ischemia, severe pump failure, or endogenous factors such as abnormal potassium levels, autonomic imbalances, hypoxia, and acid-base disturbances, that may require corrective measures. The need for arrhythmias

VF occurring within 48 hours of the onset of STEMI has been related to higher in-hospital mortality, but not to long-term mortality. The major determinants of risk of sudden death are related more to the severity of the cardiac disease and less to frequency of classification

Ventricular ectopic beats are common during the initial phase of STEMI. Irrespective of their complexity (multiform QRS complex beats, short runs of ventricular beats or the R-on-T

Either not sustained VT (lasting <30s) not accelerated idioventricular rhythm (usually a harmless consequence of reperfusion with a ventricular rate<120beats), occurring in the setting of STEMI, serve as a reliably predictive marker of early VF. Sustained and/or haemodinamically compromising VT (occurring in 3%) requires suppressive therapy, and outlined in the guidelines for ventricular arrythmias (Zipes et al., 2006). Pulsless VT and VF

Atrial fibrillation (AF), which complicates 10-20% of STEMI is more prevalent in older patients and in those with severe LV damage and heart failure. AF is associated with increased in-hospital mortality (Fuster et al., 2006). In many cases this arrhythmia is well tolerated and no specific treatment is required. In other instances, the high ventricular

platelet and anti-thrombin agents with each reperfusion choice.

**4.1 Ventricular arrhythmias** 

**4.2 Ventricular ectopic rhythms** 

**4.4 Supraventricular arrhythmias** 

of ventricular arrhythmias (Huikuri et al., 2001).

phenomenon) their value, as predictor of VF, is questionable.

should be managed according to the resuscitation guidelines.

response contributes to heart failure and prompt treatment is needed.

**4.3 Ventricular tachycardia and Ventricular fibrillation** 

**4. Arrhythmias and conduction disturbances in the acute phase** 

treatment depends mainly upon the hemodynamic impact of the rhythm disorder.

currents across the myocites membrane. Thus a prompt decrease in ST-segment elevation is a powerful predictor of reperfusion (Richardson et al., 1988), whereas the persistence of STsegment elevation represent a marker of unsuccessful reperfusion therapy and is an independent determinant of major adverse cardiac event (Claeys et al., 1999). Interestingly, a decrease in ST-segment elevation by at least 50% seems to be associated with 94% positive predictive value for complete reperfusion (Krucoff et al., 1993). Indeed, studies have also found that even after a complete and sustained patency of epicardial infarct-related artery obtained by pharmacological or mechanical recanalization, about one-third of patients still show a persistent ST- segment elevation, as result of unsuccessful reperfusion of the microvasculature (De Lemos & Brunwald, 2001). This condition is known as a no-reflow phenomenon, and has been related to a higher mortality and worse clinical outcome after myocardial infarction (Poli et al., 2002). Thus it is important to remark that normalization of the ST-segment indicates adequate perfusion throughout the myocardial microvasculature rather than epicardial coronary patency.

### **3. Choice of reperfusion strategy**

Primary percutaneous coronary intervention and thrombolysis remain therapies of choice for patients presenting with evolving STEMI. However, clinical outcome after STEMI is mainly related to complete and sustained myocardial reperfusion, but strongly influenced by delay in achieving reperfusion. In fact, the extension of necrosis is time dependent, with a wave front developing from the subendocardium and extending transmurally to the epicardium over time. For every 30 minutes duration of ischemia, there is an 8-10% increase in mortality (Pinto et al., 2006). Reperfusion therapy, with dissolution or removal of the intracoronary thrombus, provides the best chance for mortality reduction. The Focused Update gives primary percutaneous coronary intervention (P-PCI) a Class IA recommendation for reperfusion, as long as it can be accomplished with a first medical contact to balloon inflation time of 90 minutes or less (Antman et al., 2008). Fibrinolysis, which is less effective than P-PCI in head-to-head trials, is given a Class IB rating as an alternative to P-PCI, as long as P-PCI can't be accomplished within 90 minutes. Although P-PCI is commonly more effective than thrombolytic therapy (TT) for the treatment of patients with STEMI, the mortality benefit of P-PCI over TT is risk and time-dependent (Antman et al., 2008; Keeley et al., 2003; Tarantini et al., 2005; Thune et al., 2005; Cannon et al., 2000; De Luca et al., 2003). As the time delay for performing P-PCI increases, the mortality benefit of P-PCI compared with fibrinolysis decreases. The P-PCI strategy may not reduce mortality when the delay is 60 min compared with immediate administration of a fibrin-specific lytic agent (Nallamothu & Bates, 2003). However, the value of 60 min is still controversial and should not be stated so categorically; other authors, for example, found that longer P-PCIrelated delays do not negate the survival benefit of PPCI even when the delay is up to 3 h (Boersma et al., 2006; Stenestrand et al., 2006; Betriu & Masotti, 2005). Moreover, a recent evaluation of registry data has shown that the acceptable P-PCI-related delay depends upon the risk of the patient (Pinto et al., 2006). It has been explored the relationship between risk and P-PCI delay, adjusted for the delay at presentation, which leads to equivalent 30-day mortality between P-PCI and fibrin-specific thrombolytic therapy. Baseline mortality risk of STEMI patients is a major determinant of the acceptable time delay to choose the most appropriate therapy. Although a longer delay lowers the survival advantage of P-PCI, a longer P-PCI-related delay could be acceptable in high-risk STEMI patients (Tarantini et al.,

currents across the myocites membrane. Thus a prompt decrease in ST-segment elevation is a powerful predictor of reperfusion (Richardson et al., 1988), whereas the persistence of STsegment elevation represent a marker of unsuccessful reperfusion therapy and is an independent determinant of major adverse cardiac event (Claeys et al., 1999). Interestingly, a decrease in ST-segment elevation by at least 50% seems to be associated with 94% positive predictive value for complete reperfusion (Krucoff et al., 1993). Indeed, studies have also found that even after a complete and sustained patency of epicardial infarct-related artery obtained by pharmacological or mechanical recanalization, about one-third of patients still show a persistent ST- segment elevation, as result of unsuccessful reperfusion of the microvasculature (De Lemos & Brunwald, 2001). This condition is known as a no-reflow phenomenon, and has been related to a higher mortality and worse clinical outcome after myocardial infarction (Poli et al., 2002). Thus it is important to remark that normalization of the ST-segment indicates adequate perfusion throughout the myocardial microvasculature

Primary percutaneous coronary intervention and thrombolysis remain therapies of choice for patients presenting with evolving STEMI. However, clinical outcome after STEMI is mainly related to complete and sustained myocardial reperfusion, but strongly influenced by delay in achieving reperfusion. In fact, the extension of necrosis is time dependent, with a wave front developing from the subendocardium and extending transmurally to the epicardium over time. For every 30 minutes duration of ischemia, there is an 8-10% increase in mortality (Pinto et al., 2006). Reperfusion therapy, with dissolution or removal of the intracoronary thrombus, provides the best chance for mortality reduction. The Focused Update gives primary percutaneous coronary intervention (P-PCI) a Class IA recommendation for reperfusion, as long as it can be accomplished with a first medical contact to balloon inflation time of 90 minutes or less (Antman et al., 2008). Fibrinolysis, which is less effective than P-PCI in head-to-head trials, is given a Class IB rating as an alternative to P-PCI, as long as P-PCI can't be accomplished within 90 minutes. Although P-PCI is commonly more effective than thrombolytic therapy (TT) for the treatment of patients with STEMI, the mortality benefit of P-PCI over TT is risk and time-dependent (Antman et al., 2008; Keeley et al., 2003; Tarantini et al., 2005; Thune et al., 2005; Cannon et al., 2000; De Luca et al., 2003). As the time delay for performing P-PCI increases, the mortality benefit of P-PCI compared with fibrinolysis decreases. The P-PCI strategy may not reduce mortality when the delay is 60 min compared with immediate administration of a fibrin-specific lytic agent (Nallamothu & Bates, 2003). However, the value of 60 min is still controversial and should not be stated so categorically; other authors, for example, found that longer P-PCIrelated delays do not negate the survival benefit of PPCI even when the delay is up to 3 h (Boersma et al., 2006; Stenestrand et al., 2006; Betriu & Masotti, 2005). Moreover, a recent evaluation of registry data has shown that the acceptable P-PCI-related delay depends upon the risk of the patient (Pinto et al., 2006). It has been explored the relationship between risk and P-PCI delay, adjusted for the delay at presentation, which leads to equivalent 30-day mortality between P-PCI and fibrin-specific thrombolytic therapy. Baseline mortality risk of STEMI patients is a major determinant of the acceptable time delay to choose the most appropriate therapy. Although a longer delay lowers the survival advantage of P-PCI, a longer P-PCI-related delay could be acceptable in high-risk STEMI patients (Tarantini et al.,

rather than epicardial coronary patency.

**3. Choice of reperfusion strategy** 

2010). Generally factors which preclude "waiting for PCI" include young age, anterior MI, and early (<3 hrs of pain) presentation. Factors which make delayed P-PCI the preferred strategy include contraindications to fibrinolysis, cardiogenic shock, advanced age, inferior MI, and delayed presentation (Antman et al., 2004). Whichever reperfusion strategy is chosen, it is important to maximize the effectiveness of that therapy by applying not only prompt, but also appropriate anti-platelet and anti-thrombin adjuncts. The recommendations for these therapies differ with the reperfusion method chosen. Appropriate protocol development demands maximization of the effectiveness of antiplatelet and anti-thrombin agents with each reperfusion choice.

### **4. Arrhythmias and conduction disturbances in the acute phase**

Ventricular tachycardia (VT), Ventricular Fibrillation (VF) and complete atrio-ventricular (BAVC) block, may be the first manifestation of ischemia and requires immediate correction, since these arrhythmias may cause sudden cardiac death. VF and VT have been reported up to 20% of patients with STEMI (Henkel et al., 2006). Often arrhythmias are the manifestation of a serious underlying disorder, such as persisting ischemia, severe pump failure, or endogenous factors such as abnormal potassium levels, autonomic imbalances, hypoxia, and acid-base disturbances, that may require corrective measures. The need for arrhythmias treatment depends mainly upon the hemodynamic impact of the rhythm disorder.

### **4.1 Ventricular arrhythmias**

VF occurring within 48 hours of the onset of STEMI has been related to higher in-hospital mortality, but not to long-term mortality. The major determinants of risk of sudden death are related more to the severity of the cardiac disease and less to frequency of classification of ventricular arrhythmias (Huikuri et al., 2001).

### **4.2 Ventricular ectopic rhythms**

Ventricular ectopic beats are common during the initial phase of STEMI. Irrespective of their complexity (multiform QRS complex beats, short runs of ventricular beats or the R-on-T phenomenon) their value, as predictor of VF, is questionable.

### **4.3 Ventricular tachycardia and Ventricular fibrillation**

Either not sustained VT (lasting <30s) not accelerated idioventricular rhythm (usually a harmless consequence of reperfusion with a ventricular rate<120beats), occurring in the setting of STEMI, serve as a reliably predictive marker of early VF. Sustained and/or haemodinamically compromising VT (occurring in 3%) requires suppressive therapy, and outlined in the guidelines for ventricular arrythmias (Zipes et al., 2006). Pulsless VT and VF should be managed according to the resuscitation guidelines.

### **4.4 Supraventricular arrhythmias**

Atrial fibrillation (AF), which complicates 10-20% of STEMI is more prevalent in older patients and in those with severe LV damage and heart failure. AF is associated with increased in-hospital mortality (Fuster et al., 2006). In many cases this arrhythmia is well tolerated and no specific treatment is required. In other instances, the high ventricular response contributes to heart failure and prompt treatment is needed.

ECG in Acute Myocardial Infarction in the Reperfusion Era 123

Many studies, evaluating the outcomes of primary angioplasty in STEMI, found that persistent ST-segment elevation after coronary flow restoration, is one of the independent determinant of adverse cardiac event (Schröder et al., 1994; de Lemos & Braunwald, 2001). In fact, patients with persistent ST-segment elevation, even after a successful restoration of normal antegrade coronary flow in the epicardial artery, show absent or inadequate flow at level of microvasculature (van't Hof et a., 1997). This phenomenon, known as a no-reflow phenomenon, has been described in animal and clinical studies, involving about one third of patients who underwent successful recanalization of the infarct-related artery. This condition, has been related to larger necrosis, adverse ventricular remodeling and higher morbidity/mortality at short and long-term follow-up. Otherwise, a resolution of STsegment elevation by at least 50% is associated with a high positive predictive value for successful myocardial reperfusion. In this setting, the analysis of ST-segment evolution during and after coronary recanalization represents an useful tool to guide further pharmacological treatments, as well as more aggressive management of these patients. Different methods, cut-offs, and timing have been proposed to evaluate ST-segment resolution. In most studies, resolution of ST-segment elevation has been expressed as percentage of resolution of the sum of ST-segment elevation in all leads (van't Hof et a., 1997; Schröder et al., 1994; de Lemos JA & Braunwald, 2001; Zeymer et al., 2003). To this purpose, ST sum should take into account not only the ST shift in all leads showing ST elevation, but also the reciprocal ST deviation in leads showing ST depression. However, measuring ST resolution from all leads is time consuming and may be influenced by patient's position and by changes in position of lead electrodes. In order to simplify ST resolution assessment, other authors have proposed an alternative method based on measurement of ST resolution in only the single lead showing the maximum deviation before reperfusion: "the single lead ST resolution" (Schröder et al., 2001). In the single lead method, ST resolution is measured by comparing one ECG lead with the most prominent ST-segment shift at baseline and at a given time-point after reperfusion therapy, irrespective of the ECG lead measure at baseline. This method resulted as simple as accurate when compared to conventional model of sum ST resolution model. The optimal cut-off for defining reperfusion effectiveness and then mortality risk groups were assessed by statistical methods. Applying 2 cut-offs provides the most powerful stratification of high and low mortality risk group. To this purpose sum ST resolution is conventionally categorized as complete (≥ 70%), partial (<70% to 30%), and no (≤ 30%) ST-segment elevation resolution (Schroder et al., 1994). Although different time points have been reported across the studies evaluating the relationship between ST resolution and outcome, such as 30, 60, or even 90 minutes after reperfusion therapy, no significant differences between various time points were found, and the ideal time of measuring ST resolution remains unclear. However, these studies were mostly conducted in patients receiving fibrinolytic therapy, and may not directly applicable when assessing success of primary angioplasty. In fact, after fibrinolysis for STEMI the ECG for measuring ST resolution is usually taken 60 to 90 minutes after the onset of therapy, in order to detect by means of noninvasive tool the patency of epicardial vessel, rather than the reperfusion at microvascular level. In studies assessing the ST resolution on ECG, the timing of ECG after primary angioplasty was highly variable, ranging from 30 minutes after angioplasty to 60 minutes, or even several hours later. Strong evidences showed that ST resolution as evaluated 30

**6. ECG in mechanical reperfusion - implication for prognosis** 

### **4.5 Sinus bradycardia and heart block**

*Sinus bradicardya*: is common (9-25%) in the first hour, particularly in inferior infarction (Goldestein et al 2005). If associated with hemodynamic compromise it should be treated. *AV block:* Data from four large, randomized trials suggest that AV block occurs in almost 7% (Meine et al., 2005) and persistent LBBB in up to 5.3% of cases of STEMI (Newby et al., 1996). Patients with peri-infarction AV block have an higher in hospital mortality than those with preserved AV conduction (Meine et al., 2005). The increased mortality seems related to the extensive myocardial damage required to develop heart block rather than to heart block itself. AV block associated with inferior wall infarction is usually transient, whereas AV block related to anterior wall infarction is more often located below the AV node and associated with an unstable, wide QRS escape rhythm due to extensive myocardial necrosis. A new LBBB usually indicated extensive anterior infarction with high probability to develop complete AV block and pump failure. The preventive placement of a temporary pacing electrode may be warranted. Raccomandations for permanent cardiac pacing for persistent conduction disturbances (>14 days) due to STEMI are outlined in the ESC Guidelines for cardiac pacing.

### **5. ECG in pharmacological reperfusion - implications for adjunctive therapies**

As a tool to identify epicardial reperfusion all methods of ST resolution, assessed by either continuous monitoring or static ECG recording, have the limitation that ST-segment changes integrate both epicardial and myocardial reperfusion. A resolution of ST-segment elevation of more than 70% of the initial value at 60 to 90 minutes after the initiation of therapy, is a powerful predictor of successful myocardial reperfusion and is therefore associated with enhanced recovery of LV function, reduced infarct size, and improved prognosis (de Lemos et al., 2000; Zeymer et al., 2001). Thus patients with complete ST-resolution at 90 minutes after fibrinolysis have a > 90% probability of a patent infarct-related artery associated with a successful reperfusion at the microvascular level. However, approximately 50% of patients with no ST-segment resolution after fibrinolysis still show a patent epicardial infarct artery. In fact in these patients the lack of ST resolution is caused by the failure of reperfusion at the level of microvasculature rather than at epicardial vessel. Thus, ST resolution represents a powerful predictor of infarct-related artery patency, but it is less accurate for predicting the persistence of epicardial vessel occlusion after fibrinolysis (Schröder et al., 2004). Therefore, in order to judge the need for adjunctive mechanical reopening of the infarct-related artery after failed fibrinolysis, by the so called "rescue angioplasty", it is important to integrate clinical and ECG data. According to the ACC/AHA guidelines, it is reasonable to monitor the pattern of ST-segment elevation, cardiac rhythm, and clinical symptoms during the 60 to 90 minutes after the initiation of fibrinolytic therapy. Non-invasive findings suggesting for a successful reperfusion include relief of symptoms, maintenance or restoration of hemodynamic and electrical stability, and a reduction of at least 50% in the initial STsegment elevation. In this scenario, the presence of particular arrhythmias, such as not rapid ventricular tachycardia, idioventricular rhythm or not-sustained bradycardia, early after fibrinolytic administration, represents a highly specific marker of reperfusion. Otherwise, persistence of ischemic chest pain, absence of resolution of the qualifying ST-segment elevation, and hemodynamic or electrical instability are generally predictors of failed pharmacological reperfusion, needing rescue angioplasty.

*Sinus bradicardya*: is common (9-25%) in the first hour, particularly in inferior infarction (Goldestein et al 2005). If associated with hemodynamic compromise it should be treated. *AV block:* Data from four large, randomized trials suggest that AV block occurs in almost 7% (Meine et al., 2005) and persistent LBBB in up to 5.3% of cases of STEMI (Newby et al., 1996). Patients with peri-infarction AV block have an higher in hospital mortality than those with preserved AV conduction (Meine et al., 2005). The increased mortality seems related to the extensive myocardial damage required to develop heart block rather than to heart block itself. AV block associated with inferior wall infarction is usually transient, whereas AV block related to anterior wall infarction is more often located below the AV node and associated with an unstable, wide QRS escape rhythm due to extensive myocardial necrosis. A new LBBB usually indicated extensive anterior infarction with high probability to develop complete AV block and pump failure. The preventive placement of a temporary pacing electrode may be warranted. Raccomandations for permanent cardiac pacing for persistent conduction disturbances (>14 days) due to STEMI are outlined in the ESC Guidelines for

**5. ECG in pharmacological reperfusion - implications for adjunctive therapies**  As a tool to identify epicardial reperfusion all methods of ST resolution, assessed by either continuous monitoring or static ECG recording, have the limitation that ST-segment changes integrate both epicardial and myocardial reperfusion. A resolution of ST-segment elevation of more than 70% of the initial value at 60 to 90 minutes after the initiation of therapy, is a powerful predictor of successful myocardial reperfusion and is therefore associated with enhanced recovery of LV function, reduced infarct size, and improved prognosis (de Lemos et al., 2000; Zeymer et al., 2001). Thus patients with complete ST-resolution at 90 minutes after fibrinolysis have a > 90% probability of a patent infarct-related artery associated with a successful reperfusion at the microvascular level. However, approximately 50% of patients with no ST-segment resolution after fibrinolysis still show a patent epicardial infarct artery. In fact in these patients the lack of ST resolution is caused by the failure of reperfusion at the level of microvasculature rather than at epicardial vessel. Thus, ST resolution represents a powerful predictor of infarct-related artery patency, but it is less accurate for predicting the persistence of epicardial vessel occlusion after fibrinolysis (Schröder et al., 2004). Therefore, in order to judge the need for adjunctive mechanical reopening of the infarct-related artery after failed fibrinolysis, by the so called "rescue angioplasty", it is important to integrate clinical and ECG data. According to the ACC/AHA guidelines, it is reasonable to monitor the pattern of ST-segment elevation, cardiac rhythm, and clinical symptoms during the 60 to 90 minutes after the initiation of fibrinolytic therapy. Non-invasive findings suggesting for a successful reperfusion include relief of symptoms, maintenance or restoration of hemodynamic and electrical stability, and a reduction of at least 50% in the initial STsegment elevation. In this scenario, the presence of particular arrhythmias, such as not rapid ventricular tachycardia, idioventricular rhythm or not-sustained bradycardia, early after fibrinolytic administration, represents a highly specific marker of reperfusion. Otherwise, persistence of ischemic chest pain, absence of resolution of the qualifying ST-segment elevation, and hemodynamic or electrical instability are generally predictors of failed

pharmacological reperfusion, needing rescue angioplasty.

**4.5 Sinus bradycardia and heart block** 

cardiac pacing.

### **6. ECG in mechanical reperfusion - implication for prognosis**

Many studies, evaluating the outcomes of primary angioplasty in STEMI, found that persistent ST-segment elevation after coronary flow restoration, is one of the independent determinant of adverse cardiac event (Schröder et al., 1994; de Lemos & Braunwald, 2001). In fact, patients with persistent ST-segment elevation, even after a successful restoration of normal antegrade coronary flow in the epicardial artery, show absent or inadequate flow at level of microvasculature (van't Hof et a., 1997). This phenomenon, known as a no-reflow phenomenon, has been described in animal and clinical studies, involving about one third of patients who underwent successful recanalization of the infarct-related artery. This condition, has been related to larger necrosis, adverse ventricular remodeling and higher morbidity/mortality at short and long-term follow-up. Otherwise, a resolution of STsegment elevation by at least 50% is associated with a high positive predictive value for successful myocardial reperfusion. In this setting, the analysis of ST-segment evolution during and after coronary recanalization represents an useful tool to guide further pharmacological treatments, as well as more aggressive management of these patients. Different methods, cut-offs, and timing have been proposed to evaluate ST-segment resolution. In most studies, resolution of ST-segment elevation has been expressed as percentage of resolution of the sum of ST-segment elevation in all leads (van't Hof et a., 1997; Schröder et al., 1994; de Lemos JA & Braunwald, 2001; Zeymer et al., 2003). To this purpose, ST sum should take into account not only the ST shift in all leads showing ST elevation, but also the reciprocal ST deviation in leads showing ST depression. However, measuring ST resolution from all leads is time consuming and may be influenced by patient's position and by changes in position of lead electrodes. In order to simplify ST resolution assessment, other authors have proposed an alternative method based on measurement of ST resolution in only the single lead showing the maximum deviation before reperfusion: "the single lead ST resolution" (Schröder et al., 2001). In the single lead method, ST resolution is measured by comparing one ECG lead with the most prominent ST-segment shift at baseline and at a given time-point after reperfusion therapy, irrespective of the ECG lead measure at baseline. This method resulted as simple as accurate when compared to conventional model of sum ST resolution model. The optimal cut-off for defining reperfusion effectiveness and then mortality risk groups were assessed by statistical methods. Applying 2 cut-offs provides the most powerful stratification of high and low mortality risk group. To this purpose sum ST resolution is conventionally categorized as complete (≥ 70%), partial (<70% to 30%), and no (≤ 30%) ST-segment elevation resolution (Schroder et al., 1994). Although different time points have been reported across the studies evaluating the relationship between ST resolution and outcome, such as 30, 60, or even 90 minutes after reperfusion therapy, no significant differences between various time points were found, and the ideal time of measuring ST resolution remains unclear. However, these studies were mostly conducted in patients receiving fibrinolytic therapy, and may not directly applicable when assessing success of primary angioplasty. In fact, after fibrinolysis for STEMI the ECG for measuring ST resolution is usually taken 60 to 90 minutes after the onset of therapy, in order to detect by means of noninvasive tool the patency of epicardial vessel, rather than the reperfusion at microvascular level. In studies assessing the ST resolution on ECG, the timing of ECG after primary angioplasty was highly variable, ranging from 30 minutes after angioplasty to 60 minutes, or even several hours later. Strong evidences showed that ST resolution as evaluated 30

ECG in Acute Myocardial Infarction in the Reperfusion Era 125

and type of myocardial damage have been reported. Particularly, the presence of persisting ST-elevation seems related to the presence of large microvascular damage in the context of transmural necrosis (Figure 3). These findings suggest that in this scenario late persistence of ST elevation indicates not only, as predictable, a greater extent of myocardial necrosis, but also, and more interestingly, the presence of severe microvascular damage as shown by cardiac magnetic resonance. Patients exhibiting persistent ST elevation showed more frequently left ventricular aneurysm, even though this difference did not achieve a statistical significance. Taking into account the findings of previous studies, these observations lead to the criticism about wall motion abnormalities as mechanism of electrocardiographic alterations. Recently, Li et al provided direct evidence in animals that opening of sarcolemmal KATP channels underlies ST elevation during ischemia (Li RA et al., 2000). It has also been demonstrated in a swine model that mechanical stimuli can induce marked ST elevation , by producing the stretching activation of KATP channel (Link et al., 1999). On these basis it has been hypothesized that outward bulging of myocardial necrotic wall, producing an abnormal stretch on the adjacent tissue, may alter cellular activity, generating injury currents at this level responsible for the ST elevation (Gussak et al., 2000). Thus patients exhibiting persistence of ST elevation had not only more severe myocardial

**Panel A**: ECG shows neither ST-segment elevation nor pathological Q-wave; the ce-MRI detects nontrasmural necrosis (middle and apical segments) of anterolateral wall, without either persistent microvascular obstruction or left ventricular aneurysm. **Panel B**: ECG shows pathological Q-wave in leads V4 to V6, with persistent ST elevation; the corresponding ce-MRI shows transmural necrosis of the septum, anterolateral wall (middle and apical segments), and apex, with evidence of persistent microvascular obstruction in the setting of necrotic core, without aneurysm. **Panel C :** ECG shows Qwave in leads V1 through V6, DI, aVL, and STE in leads V1 through V6. The corresponding ce-MRI shows a large trasmural necrosis in the septum and anterolateral wall (middle and apical segments), and of the apical segments of inferior wall, with evidence of persistent microvascular obstruction in the

Fig. 3. Different patterns of myocardial structural abnormalities detected by contrast-

enhanced magnetic resonance imaging (ce-MRI) and corresponding 12-leads

necrotic core.

electrocardiogram (ECG).

minutes after angioplasty correlated better with other markers of myocardial perfusion than ST resolution at 60 to 90 minutes. Indeed, recent evidences have shown that early complete ST recovery, as assessed immediately after last contrast injection in the catheterization laboratory, have a better preserved left ventricular ejection fraction and smaller infarct at magnetic resonance than patients showing ST resolution at 30 minutes or later (Haeck et al., 2011). These findings are not only consistent with the hypothesis that ST resolution implies effective microvascular and tissue reperfusion, but also relate the recovery of electrocardiografic changes to salvage of viable myocardium. Indeed, early assessment of ST recovery may represents the appropriate time to identify patients at higher risk of adverse events potentially benefit from additional novel therapies, ideally starting already at the catheterization laboratory.

### **7. ECG in stabilized myocardial infarction**

The ECG in the stabilized phase of STEMI, after reperfusion therapy, represents a simply and universally applicable diagnostic tool to understand the prognosis and to guide further interventions. One method for determining the presence of pathological Q-waves related to myocardial infarction has been the Minnesota Code (Blackburn et a., 1960). This method was developed for diagnosis of infarction rather than the quantification of its size and correlates poorly with anatomically measured infarct size (Pahlm et al., 1998). An improved correlation of changes in the QRS complex with infarct size was the development of a QRS scoring system by Selvester et al. The Selvester QRS scoring system included 54 criteria from the QRS complexes in 10 of the standard leads, which totaled 32 points, each equivalent to approximately 3% of the left ventricular wall (Startt-Selvester et al., 1989). Recently, studies using cardiac magnetic resonance have show that Q-wave predict the location and size of myocardial infarction (Wu E et al., 2001.). Historically the presence of Q-wave on ECG after myocardial infarction has been used in clinical practice to stratify patients in Q-wave and non-Q-wave myocardial infarction, according to larger necrosis and worse outcome discovered in Q-wave infarctions (Stone PH et al., 1988). On these basis, for many years after the original report by Prinzmetal in animal model (Prinzmetal et al., 1954), the presence of Q-wave has been related to transmural infarction, whereas its absence was categorize as non-transmural infarction. Recently, studies based on cardiac magnetic resonance have clarified that, even if this distinction still appears useful to stratify the risk after myocardial infarction, the presence of Q-wave on surface ECG is determined by the total size of necrosis rather than transmural extent of underlying myocardial infarction (Moon et al., 2004). A relative small number of patients after myocardial infarction still show persistence of ST-segment elevation even days and month after the acute event. Historically, this late persistence of ST-segment elevation has been ascribed to left ventricular aneurysm or impending rupture of free wall or ventricular septum, identifying patients at very high risk for heart failure and death (Chon et al., 1967 ). However, this association is among the most controversial in electrocardiography, since previous studies, including echocardiography and angiography, clearly showed a more severe systolic dysfunction and wall motion abnormalities in patients with persistent STE, but failed to demonstrate a definite relationship between this electrocardiographic pattern and left ventricular aneurysm. Moreover, the explanation of the underlying mechanism of persistent STE and its pathological correlates are still unclear (Bar et al., 1984 & Lidsay J et al., 1984 ; Bhatnagar, 1994). Recently, using cardiac magnetic resonance, correlations between this ECG pattern

minutes after angioplasty correlated better with other markers of myocardial perfusion than ST resolution at 60 to 90 minutes. Indeed, recent evidences have shown that early complete ST recovery, as assessed immediately after last contrast injection in the catheterization laboratory, have a better preserved left ventricular ejection fraction and smaller infarct at magnetic resonance than patients showing ST resolution at 30 minutes or later (Haeck et al., 2011). These findings are not only consistent with the hypothesis that ST resolution implies effective microvascular and tissue reperfusion, but also relate the recovery of electrocardiografic changes to salvage of viable myocardium. Indeed, early assessment of ST recovery may represents the appropriate time to identify patients at higher risk of adverse events potentially benefit from additional novel therapies, ideally starting already at the

The ECG in the stabilized phase of STEMI, after reperfusion therapy, represents a simply and universally applicable diagnostic tool to understand the prognosis and to guide further interventions. One method for determining the presence of pathological Q-waves related to myocardial infarction has been the Minnesota Code (Blackburn et a., 1960). This method was developed for diagnosis of infarction rather than the quantification of its size and correlates poorly with anatomically measured infarct size (Pahlm et al., 1998). An improved correlation of changes in the QRS complex with infarct size was the development of a QRS scoring system by Selvester et al. The Selvester QRS scoring system included 54 criteria from the QRS complexes in 10 of the standard leads, which totaled 32 points, each equivalent to approximately 3% of the left ventricular wall (Startt-Selvester et al., 1989). Recently, studies using cardiac magnetic resonance have show that Q-wave predict the location and size of myocardial infarction (Wu E et al., 2001.). Historically the presence of Q-wave on ECG after myocardial infarction has been used in clinical practice to stratify patients in Q-wave and non-Q-wave myocardial infarction, according to larger necrosis and worse outcome discovered in Q-wave infarctions (Stone PH et al., 1988). On these basis, for many years after the original report by Prinzmetal in animal model (Prinzmetal et al., 1954), the presence of Q-wave has been related to transmural infarction, whereas its absence was categorize as non-transmural infarction. Recently, studies based on cardiac magnetic resonance have clarified that, even if this distinction still appears useful to stratify the risk after myocardial infarction, the presence of Q-wave on surface ECG is determined by the total size of necrosis rather than transmural extent of underlying myocardial infarction (Moon et al., 2004). A relative small number of patients after myocardial infarction still show persistence of ST-segment elevation even days and month after the acute event. Historically, this late persistence of ST-segment elevation has been ascribed to left ventricular aneurysm or impending rupture of free wall or ventricular septum, identifying patients at very high risk for heart failure and death (Chon et al., 1967 ). However, this association is among the most controversial in electrocardiography, since previous studies, including echocardiography and angiography, clearly showed a more severe systolic dysfunction and wall motion abnormalities in patients with persistent STE, but failed to demonstrate a definite relationship between this electrocardiographic pattern and left ventricular aneurysm. Moreover, the explanation of the underlying mechanism of persistent STE and its pathological correlates are still unclear (Bar et al., 1984 & Lidsay J et al., 1984 ; Bhatnagar, 1994). Recently, using cardiac magnetic resonance, correlations between this ECG pattern

catheterization laboratory.

**7. ECG in stabilized myocardial infarction** 

and type of myocardial damage have been reported. Particularly, the presence of persisting ST-elevation seems related to the presence of large microvascular damage in the context of transmural necrosis (Figure 3). These findings suggest that in this scenario late persistence of ST elevation indicates not only, as predictable, a greater extent of myocardial necrosis, but also, and more interestingly, the presence of severe microvascular damage as shown by cardiac magnetic resonance. Patients exhibiting persistent ST elevation showed more frequently left ventricular aneurysm, even though this difference did not achieve a statistical significance. Taking into account the findings of previous studies, these observations lead to the criticism about wall motion abnormalities as mechanism of electrocardiographic alterations. Recently, Li et al provided direct evidence in animals that opening of sarcolemmal KATP channels underlies ST elevation during ischemia (Li RA et al., 2000). It has also been demonstrated in a swine model that mechanical stimuli can induce marked ST elevation , by producing the stretching activation of KATP channel (Link et al., 1999). On these basis it has been hypothesized that outward bulging of myocardial necrotic wall, producing an abnormal stretch on the adjacent tissue, may alter cellular activity, generating injury currents at this level responsible for the ST elevation (Gussak et al., 2000). Thus patients exhibiting persistence of ST elevation had not only more severe myocardial

**Panel A**: ECG shows neither ST-segment elevation nor pathological Q-wave; the ce-MRI detects nontrasmural necrosis (middle and apical segments) of anterolateral wall, without either persistent microvascular obstruction or left ventricular aneurysm. **Panel B**: ECG shows pathological Q-wave in leads V4 to V6, with persistent ST elevation; the corresponding ce-MRI shows transmural necrosis of the septum, anterolateral wall (middle and apical segments), and apex, with evidence of persistent microvascular obstruction in the setting of necrotic core, without aneurysm. **Panel C :** ECG shows Qwave in leads V1 through V6, DI, aVL, and STE in leads V1 through V6. The corresponding ce-MRI shows a large trasmural necrosis in the septum and anterolateral wall (middle and apical segments), and of the apical segments of inferior wall, with evidence of persistent microvascular obstruction in the necrotic core.

Fig. 3. Different patterns of myocardial structural abnormalities detected by contrastenhanced magnetic resonance imaging (ce-MRI) and corresponding 12-leads electrocardiogram (ECG).

ECG in Acute Myocardial Infarction in the Reperfusion Era 127

Bar, FW. (1984). Prognostic value of Q waves, R/S ratio, loss of R wave voltage, ST-T

Barbagelata, A. (1997). Thrombolysis and Q wave versus non-Q wave first acute myocardial infarction: a GUSTO-I substudy. *J Am Coll Cardiol,* Vol 29, pp. 770-7, 0735-1097 Bayes de Luna, A. (2006a). Concordance of electrocardiographic patterns and healed

Bayes de Luna, A. (2006b). A new terminology for the left ventricular walls and location of

Betriu, A. (2005). Comparison of mortality rates in acute myocardial infarction treated by

Bhatnagar, SK. (1994). Observation of the relationship between left ventricular aneurysm

Birnbaum, Y. (1993). Prediction of the level of left descending coronary artery obstruction

Blackburn, H. (1960). Electrocardiogram in population studies: a classification system.

Boersma, E. (2006). Does time matter? A pooled analysis of randomized clinical trials

Brady, W.J. (2001). Electrocardiographic STsegment elevation: the diagnosis of acute

Brener, S.J. (1998). Randomized, placebo-controlled trial of platelet glycoprotein IIb/IIIa

Cannon, C.P. (2000). Relationship of symptom- onset-to-balloon time and door-to-balloon

Cerqueira, M.D. (2002). Standardized myocardial segmentation and nomenclature for tomographic imaging of the heart. *Circulation*, Vol.105, pp. 539-542, 0009-7322 Chon, K. (1967). Use of electrocardiogram as an aid in screening for left ventricular

Chong, E. (2010). Two-year clinical registry follow-up of endothelial progenitor cell capture

resonance imaging. *Circulation*, Vol114, pp. 1755-1760, 0009-7322

infarction. *Eur Heart,* Vol.15, pp. 1500-1504, 1522-9645

*Circulation,* Vol.21, pp. 1160-75, 0009-7322

*Circulation,* Vol. 98, pp. 734-41, 0009-7322.

*Med,* Vol.277, pp. 222-232, 1533-4406

infarction. *JAMA,* Vol 283, pp. 2941–2947, 0098-7487

electrocardiogram. *Am J Cardiol*, Vol.72, pp 823-826, 0002-9149

1097

Vol.97, pp. 443-451, 0002-9149

100–101, 0002-9149

788, 1522-9645

Vol.8, pp. 961-967, 1069-6563

segment abnormalities, electrical axis, low voltage and notching: correlation of electrocardiogram and left ventriculogram. *J Am Coll Cardiol,* Vol.4, pp. 17-27, 0735-

myocardial infarction detected by cardiovascular magnetic resonance. *Am J Cardiol*,

myocardial infarcts that present Q-wave on the standard of cardiac magnetic

percutaneous coronary intervention versus fibrinolysis. *Am J Cardiol,* Vol.95, pp.

and ST segment elevation in patients with a first acute anterior Q wave myocardial

during anterior wall acute myocardial infarction by the admission

comparing primary percutaneous cor- onary intervention and in-hospital fibrinolysis in acute myocardial infarction patients. *Eur Heart J* , Vol.27, pp. 779–

myocardial infarction by morphologic analysis of the ST segment. *Acad Emerg Med*,

blockade with primary angioplasty for acute myocardial infarction. ReoPro and Primary PTCA Organization and Randomized Trial (RAPPORT) Investigators.

time with mortality in patients undergoing angioplasty for acute myocardial

aneurysm. J Electrocardiol. 1976;9:53-58. Herman MV, et al. Localized disorders in myocardial contraction. Asynergy and its role in congestive heart failure. *N Engl J* 

stent versus sirolimus-eluting bioabsorbable polymer-coated stent versus bare

damage, but also more frequently coexistence of microvascular damage within it, that could account for diffuse alterations in myocardial skeletal favoring myocardial bulging and mechanical activation of KATP channels in the adjacent tissue. Finally, these findings may also explain the temporal discrepancy between developing of aneurysm and ECG alterations.

### **8. Conclusion**

The ECG is the most important diagnostic tool in the diagnosis of evolving ST-segment elevation myocardial infarction, influencing therapeutic strategies and management. Moreover, ECG remains a simple but valuable method to estimate the risk of STEMI patients either before and after reperfusion therapy. Finally the value of ECG in the prognostic stratification after stabilized STEMI have still a role in current management of these patients.

### **9. References**


damage, but also more frequently coexistence of microvascular damage within it, that could account for diffuse alterations in myocardial skeletal favoring myocardial bulging and mechanical activation of KATP channels in the adjacent tissue. Finally, these findings may also explain the temporal discrepancy between developing of aneurysm and ECG

The ECG is the most important diagnostic tool in the diagnosis of evolving ST-segment elevation myocardial infarction, influencing therapeutic strategies and management. Moreover, ECG remains a simple but valuable method to estimate the risk of STEMI patients either before and after reperfusion therapy. Finally the value of ECG in the prognostic stratification after stabilized STEMI have still a role in current management of

Aldrich, H.R. (1988). Use of initial ST-segment deviation for prediction of final

American College Cardiology (ACC)/American Heart Association (AHA) (2009).

Antman, E.M. (2004). ACC/AHA guidelines for the management of patients with ST-

Antman, E.M. (2006). Enoxaparin versus unfractionated heparin with fibrinolysis for ST

Antman, E.M. (2008). Focused Update of the ACC/AHA 2004 Guidelines for the

Antman, E.M. (2008). Focused update of the ACC/AHA 2004 guidelines for the

Antoniucci, D. (2004). Abciximab-supported infarct artery stent implantation for acute

Bairey, C.N. (1987). Electrocardiographic differentiation of occlusion of the left circumflex

Bär, F.W. (1996). Development of ST- segment elevation and Q-and R-wave changes in acute

Practice Guidelines. *J Am Coll Cardiol,* Vol.51, pp. 210-47, 0735-1097

*Coll Cardiol*, Vol. 34, pp 1890 –1911, 0735-1097

alone. *Circulation,* Vol.109, pp.1704–6, 0009-7322

*Am J Cardiol*, Vol.60, pp. 456-69, 0002-9149

Vol.77, pp.337-43, 0002-9149

electrocardiographic size of acute myocardial infarcts. *Am J Cardiol*, Vol.61, pp. 749-

Guidelines for the management of patients with acute myocardial infarction. *J Am* 

elevation myocardial infarction--executive summary. *J Am Coll Cardiol,* Vol.44, pp.

elevation MI (EXTRACT TIMI 25 trial). *N Eng J Med,* Vol.354, pp. 1477-1488, 1533-

Management of Patients With ST-Elevation Myocardial Infarction. *JACC,* Vol.51, 2,

management of patients with ST-elevation myocardial infarction: a report of the American College of Cardiology/American Heart Association Task Force on

myocardial infarction and long-term survival: A prospective, multicenter, randomized trial comparing infarct artery stenting plus abciximab with stenting

versus the right coronary artery as a cause of inferior acute myocardial infarction.

myocardial infarction and the influence of thrombolytic therapy. *Am J Cardiol,* 

alterations.

**8. Conclusion** 

these patients.

**9. References** 

753, 0002-9149

671-719, 0735-1097

pp. 210-247, 0735-1097

4406


ECG in Acute Myocardial Infarction in the Reperfusion Era 129

Goldstein, JA. (2005). Patterns of coronary compromise leading to bradyarrhytmias and

Gurm, HS. (2008). The relative safety and efficacy of abciximab and eptifibatide in patients

Gussak, I. (2000). Exercise induced ST segment elevation in Q wave leads in postinfarction

Haeck, JDE. (2011). Impact of early, late, and no ST-segment resolution measured by

Haines, D.E. (1983). Anatomic and prognostic significance of new T-wave inversion in

Hamby, R.I. (1983). Left bundle branch block: a predictor of poor left ventricular function in

Henkel, DM. (2006). Ventricular arrhytmias after acute myocardial infarction: a 20 year

Herz, M. (1997). New electrocardiographic criteria for predicting either the right or left

Huikuri, H. (2001). Sudden death due to cardiac arrhytmias. *N Engl J Med,* Vol.345, pp. 1473-

Keeley, E.C. (2003). Primary angioplasty versus intravenous throm- bolytic therapy for acute

Krucoff, M.W. (1993). Continuous 12-lead ST- segment recovery analysis in the TAMI 7

Lee, K.L. (1995). Predictors of 30-day mortality in the era of reperfusion for acute myocardial

Lee, Y.P. (2010). Endothelial progenitor cell capture stent implantation in patients with ST-

Lemos, P.A. (2004). Unrestricted utilization of sirolimus-eluting stents compared with

Li, RA. (2000). Molecular basis of electrocardiographic ST-segment elevation. *Circ Res,* 

Lidsay, J Jr. (1984). Relation of ST-segment elevation after healing of acute myocardial

myocardial reperfusion. *Circulation,* Vol.88, pp. 437-46, 0009-7322.

circumflex artery as the culprit coronary artery in inferior wall acute myocardial

myocardial infarction a quantitative review of 23 ran- domized trials. *Lancet,*

study. Performance of a non invasive method for real-time detection of failed

segment elevation acute myocardial infarction: one year follow-up.

conventional bare stent implantation in the "real world": the Rapamycin- Eluting Stent Evaluated at Rotterdam Cardiology Hospital (RESEARCH) registry.

infarction to the presence of left ventricular aneurysm. *Am J Cardiol,* Vol.54, pp. 84-

coronary heart disease. *Am Heart J*, Vol.106, pp. 471-477, 0002-8703

community study. *Am Heart J,* Vol.151, pp. 806-812, 0002-8703

unstable angina. *Am J Cardiol,* Vol.52, pp.14-8, 0002-9149

infarction. *Am J Cardiol*, Vol.80, pp. 1343-1345, 0002-9149

infarction. *Circulation*, Vol. 91, pp. 1659-1568, 0009-7322

*Eurointervention,* Vol.5, pp. 698-702, 1969-6213

*Circulation,* Vol.109, pp. 190-5, 0009-7322

265-274

205-209

Vol.44, pp. 36-41

1482, 1533-4406

Vol.87, pp. 837-39

6, 0002-9149.

Vol.361, pp 13–20, 0140-6736

*Cardiol* , Vol 51, pp. 529–35, 0735-1097

hypotension in inferior myocardial infarction. *Coron Artery Dis* 2005, Vol.16, pp.

undergoing primary percutaneous coronary intervention: insights from a large regional registry of contemporary percutaneous coronary intervention. *J Am Coll* 

patients: defining its meaning and utility in today's practice. *Cardiology,* Vol.93, pp.

continuous ST Holter monitoring on left ventricular ejection fraction and infarct size as determined by cardiovascular magnetic resonance imaging. *J Electrocardiol,* 

metal stents in patients undergoing primary percutaneous coronary intervention for ST elevation myocardial infarction. *J Inter Cardiol,* Vol 23, pp.101-8, 0167-5273


Co, M. (2008). Use of endothelial progenitor cell capture stent (Genous Bio-Engineered R

Correale, E. (1999). Electrocardiographic patterns in acute inferior myocardial infarction

Cropp, G.J. (1960). Manning GW. Electrocardiographic changes simulating myocardial

Daemen, J. (2007). Comparison of three-year clinical outcome of sirolimus- and paclitaxel-

De Lemos, J.A. (2001). ST segment resolution as a tool for assessing the efficacy of reperfusion therapy. *J Am Coll Cardiol,* Vol 38, pp. 1283-94, 0735-1097 De Lemos, JA. (2000) . ST-segment resolution and infarct-related artery patency and flow

De Lemos, JA. (2001). ST segment resolution as a tool for assessing the efficacy of reperfusion therapy. *J Am Coll Cardiol,* Vol.38, pp. 1283-1294, 0735-1097 De Lemos, JA. (2001). ST-segment resolution as a tool for assessing the efficacy of reperfusion therapy. *J Am Coll Cardiol,* Vol.38, pp. 1283-1294, 0735-1097 De Luca, G. (2003). Myocardial Infarction Study Group. Symptom onset to balloon time and

Dressler, W. (1947). High T waves in the earliest stage of myocardial infarction. *Am Heart J*,

Edwards, J. (2005). The COMMIT trial investigators: Addition of clopidogrel to aspirin in

Engelen, D.J. (1999). Value of electrocardiogram in localizing the occlusion site in the left

Essen, R. (1979). Spontaneous course of ST-segment elevation in acute anterior myocardial

Fuster, V. (2006). ACC/AHA/ESC 2006 Guidelines for the Management of Patients with

Goldberger A.L. 4th ed. (1991). *Myocardial infarction: electrocardiographic differential diagnosis*,):

Goldschlager, N. (1989). Principles of clinical electrocardiography, In: *Appleton and Lange,* 

Atrial Fibrillation. *Circulation,* Vol.114, pp. e257–e354, 0009-7322

infarction. *American Heart Journal,* Vol.155, pp. 128-32, 0002-8703

prognostic value, masking effect. *Clin Cardiol*, Vol.22, pp. 37-44,

myocardial infarction. *Am J Cardiol,* Vol.99, pp 1027–32, 0002-9149

angioplasty. *J Am Coll Cardiol,* Vol.42, pp. 991–997, 0735-1097

*Circulation,* Vol 22, pp. 25-38, 0009-7322

0009-7322

304, 0002-9149

Vol.34, pp. 627-645, 0002-8703

*Cardiol*, Vol.34, pp 389-395, 0735-1097

Norwalk Conn, pp. 110-2. 13th ed

infarction. *Circulation,* Vol.59, pp. 105-12, 0009-7322

pp. 1607-1621, 0140-6736

Mosby; St. Louis

metal stents in patients undergoing primary percutaneous coronary intervention for ST elevation myocardial infarction. *J Inter Cardiol,* Vol 23, pp.101-8, 0167-5273 Claeys, M.J. (1999). Determinants and prognostic implications of persistent ST-segment

elevation after primary angio- plasty for acute myocardial infarction: Importance of microvascular reperfusion injury on clinical outcome. *Circulation,* Vol.99, pp.1972-7,

Stent) during primary percutaneous coronary intervention in acute myocardial

with and without right ventricular involvement: classification, diagnostic and

ischemia and infarction associated with spontaneous intracranial hemorrhage.

eluting stents versus bare metal stents in patients with ST-segment elevation

after thrombolytic therapy. TIMI-14 14 Investigators. *Am J Cardiol* , Vol.85, pp. 299-

mortality in patients with acute myocardial infarction treated by primary

45,852 patients with AMI: a randomized placebo controlled trial. *Lancet,* Vol.366,

anterior descending coronary artery in acute myocardial infarction. *J Am Coll* 


ECG in Acute Myocardial Infarction in the Reperfusion Era 131

Prinzmetal, M. (1954).Studies on the mechanism of ventricular activity. The depolarization

Raitt, M.H. (1995). Appearance of abnormal Q waves early in the course of acute myocardial

Richardson, S.G. (1988). Relation of coronary arterial patency and left ventricular function to

Sabatine, M.S. (2005). Addition of clopidogrel to aspirin and fibrinolytic therapy for STEMI.

Schröder R. (2004). Prognostic Impact of early ST-segment resolution in acute ST-elevation myocardial infarction. *Circulation,* Vol 110, pp. e506-e510, 0009-7322 Schröder, K. (1994). Extent of early ST segment elevation resolution: a simple but strong

Schröder, K. (2001). Extent of ST deviation in the single ECG lead of maximum deviation

Sgarbossa, E.B. (1996). Early electrocardiographic diagnosis of acute myocardial infarction in

Sgarbossa, E.B. (1998). Electrocardiographic diagnosis of evolving acute myocardial

Smith, S.W. (2005). Acute coronary syndromes: acute myocardial infarction and ischemia.

Startt-Selvester, RH. (1989). Myocardial infarction. In: *Comprehensive Electrocardiology: theory* 

Stenestrand, U. (2006). RIKS-HIA Registry. Long-term outcome of primary percutaneous

Stone, PH (1988). Prognostic significance of location and type of myocardial infarction. *J Am* 

Tamura, A. (1999). Significance of spontaneous normalization of negative T waves in infarct-

Smith, S.W. (2006). Acute coronary syndromes. *Emerg Med Clin North,* Vol 24, pp. 53-89. Somers, M.P. (2002). The prominant T wave: electrocardiographic differential diagnosis. *Am* 

in acute myocardial infarction. *Z Kardiol 2001,* Vol.90, pp. 557-567

normal electrocardiogram. *Am J Med,* Vol.16, pp. 469-88

myocardial infarction. *Am J Cardiol,* Vol.61, 0002-9149

editors, 1st edition, pp. (151-63). Mosby, Philadelphia

*J Emerg Med*, Vol 20, pp. 243-251, 0735-6757

Pergamon Press, New York

*Med,* Vol.358, pp. 2218-30. 1533-4406

*Cardiol,* Vol*.*84, pp.1341-4, 0002-9149

*Coll Cardiol,* Vol 11, pp. 453-63, 1735-1097

*N Engl J Med,* Vol.352, pp. 1179-1189. 1533-4406

25, pp.1084-8, 0735-1097

Vol.24, pp. 384-391, 0735-1097

9149

87, 1533-4406

complex in pure subendocardial infarction: role of subendocardial region in the

infarction: implications for efficacy of thrombolytic therapy. *J Am Coll Cardiol,* Vol

electrocardiographic changes after streptokinase treatment during acute

predictor of outcome in patients with acute myocardial infarction. *J Am Coll Cardiol,* 

present 90 or 180 minutes after start of thrombolytic therapy best predicts outcome

the presence of ventricular paced rhythm. *Am J Cardiol*, Vol.77, pp. 423–424, 0002-

infarction in the presence of left bundle-brach block. *N Engl J Med*, Vol.334, pp. 81–

In: *ECG in emergency medicine and acute care,* Chan TC, Brady WJ, Harrigan RA,

*and practice in health disease,* Macfarlane PW Lawrie TDV editors, pp. 565-629,

coronary intervention vs prehospital and in-hospital thrombolysis for patients with ST-elevation myocardial infarction. *JAMA,* Vol.296, pp. 1749–1756, 0098-7484 Stone, G.W. (2008). Bivalirudin during primary PCI in acute myocardial infarction. *N Engl J* 

related leads during healing of anterior wall acute myocardial infarction. *Am J* 


Link, MS. (1999). Selective activation of the KATP channel is mechanism by which sudden

Mandel, W.J.(1968). Analysis of T-wave abnormalities associated with myocardial infarction

Meine, TJ. (2005). Incidence, predictors, and outcomes ESC Guidelines of high-degree

Menown, I.B. (2000). Optimizing the initial 12-lead electrocardiographic diagnosis of acute

Mills, R.M. (1975). Natural history of S-T segment elevation after acute myocardial

Montalescot, G. (2001). Platelet glycoprotein IIb/IIIa inhibition with coronary stenting for acute myocardial infarction. *N Engl J Med,* Vol.344, pp. 1895–903, 1533-4406 Moon, JCC. (2004). The Pathologic basis of Q-wave and non-Q wave myocardial infarction.

Nallamothu, B.K. (2003). Percutaneous coronary intervention versus fibrinolytic therapy in

Newby, KH. (1996). Incidence and clinical relevance of the occurrence of bundle-branch

Oliva, P.B. (1993). Electrocardiographic diagnosis of postinfarction regional pericarditis.

Opolski, G. The effect of infarct size on atrioventricular and intraventricular conduction disturbances in acute myocardial infarction. *Int J Cardiol*, Vol.10, pp. 141-147 Pahlm, US. (1998). Comparison of various electrocardiografic scoring codes for estimating

Parikh, A. 2nd ed. (1997). New insights into the electrocardiogram of acute myocardial

Perera, D. (2004). Dynamics of ST segment in ischaemic heart disease, In: *Dynamic electrocardiography,* Malik M, Camm AJ, editors.. 1st ed. Elmsford (NY) Pinto, D.S. (2006). Hospital delays in reperfusion for ST-elevation myocardial infarction.

Pinto, D.S. (2007). Hospital delays in reperfusion for ST-elevation myocardial infarction:

Poli, A. (2002). Integrated analysis of myocardial blush and ST-segment elevation recovery

acute myocardial infarction is timing (almost) everything? *Am J Cardiol,* Vol.92:, pp.

block in patients treated with thrombolytic therapy. *Circulation,* Vol.94, pp. 2424–

Ancillary observations regard- ing the effect of reperfusion on the rapidity and amplitude of T wave inversion after acute myocardial infarction. *Circulation,* Vol 88,

anatomically documented size and single and multiple infarcts of the left ventricle.

infarction, In: *Acute myocardial infarction,* Gersh BJ Rahimtoola SH editors,

Implications when selecting a reperfusion strategy. *Circulation,* Vol.114, pp. 2019–

implications when selecting a reperfusion strategy. *Circulation,* Vol.114, pp. 2019-

after successful primary angioplasty: real-time grading of microvascular reperfusion and prediction of early and late recovery of left ventricular function.

using a theoretic model. *Circulation,* Vol.38, pp. 178-88, 0009-7322

thrombolytic therapy. *Am Heart J ,* Vol.149, pp. 670–674, 0002-8703

myocardial infarction. *EurHeart J*, Vol.21, pp. 275-283, 1522-9645

infarction. *Am J Cardiol,* Vol 35, pp. 609-14, 0002-9149

*JAm Coll Cardiol,* Vol 44, pp. 554-60, 0735-1097

*Am J Cardiol,* Vol.81, pp. 809-15, 0002-9149

*Circulation,* Vol 106, pp. 313-8, 0009-7322

Chapman and Hall, New York

418, 0009-7322.

824–826, 0002-9149

2428, 0009-7322.

2025, 0009-7322.

2025, 0009-7322

pp. 896-904, 0009-7322

death is produced by low energy chest wall impact. *Circulation,* Vol 100, pp. 413-

atrioventricular block complicating acute myocardial infarction treated with


**9** 

*Nanning, China* 

**Mechanisms of Postinfarction** 

Guoqiang Zhong, Jinyi Li, Honghong Ke, Yan He, Weiyan Xu and Yanmei Zhao

**Electrophysiological Abnormality:** 

**Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling** 

*Department of Cardiology, The First Affiliated Hospital of Guangxi Medical University,* 

Of various functional impairments of electrical events in the heart, ventricular arrhythmias underlie the majority of deaths in patients with left ventricular dysfunction and heart failure after myocardial ischemia and infarction. As heart failure develops, pathophysiological remodeling of cardiac function occurs at multiple levels, spanning the spectrum from molecular and subcellular changes to those occurring at the organ system levels (Jin et al.,

Although advances in anti-arrhythmic agents and implantation of direct-current defibrillator have resulted in improved prevention of death due to arrhythmia in myocardial infarction, morbidity and mortality due to arrhythmias are still high in all over the world. In addition, in patients with severe congestive heart failure, ventricular arrhythmia is also a critical determinant of prognosis, because conservative anti-arrhythmia therapies are not very effective. Therapeutic strategies for arrhythmias have been focused mainly on electrophysiological aspects with little consideration of structural or cellular bases for arrhythmogenesis. Thus, the exact cellular mechanism underlying lethal arrhythmias is undetermined. Identification of arrhythmogenic substrates from viewpoints other than

Despite decades of investigation, the precise mechanisms that underlie the electrophysiological abnormality remain elusive. In this chapter we therefore focus on three main issues with an emphasis on the mechanisms responsible for these adaptations:

Cardiac innervation comes from both extrinsic and intrinsic sources. The extrinsic sympathetic innervation arises from the stellate ganglia and paravertebral sympathetic

sympathetic neural remodeling, electrical remodeling and gap junction remodeling.

electrophysiological ones is essential (Takamatsu, 2008).

**2. Cardiac sympathetic neural remodeling 2.1 The sympathetic nervous system in the heart** 

**1. Introduction** 

2008).


## **Mechanisms of Postinfarction Electrophysiological Abnormality: Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling**

Guoqiang Zhong, Jinyi Li, Honghong Ke, Yan He, Weiyan Xu and Yanmei Zhao *Department of Cardiology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China* 

### **1. Introduction**

132 Advances in Electrocardiograms – Clinical Applications

Tanimoto, S. (2006). Drug-eluting stent implantation in acute myocardial infarction. Do we

Tarantini, G. (2005). Expla-nation for the survival benefit of primary angioplasty over

Tarantini, G. (2010). Acceptable reperfusion delay to prefer primary angioplasty over fibrin-

Tcheng, J.E. (2003). Benefits and risks of abciximab use in primary angioplasty for acute

Thune, J.J. (2005). DANAMI-2 Investigators. Simple risk stratification at the admission to

Valgimigli, M. (2008). Comparison of angioplasty with infusion of tirofiban or abciximab

Van't Hof, AW. (1997). Clinical value of 12-lead electrocardiogram after successful

Verouden, N.J. (2209). Distinguishing the right coronary artery from the left circumflex

Wagner, G.S. (2009). Electrocardiography and Arrhythmias Committee. AHA/ACCF/HRS

Zeymer, U. (2001).Non-invasive detection of early infarct vessel patency by resolution of ST-

Zeymer, U. (2003). Primary percutaneous transluminal coronary angioplasty accelerates

Zipes, DP. (2006). ACC/AHA/ESC 2006 guidelines for management of patients with

electrocardiogram. *J Am Coll Cardiol*, Vol.53, pp. 1003-1011, 0735-1097 Wu, E. (2001). Visualization of presence, location, and transmural extent of healed Q-wave and non-Q-wave myocardial infarction. *Lancet,* Vol.357, pp 21-8, 0140-6736 Yusuf, S. (2006). Effects of fondaparinux on mortality and reinarction in patients with acute

does not fit all. *Eur Heart J,* Vol 31, pp. 676–683, 1522-9645.

*EuroIntervention,* Vol.2, pp. 23-7, 1969-6213

*J Cardiol,* Vol.96, pp. 1503–1505, 0002-9149

1316–23, 0009- 7322

0098-7484

0140- 6736

Vol.112, pp. 2017–2021, 0009-7322.

*Europace*, Vol.11, pp 1517-1521,

Vol.295, pp.1519-30, 0098-7484

*Eur Heart J,* Vol .2, pp. 769-775, 1522-9645.

AMI. *Am Heart J,* Vol.146, pp. 686-691, 0002-8703

summary. *Eur Heart J,* Vol.27, pp. 2099–2140, 1522-9645

need another randomized trial? (TYPHOON, PASSION and HORIZONS trials).

thrombolytic therapy in patients with ST-elevation acute myocardial infarction. *Am* 

specific thrombolytic therapy is affected (mainly) by the patient's mortality risk: 1 h

myocardial infarction: the Controlled Abciximab and Device Investigation to Lower Late Angioplasty Complications (CADILLAC) trial. *Circulation,* Vol.108, pp.

identify patients with reduced mortality from primary angioplasty. *Circulation,* 

and with implantation of sirolimus-eluting or uncoated stents for acute myocardial infarction, the MULTISTRATEGY randomized trial. *JAMA,* Vol.299, pp. 1788–99,

reperfusion therapy for acute myocardial infarction. *Lancet,* Vol.350, pp. 615-619,

coronary artery as the infarct-related artery in patients undergoing primary percutaneous coronary intervention for acute inferior myocardial infarction.

Recommendations for the standardization and interpretation of the

ST-segment elevation myocardial infarction. The OASIS-6 randomized trial. *JAMA,* 

segment elevation in patients with thrombolysis for acute myocardial infarction.

early myocardial reperfusion compared to thrombolytic therapy in patients with

ventricular arrhythmias and the prevention of sudden cardiac death—executive

Of various functional impairments of electrical events in the heart, ventricular arrhythmias underlie the majority of deaths in patients with left ventricular dysfunction and heart failure after myocardial ischemia and infarction. As heart failure develops, pathophysiological remodeling of cardiac function occurs at multiple levels, spanning the spectrum from molecular and subcellular changes to those occurring at the organ system levels (Jin et al., 2008).

Although advances in anti-arrhythmic agents and implantation of direct-current defibrillator have resulted in improved prevention of death due to arrhythmia in myocardial infarction, morbidity and mortality due to arrhythmias are still high in all over the world. In addition, in patients with severe congestive heart failure, ventricular arrhythmia is also a critical determinant of prognosis, because conservative anti-arrhythmia therapies are not very effective. Therapeutic strategies for arrhythmias have been focused mainly on electrophysiological aspects with little consideration of structural or cellular bases for arrhythmogenesis. Thus, the exact cellular mechanism underlying lethal arrhythmias is undetermined. Identification of arrhythmogenic substrates from viewpoints other than electrophysiological ones is essential (Takamatsu, 2008).

Despite decades of investigation, the precise mechanisms that underlie the electrophysiological abnormality remain elusive. In this chapter we therefore focus on three main issues with an emphasis on the mechanisms responsible for these adaptations: sympathetic neural remodeling, electrical remodeling and gap junction remodeling.

### **2. Cardiac sympathetic neural remodeling**

### **2.1 The sympathetic nervous system in the heart**

Cardiac innervation comes from both extrinsic and intrinsic sources. The extrinsic sympathetic innervation arises from the stellate ganglia and paravertebral sympathetic

Electrophysiological Abnormality:

(Figure 4) (Oh et al., 2006).

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 135

In ischemic hearts, regional heterogeneity of sympathetic innervation usually occurs as a result of ischemia- or infarction-induced neural axons necrosis and nerve sprouting (Zipes, 1990). Sympathetic neural remodeling characterized by heterogeneous cardiac nerve sprouting and

The density of nerve fibers immunopositive for Growth-associated protein-43 upregulated after myocardial infarction at different periods, indicating nerve fibers undergoing sprouting activity shortly after myocardial infarction (Oh et al., 2006; Zhou et al., 2004). However, sprouting nerve fibers may regress unless nerve terminals can form synaptic contacts with target cells. Therefore, the density of growth-associated protein-43 positive nerve fibers is a measurement for nerve sprouting activity instead of a measurement for stable innervation. Myocardial infarction induced nerve sprouting activity in both peri-infarct and remote areas. Nerve sprouting, which resulted in sympathetic (but not parasympathetic) hyperinnervation, was greater in the outer loop of the heart. The difference in the outer and inner loops started within 3 hours and persisted for 2 months after myocardial infarction

Fig. 4. Nerve fiber densities in the outer versus the inner loop of normal mouse heart. A, Cross-section of a normal mouse ventricle. The fiber orientation in the inner loop is roughly

ventricles. B, C: Growth-associated protein-43 immunopositive nerves in the inner and outer loops, respectively. More nerve fibers are present in the outer loop than the inner loop.

perpendicular, whereas the outer loop fibers are parallel to the circumference of the

Arrows point to brown nerve twigs (20×objective) (Oh et al., 2006).

**2.2 Sympathetic neural remodeling after myocardial infarction** 

Fig. 3. Neural remodeling and nerve sprouting (Cao et al., 2000).

sympathetic hyperinnervation was also observed; see Figure 3 (Cao et al., 2000).

ganglia. The vagal nerves are sources of extrinsic parasympathetic nerves that innervate the heart. In addition to the extrinsic cardiac nervous system, there is also an extensive intrinsic cardiac nervous system that includes collections of ganglionated plexuses (Armour, 2010). The major neurotransmitter mediating sympathetic response is norepinephrine; of note, epinephrine release during activation is negligible (Fig 1) (Esler et al., 1990).

Fig. 1. Cardiac sympathetic control. NE, norepinephrine; Ach, acetylcholine; E, epinephrine (Esler et al., 1990).

Each ganglionated plexuses contains both sympathetic and parasympathetic neurons that are associated with complex synaptology. The sympathetic innervation to the ventricles follows a course along the common pulmonary artery into the plexus supplying the main left coronary artery. The sympathetic nerves are distributed to the myocardium in superficial epicardial layers. They penetrate the myocardium along with the coronary arteries (Figure 2) (Zipes, 1990). Immunocytochemical techniques have allowed investigators to stain many nerve cells and nerve tissues including the Schwann cells and autonomic nerves using different antibodies with different stains (Oki et al., 1995; Gulbenkian et al., 1993; Chow et al., 1998).

Fig. 2. Schematic of sagittal view of left ventricular wall showing pathways of vagal and sympathetic afferent and efferent nerves. Postganglionic sympathetic axons are located superficially in periadventia of coronary arteries; postganglionic vagal axons cross the atrioventricular groove in subepicardium but are located in subendocardium. Cx, circumflex coronary artery; LAD, left ventricular descending coronary artery (Zipes, 1990).

### **2.2 Sympathetic neural remodeling after myocardial infarction**

134 Advances in Electrocardiograms – Clinical Applications

ganglia. The vagal nerves are sources of extrinsic parasympathetic nerves that innervate the heart. In addition to the extrinsic cardiac nervous system, there is also an extensive intrinsic cardiac nervous system that includes collections of ganglionated plexuses (Armour, 2010). The major neurotransmitter mediating sympathetic response is norepinephrine; of note,

Fig. 1. Cardiac sympathetic control. NE, norepinephrine; Ach, acetylcholine; E, epinephrine

Each ganglionated plexuses contains both sympathetic and parasympathetic neurons that are associated with complex synaptology. The sympathetic innervation to the ventricles follows a course along the common pulmonary artery into the plexus supplying the main left coronary artery. The sympathetic nerves are distributed to the myocardium in superficial epicardial layers. They penetrate the myocardium along with the coronary arteries (Figure 2) (Zipes, 1990). Immunocytochemical techniques have allowed investigators to stain many nerve cells and nerve tissues including the Schwann cells and autonomic nerves using different antibodies with different stains (Oki et al., 1995; Gulbenkian et al., 1993; Chow et al., 1998).

Fig. 2. Schematic of sagittal view of left ventricular wall showing pathways of vagal and sympathetic afferent and efferent nerves. Postganglionic sympathetic axons are located superficially in periadventia of coronary arteries; postganglionic vagal axons cross the atrioventricular groove in subepicardium but are located in subendocardium. Cx, circumflex

coronary artery; LAD, left ventricular descending coronary artery (Zipes, 1990).

(Esler et al., 1990).

epinephrine release during activation is negligible (Fig 1) (Esler et al., 1990).

In ischemic hearts, regional heterogeneity of sympathetic innervation usually occurs as a result of ischemia- or infarction-induced neural axons necrosis and nerve sprouting (Zipes, 1990). Sympathetic neural remodeling characterized by heterogeneous cardiac nerve sprouting and sympathetic hyperinnervation was also observed; see Figure 3 (Cao et al., 2000).

Fig. 3. Neural remodeling and nerve sprouting (Cao et al., 2000).

The density of nerve fibers immunopositive for Growth-associated protein-43 upregulated after myocardial infarction at different periods, indicating nerve fibers undergoing sprouting activity shortly after myocardial infarction (Oh et al., 2006; Zhou et al., 2004). However, sprouting nerve fibers may regress unless nerve terminals can form synaptic contacts with target cells. Therefore, the density of growth-associated protein-43 positive nerve fibers is a measurement for nerve sprouting activity instead of a measurement for stable innervation.

Myocardial infarction induced nerve sprouting activity in both peri-infarct and remote areas. Nerve sprouting, which resulted in sympathetic (but not parasympathetic) hyperinnervation, was greater in the outer loop of the heart. The difference in the outer and inner loops started within 3 hours and persisted for 2 months after myocardial infarction (Figure 4) (Oh et al., 2006).

Fig. 4. Nerve fiber densities in the outer versus the inner loop of normal mouse heart. A, Cross-section of a normal mouse ventricle. The fiber orientation in the inner loop is roughly perpendicular, whereas the outer loop fibers are parallel to the circumference of the ventricles. B, C: Growth-associated protein-43 immunopositive nerves in the inner and outer loops, respectively. More nerve fibers are present in the outer loop than the inner loop. Arrows point to brown nerve twigs (20×objective) (Oh et al., 2006).

Electrophysiological Abnormality:

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 137

Table 1. Neurotrophic factors gene expression in the myocardium after myocardial infarction Numbers indicate ratio to normal control. Bold numbers indicate those>3×of control in at least two consecutive time points. Bold numbers in parentheses are obtained with quantitative reverse-transcriptase polymerase chain reaction. Other numbers are obtained using DNA microarray. BDNF = brain-derived neurotrophic factor; EGF = epidermal growth factor; FGF = fibroblast growth factor; GDNF = glial cell-derived neurotrophic factor; IGF = insulin-like growth factor; IL = interleukin; LIF = leukemia inhibitory factor; NT-3 = neutrophin-3; NGF, nerve growth factor; TGF = transforming

Increase of neurotrophic factors sometimes can be injurious. Thus, both exogenous and endogenous nerve growth factor can induce cardiac nerve sprouting, but they also can increase the incidence of ventricular arrhythmias and sudden death in canine models (Cao et al., 2000a). Increased neurotrophic factor expression and heterogeneous innervation also could induce side effects that contribute to the increased risk for sudden death after

**2.4 The interaction between sympathetic neural remodeling and electrical remodeling**  Regional heterogeneity of sympathetic innervation is also closely related to electrical inhomogeneity. Previous study revealed that electrical heterogeneity is exacerbated after initial nerve injury by sympathetic nerve sprouting and subsequent regional myocardial hyperinnervation (Figure 6) (Rubart & Zipes, 2005). The enhanced spatial inhomogeneity in cardiac sympathetic innervation could amplify the spatial inhomogeneity of these electrophysiological properties and therefore facilitate the initiation of ventricular

Nerve growth factor overexpression and nerve sprouting affected the expressions and functions of certain potassium channels and thus increases the susceptibility to ventricular tachyarrhythmia. Nerve sprouting suppressed the expressions and functions of myocardial transient outward current (Ito) and inward rectifier current (IK1) channels. Myocardial necrotic injury plus intensified sympathetic nerve sprouting further decreased Kir2.1 expression and IK1 current density. All of these changes affected the repolarization of myocardial cells and

hence increased the vulnerability to ventricular arrhythmia (Ren et al., 2008).

growth factor; TNF = tumor necrosis factor (Oh et al., 2006).

myocardial infarction (Luisi et al., 2002; Canty et al., 2004).

arrhythmias (Cao et al., 2000b).

Necrotic injury to the rat myocardium results in denervation followed by proliferative regeneration of schwann cells and axons (Nori et al., 1995). Abnormal patterns of neurilemma proliferation have been documented in infarsitescted human and rat hearts (Vracko, 1990, 1991). Rencently, Huazhi et al (Huazhi et al., 2010) showed that sympathetic nerve sprouting at the peri-infarct zone in infarcted hearts at 8 weeks post myocardial infarction was more excessive and heterogeneous than that in the corresponding zone in normal hearts. Excessive nerve sprouting may result in abnormal patterns of myocardial innervation and may potentially increase cardiac arrhythmia. Wei et al (Wei et al., 2004) identified a number of changes, including substantial denervation of the left ventricular and areas of apparent hyperinnervation at the border of innervated and denervated myocardium after myocardial infarction.

### **2.3 Mechanisms of sympathetic neural remodeling after myocardial infarction**

The sympathetic innervation of the heart regulates cardiac function by stimulating heart rate, contractility, and conduction velocity through the release of norepinephrine and the activation of cardiac β1-adrenergic receptors. Sympathetic innervation of the heart is sculpted during development by chemoattractive and chemorepulsive factors (Ieda et al., 2007). For example, nerve growth factor supports sympathetic neuron survival and promotes cardiac axon outgrowth during development (Crowley et al., 1994), whereas the chemorepulsive factorsemaphorin 3a (Sema3a) attenuates sympathetic axon extension in the heart (Ieda et al., 2007) and in the peripheral vasculature (Long et al., 2009).

The nerve fiber regeneration process is triggered by up-regulation of nerve growth factor or other neurotrophic factor genes in the non-neuronal cells around the site of injury. Upregulation of nerve growth factor was more prominent in the area near the infarct than in the area remote to the infarct (Oh et al., 2006). Nerve growth factor mRNA was significantly up-regulated within 3 hours after myocardial infarction and persisted for 1 month in both the infarcted myocardium and the noninfarcted left ventricular free wall (Oh et al., 2006; Zhou et al., 2004).

Neurotrophins such as nerve growth factor act through two distinct types of receptors: tropomyosin-related tyrosine kinase (Trk) receptors and the lower-affinity p75 neurotrophin receptor (p75NTR) (Kuruvilla et al., 2004; Kaplan & Miller, 2003). Nerve growth factor acts through TrkA to promote the extension of sympathetic axons into the heart (Kuruvilla et al., 2004), whereas p75NTR modulates signaling by coreceptors that can either stimulate or inhibit axon outgrowth (Kaplan & Miller, 2003). p75NTR has a great functional impact on the cardiovascular system and cardiac rhythm stability (Christina et al., 2010), which were particularly relevant to myocardial infarction and heart failure where heterogeneous sympathetic innervation was correlated with altered neurotrophin expression and the development of ventricular arrhythmias (Rubart & Zipes, 2005).

In addition to nerve growth factor, other neurotrophic factors are also up-regulated after myocardial infarction. For example, Leukemia inhibitory factor protein was increased by 40% at 48 hours after myocardial infarction in mice (Fuchs et al., 2003). IL-1α was increased after myocardial infarction, primarily at peri-infarct sites. And IL-1, IL-1β, and IL-6 were increased at only one time point after myocardial infarction (Oh et al., 2006; Nian et al., 2004). IL-6 mRNA was induced in myocytes at the viable border zone in dogs subjected to ischemia-reperfusion (Gwechenberger et al., 1999). Table 1 describes detailly neurotrophic factors gene expression in the myocardium after myocardial infarction.

Necrotic injury to the rat myocardium results in denervation followed by proliferative regeneration of schwann cells and axons (Nori et al., 1995). Abnormal patterns of neurilemma proliferation have been documented in infarsitescted human and rat hearts (Vracko, 1990, 1991). Rencently, Huazhi et al (Huazhi et al., 2010) showed that sympathetic nerve sprouting at the peri-infarct zone in infarcted hearts at 8 weeks post myocardial infarction was more excessive and heterogeneous than that in the corresponding zone in normal hearts. Excessive nerve sprouting may result in abnormal patterns of myocardial innervation and may potentially increase cardiac arrhythmia. Wei et al (Wei et al., 2004) identified a number of changes, including substantial denervation of the left ventricular and areas of apparent hyperinnervation at the border of innervated and denervated

**2.3 Mechanisms of sympathetic neural remodeling after myocardial infarction** 

heart (Ieda et al., 2007) and in the peripheral vasculature (Long et al., 2009).

development of ventricular arrhythmias (Rubart & Zipes, 2005).

factors gene expression in the myocardium after myocardial infarction.

The sympathetic innervation of the heart regulates cardiac function by stimulating heart rate, contractility, and conduction velocity through the release of norepinephrine and the activation of cardiac β1-adrenergic receptors. Sympathetic innervation of the heart is sculpted during development by chemoattractive and chemorepulsive factors (Ieda et al., 2007). For example, nerve growth factor supports sympathetic neuron survival and promotes cardiac axon outgrowth during development (Crowley et al., 1994), whereas the chemorepulsive factorsemaphorin 3a (Sema3a) attenuates sympathetic axon extension in the

The nerve fiber regeneration process is triggered by up-regulation of nerve growth factor or other neurotrophic factor genes in the non-neuronal cells around the site of injury. Upregulation of nerve growth factor was more prominent in the area near the infarct than in the area remote to the infarct (Oh et al., 2006). Nerve growth factor mRNA was significantly up-regulated within 3 hours after myocardial infarction and persisted for 1 month in both the infarcted myocardium and the noninfarcted left ventricular free wall (Oh et al., 2006;

Neurotrophins such as nerve growth factor act through two distinct types of receptors: tropomyosin-related tyrosine kinase (Trk) receptors and the lower-affinity p75 neurotrophin receptor (p75NTR) (Kuruvilla et al., 2004; Kaplan & Miller, 2003). Nerve growth factor acts through TrkA to promote the extension of sympathetic axons into the heart (Kuruvilla et al., 2004), whereas p75NTR modulates signaling by coreceptors that can either stimulate or inhibit axon outgrowth (Kaplan & Miller, 2003). p75NTR has a great functional impact on the cardiovascular system and cardiac rhythm stability (Christina et al., 2010), which were particularly relevant to myocardial infarction and heart failure where heterogeneous sympathetic innervation was correlated with altered neurotrophin expression and the

In addition to nerve growth factor, other neurotrophic factors are also up-regulated after myocardial infarction. For example, Leukemia inhibitory factor protein was increased by 40% at 48 hours after myocardial infarction in mice (Fuchs et al., 2003). IL-1α was increased after myocardial infarction, primarily at peri-infarct sites. And IL-1, IL-1β, and IL-6 were increased at only one time point after myocardial infarction (Oh et al., 2006; Nian et al., 2004). IL-6 mRNA was induced in myocytes at the viable border zone in dogs subjected to ischemia-reperfusion (Gwechenberger et al., 1999). Table 1 describes detailly neurotrophic

myocardium after myocardial infarction.

Zhou et al., 2004).


Table 1. Neurotrophic factors gene expression in the myocardium after myocardial infarction Numbers indicate ratio to normal control. Bold numbers indicate those>3×of control in at least two consecutive time points. Bold numbers in parentheses are obtained with quantitative reverse-transcriptase polymerase chain reaction. Other numbers are obtained using DNA microarray. BDNF = brain-derived neurotrophic factor; EGF = epidermal growth factor; FGF = fibroblast growth factor; GDNF = glial cell-derived neurotrophic factor; IGF = insulin-like growth factor; IL = interleukin; LIF = leukemia inhibitory factor; NT-3 = neutrophin-3; NGF, nerve growth factor; TGF = transforming growth factor; TNF = tumor necrosis factor (Oh et al., 2006).

Increase of neurotrophic factors sometimes can be injurious. Thus, both exogenous and endogenous nerve growth factor can induce cardiac nerve sprouting, but they also can increase the incidence of ventricular arrhythmias and sudden death in canine models (Cao et al., 2000a). Increased neurotrophic factor expression and heterogeneous innervation also could induce side effects that contribute to the increased risk for sudden death after myocardial infarction (Luisi et al., 2002; Canty et al., 2004).

### **2.4 The interaction between sympathetic neural remodeling and electrical remodeling**

Regional heterogeneity of sympathetic innervation is also closely related to electrical inhomogeneity. Previous study revealed that electrical heterogeneity is exacerbated after initial nerve injury by sympathetic nerve sprouting and subsequent regional myocardial hyperinnervation (Figure 6) (Rubart & Zipes, 2005). The enhanced spatial inhomogeneity in cardiac sympathetic innervation could amplify the spatial inhomogeneity of these electrophysiological properties and therefore facilitate the initiation of ventricular arrhythmias (Cao et al., 2000b).

Nerve growth factor overexpression and nerve sprouting affected the expressions and functions of certain potassium channels and thus increases the susceptibility to ventricular tachyarrhythmia. Nerve sprouting suppressed the expressions and functions of myocardial transient outward current (Ito) and inward rectifier current (IK1) channels. Myocardial necrotic injury plus intensified sympathetic nerve sprouting further decreased Kir2.1 expression and IK1 current density. All of these changes affected the repolarization of myocardial cells and hence increased the vulnerability to ventricular arrhythmia (Ren et al., 2008).

Electrophysiological Abnormality:

(Zhou S, et al., 2008).

**infarction** 

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 139

Infusion of nerve growth factor accelerated and intensified the magnitude of nerve sprouting,

The left stellate ganglion is known to be important in cardiac arrhythmogenesis (Schwartz & Stone, 1980). Left cardiac sympathetic denervation, including left stellate ganglion resection, decreases the incidence of ventricular arrhythmia in patients with myocardial infarction (Schwartz et al., 1992). Increased sympathetic tone is important in the generation of ventricular arrhythmia and sudden cardiac death (Rubart & Zipes, 2005). In dogs with both complete atrioventricular block and myocardial infarction, high-amplitude spiky discharges frequently cause premature ventricular contractions or even ventricular tachycardia (Figure 7). These findings suggest that high-amplitude spiky discharges is highly arrhythmogenic in diseased hearts. However, because high-amplitude spiky discharges is also present in normal hearts, the presence of these types of discharge by itself is not pathological (Zhou S, et al., 2008).

Fig. 7. A, Electrical stimulation of left stellate ganglion lengthened the corrected QT (QTc) from 325 to 402 ms and the corrected Tpe (Tpec) from 45 to 82 ms (at 10 mA). B, Left stellate ganglion stimulation induced ventricular tachycardia (VT); C, ventricular fibrillation (VF) in the experimental group. Afterdischarges were present in left stellate ganglion recordings after electrical stimulation. BP = blood pressure; SGNA = stellate ganglion nerve activity

**2.6 Therapeutic implications of sympathetic neural remodeling after myocardial** 

ventricular tachyarrhythmias (Fig 8,Marmar & Kalyanam, 2008).

As sympathetic tone was known to be increased in cardiomyopathy patients, interventions that aim to reduce sympathetic tone could reduce the risk of sudden cardiac death and

Selective sympathetic blockade was an effective theraty, which was applied in animal models and human during myocardial ischemia (Issa et al., 2005; Nademanee et al., 2000). In a canine model, intrathecal clonidine, when delivered via a catheter at T2–T4 spinal segments, significantly reduced the occurrence of ventricular tachycardia and fibrillation during transient myocardial ischemia (Issa et al., 2005). Electrical storm, defined as recurrent multiple ventricular fibrillation (VF) episodes, often occurs in patients with recent myocardial infarction. Sympathetic blockade was proved to be superior to the antiarrhythmic therapy in treating electrical storm patients, and sympathetic blockade - not class 1 antiarrhythmic drugs - should be the treatment of choice for electrical storm

resulting in a high incidence of sudden cardiac death (Cao et al., 2000a).

Fig. 6. Factors contributing to arrhythmogenesis in hearts with heterogeneous sympathetic innervation (Rubart & Zipes, 2005).

Besides, the coexistence of sympathetic denervation zones (infarct zones), sympathetic reinnervation zones (ischemic zones) and sympathetic normal zones in the ischemic heart could increase the heterogeneity of electrical communication which is facilitative to the abnormality of cardiac autorhythmicity and triggered activity. Sympathetic neural remodeling can raises the norepinephrine concentration, which encourage the early afterdepolarization (EAD) and delayed afterdepolarization (DAD) by affect the calcium influx and repolarization potassium current, and then trigger arrhythmia. Neurotransmitters such as norepinephrine can cause focal vasoconstriction and myocardial ischemia which is facilitative to the arrhythmogenesis.

### **2.5 Relationship between sympathetic neural remodeling and ventricular arrhythmia**

The initiation of lethal arrhythmias needs a substrate and a trigger. Myocardial remodeling (structural and/or functional) is considered the substrate. In recent years, the concept "cardiac nerve remodeling" and its potentiality as a trigger for lethal arrhythmia have entered the scope of arrhythmia research (Ren et al., 2008). Abnormally increased post-injury sympathetic nerve density may be in part responsible for the occurrence of ventricular arrhythmia and sudden cardiac death in patients with severe heart failure (Cao et al., 2000b). Cardiac nerve sprouts detering occured after myocardial infarction even without exogenous nerve growth factor.

Fig. 6. Factors contributing to arrhythmogenesis in hearts with heterogeneous sympathetic

Besides, the coexistence of sympathetic denervation zones (infarct zones), sympathetic reinnervation zones (ischemic zones) and sympathetic normal zones in the ischemic heart could increase the heterogeneity of electrical communication which is facilitative to the abnormality of cardiac autorhythmicity and triggered activity. Sympathetic neural remodeling can raises the norepinephrine concentration, which encourage the early afterdepolarization (EAD) and delayed afterdepolarization (DAD) by affect the calcium influx and repolarization potassium current, and then trigger arrhythmia. Neurotransmitters such as norepinephrine can cause focal vasoconstriction and myocardial ischemia which is facilitative to the

**2.5 Relationship between sympathetic neural remodeling and ventricular arrhythmia**  The initiation of lethal arrhythmias needs a substrate and a trigger. Myocardial remodeling (structural and/or functional) is considered the substrate. In recent years, the concept "cardiac nerve remodeling" and its potentiality as a trigger for lethal arrhythmia have entered the scope of arrhythmia research (Ren et al., 2008). Abnormally increased post-injury sympathetic nerve density may be in part responsible for the occurrence of ventricular arrhythmia and sudden cardiac death in patients with severe heart failure (Cao et al., 2000b). Cardiac nerve sprouts detering occured after myocardial infarction even without exogenous nerve growth factor.

innervation (Rubart & Zipes, 2005).

arrhythmogenesis.

Infusion of nerve growth factor accelerated and intensified the magnitude of nerve sprouting, resulting in a high incidence of sudden cardiac death (Cao et al., 2000a).

The left stellate ganglion is known to be important in cardiac arrhythmogenesis (Schwartz & Stone, 1980). Left cardiac sympathetic denervation, including left stellate ganglion resection, decreases the incidence of ventricular arrhythmia in patients with myocardial infarction (Schwartz et al., 1992). Increased sympathetic tone is important in the generation of ventricular arrhythmia and sudden cardiac death (Rubart & Zipes, 2005). In dogs with both complete atrioventricular block and myocardial infarction, high-amplitude spiky discharges frequently cause premature ventricular contractions or even ventricular tachycardia (Figure 7). These findings suggest that high-amplitude spiky discharges is highly arrhythmogenic in diseased hearts. However, because high-amplitude spiky discharges is also present in normal hearts, the presence of these types of discharge by itself is not pathological (Zhou S, et al., 2008).

Fig. 7. A, Electrical stimulation of left stellate ganglion lengthened the corrected QT (QTc) from 325 to 402 ms and the corrected Tpe (Tpec) from 45 to 82 ms (at 10 mA). B, Left stellate ganglion stimulation induced ventricular tachycardia (VT); C, ventricular fibrillation (VF) in the experimental group. Afterdischarges were present in left stellate ganglion recordings after electrical stimulation. BP = blood pressure; SGNA = stellate ganglion nerve activity (Zhou S, et al., 2008).

### **2.6 Therapeutic implications of sympathetic neural remodeling after myocardial infarction**

As sympathetic tone was known to be increased in cardiomyopathy patients, interventions that aim to reduce sympathetic tone could reduce the risk of sudden cardiac death and ventricular tachyarrhythmias (Fig 8,Marmar & Kalyanam, 2008).

Selective sympathetic blockade was an effective theraty, which was applied in animal models and human during myocardial ischemia (Issa et al., 2005; Nademanee et al., 2000). In a canine model, intrathecal clonidine, when delivered via a catheter at T2–T4 spinal segments, significantly reduced the occurrence of ventricular tachycardia and fibrillation during transient myocardial ischemia (Issa et al., 2005). Electrical storm, defined as recurrent multiple ventricular fibrillation (VF) episodes, often occurs in patients with recent myocardial infarction. Sympathetic blockade was proved to be superior to the antiarrhythmic therapy in treating electrical storm patients, and sympathetic blockade - not class 1 antiarrhythmic drugs - should be the treatment of choice for electrical storm

Electrophysiological Abnormality:

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 141

The average duration of the ventricular action potential duration is reflected in the QT interval in the ECG. Factors that prolong the action potential duration (eg, a decrease in outward K+ currents or an increase in inward late Na+ current) prolong the action potential duration and the QT interval in the ECG. The QT interval of males and females is equal during early

Fig. 9. A, Membrane currents that generate the normal action potential duration. Resting (4), upstroke (0), early repolarization (1), plateau (2), and final repolarization are the 5 phases of the action potential. A decline of potential at the end of phase 3 in pacemaker cells, such as the sinus node, is shown as a broken line. The inward currents, INa, ICa, and If, are shown in

electrogenic and may generate inward or outward current. IKAch, IK1, Ito, IKur, IKr, and IKs are shown in gray boxes. The action potential duration is approximately 200ms. B, Various specialized tissues in the heart and typical corresponding action potentials. The short vertical lines indicate the time of onset of activity in the sinus atrial node for one beat. Green, slow-channel tissue (nodes); violet, fast-channel tissues (Nattel & Carlsson 2006).

Ion channels have two fundamental properties, ion permeation and gating (Hille, 1978). Ion permeation describes the movement through the open channel. The selective permeability of ion channels to specific ions is a basis of classification of ion channels (eg, Na+, K+, and Ca2+ channels). Size, valency, and hydration energy are important determinants of selectivity (Augustus, 2009). Ion channels provide multiple binding sites for ions as they traverse the membrane. Like an enzyme-substrate interaction, the binding of the permeating ion is saturable. Most ion channels are singly occupied during permeation; certain K+ channels

Gating is the mechanism of opening and closing of ion channels and is their second major property. Ion channels are also subclassified by their mechanism of gating: voltagedependent, ligand-dependent, and mechano-sensitive gating. Voltage-gated ion

yellow boxes; the sodium-calcium exchanger (NCX) is also shown in yellow. It is

**3.2 General properties of ion channels** 

may be multiply occupied (Augustus, 2009).

childhood. However, at puberty the interval of males shortens (Rautaharju et al., 1992).

(Nademanee et al., 2000). Successful treatment of recurrent ventricular tachycardia, refractory to antiarrhythmic therapy, can be achieved by neuraxial modulation at the level of the spinal cord. The benefit of thoracic epicardial anesthesia was reported in a patient with ischemic cardiomyopathy and recurrent ventricular arrhythmia refractory to intubation and sedation, with the use of 0.25% Bupicavaine at T1–T2 interspace, reducing the number of ICD shocks from 86 in 48 hours to zero (Mahajan et al., 2005).

Fig. 8. Structural and functional basis of ventricular tachycardia and fibrillation (Marmar & Kalyanam, 2008).

β-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aldosterone antagonists (Cittadini et al., 2003), statins (Gao et al., 2005), and fish oil have been shown to decrease risk of sudden cardiac death in ischemic cardiomyopathy and significantly improve mortality through modulating the autonomic nervous system to decrease sympathetic tone (Marmar & Kalyanam,2008). In a recent study, carvedilol ameliorated electrical remodeling at peri-infarct zones after myocardial infarction by improving the spatial distribution of the sympathetic nerve reinnervation (Huazhi et al., 2010). Moreover, attenuation of oxidative stress and inflammatory response after resveratrol treatment, a naturally occurring compound, significantly inhibited nerve growth factor expression, and protected against sympathetic neural remodeling (Ping et al., 2010). Ghrelin, a novel growth hormone-releasing peptide, which was showed to inhibite neural remodeling in rats with myocardial infarction, likely mediated through nerve growth factor suppression, might be used as a new potential way to treat and prevent sudden cardiac death after myocardial infarction (Ming-Jie et al., 2009).

### **3. Myocardial electrical remodelling**

### **3.1 The cardiac action potential**

The normal sequence and synchronous contraction of the atria and ventricles require the rapid activation of groups of cardiac cells. An activation mechanism must enable rapid changes in heart rate and also respond to the changes in autonomic tone. The propagating cardiac action potential fulfils these roles. The action potential is a key determinant of cardiac electrical activity and is shaped by underlying ionic currents and transporters (Nerbonne & Kass 2005). A schematic representation of a cardiac action potential and the principal currents involved in its various phases are shown in Figure 9A (Nattel & Carlsson 2006). The action potentials of pacemaker cells in the sinoatrial (SA) and atrioventricular (AV) nodes are significantly different from those in working myocardium. Figure 9B depicts action potential characteristics in different regions of the heart (Michael et al., 2009).

(Nademanee et al., 2000). Successful treatment of recurrent ventricular tachycardia, refractory to antiarrhythmic therapy, can be achieved by neuraxial modulation at the level of the spinal cord. The benefit of thoracic epicardial anesthesia was reported in a patient with ischemic cardiomyopathy and recurrent ventricular arrhythmia refractory to intubation and sedation, with the use of 0.25% Bupicavaine at T1–T2 interspace, reducing

Fig. 8. Structural and functional basis of ventricular tachycardia and fibrillation (Marmar &

β-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aldosterone antagonists (Cittadini et al., 2003), statins (Gao et al., 2005), and fish oil have been shown to decrease risk of sudden cardiac death in ischemic cardiomyopathy and significantly improve mortality through modulating the autonomic nervous system to decrease sympathetic tone (Marmar & Kalyanam,2008). In a recent study, carvedilol ameliorated electrical remodeling at peri-infarct zones after myocardial infarction by improving the spatial distribution of the sympathetic nerve reinnervation (Huazhi et al., 2010). Moreover, attenuation of oxidative stress and inflammatory response after resveratrol treatment, a naturally occurring compound, significantly inhibited nerve growth factor expression, and protected against sympathetic neural remodeling (Ping et al., 2010). Ghrelin, a novel growth hormone-releasing peptide, which was showed to inhibite neural remodeling in rats with myocardial infarction, likely mediated through nerve growth factor suppression, might be used as a new potential way to treat and prevent sudden cardiac

The normal sequence and synchronous contraction of the atria and ventricles require the rapid activation of groups of cardiac cells. An activation mechanism must enable rapid changes in heart rate and also respond to the changes in autonomic tone. The propagating cardiac action potential fulfils these roles. The action potential is a key determinant of cardiac electrical activity and is shaped by underlying ionic currents and transporters (Nerbonne & Kass 2005). A schematic representation of a cardiac action potential and the principal currents involved in its various phases are shown in Figure 9A (Nattel & Carlsson 2006). The action potentials of pacemaker cells in the sinoatrial (SA) and atrioventricular (AV) nodes are significantly different from those in working myocardium. Figure 9B depicts

action potential characteristics in different regions of the heart (Michael et al., 2009).

the number of ICD shocks from 86 in 48 hours to zero (Mahajan et al., 2005).

death after myocardial infarction (Ming-Jie et al., 2009).

**3. Myocardial electrical remodelling** 

**3.1 The cardiac action potential** 

Kalyanam, 2008).

The average duration of the ventricular action potential duration is reflected in the QT interval in the ECG. Factors that prolong the action potential duration (eg, a decrease in outward K+ currents or an increase in inward late Na+ current) prolong the action potential duration and the QT interval in the ECG. The QT interval of males and females is equal during early childhood. However, at puberty the interval of males shortens (Rautaharju et al., 1992).

Fig. 9. A, Membrane currents that generate the normal action potential duration. Resting (4), upstroke (0), early repolarization (1), plateau (2), and final repolarization are the 5 phases of the action potential. A decline of potential at the end of phase 3 in pacemaker cells, such as the sinus node, is shown as a broken line. The inward currents, INa, ICa, and If, are shown in yellow boxes; the sodium-calcium exchanger (NCX) is also shown in yellow. It is electrogenic and may generate inward or outward current. IKAch, IK1, Ito, IKur, IKr, and IKs are shown in gray boxes. The action potential duration is approximately 200ms. B, Various specialized tissues in the heart and typical corresponding action potentials. The short vertical lines indicate the time of onset of activity in the sinus atrial node for one beat. Green, slow-channel tissue (nodes); violet, fast-channel tissues (Nattel & Carlsson 2006).

### **3.2 General properties of ion channels**

Ion channels have two fundamental properties, ion permeation and gating (Hille, 1978). Ion permeation describes the movement through the open channel. The selective permeability of ion channels to specific ions is a basis of classification of ion channels (eg, Na+, K+, and Ca2+ channels). Size, valency, and hydration energy are important determinants of selectivity (Augustus, 2009). Ion channels provide multiple binding sites for ions as they traverse the membrane. Like an enzyme-substrate interaction, the binding of the permeating ion is saturable. Most ion channels are singly occupied during permeation; certain K+ channels may be multiply occupied (Augustus, 2009).

Gating is the mechanism of opening and closing of ion channels and is their second major property. Ion channels are also subclassified by their mechanism of gating: voltagedependent, ligand-dependent, and mechano-sensitive gating. Voltage-gated ion

Electrophysiological Abnormality:

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 143

Fig. 10. Changes in action potential duration measured to 50% (above left) and 90% repolarization (below left) of epicardial muscle fibers with increasing time after coronary artery occlusion. Asterisks denote values significantly different from control. At the right are shown representative transmembrane potential recordings; A, normal; B, 1 day; C, 5 days; D, 2 weeks; E, 2 months. Note that action potentials in the 1- and 5-day-old infarcts show loss of the plateau phase during repolarization. Action potential duration is decreased more

Cardiac K+ channels fall into three broad categories: Voltagegated (Ito, IKur, IKr, and IKs), inward rectifier channels (IK1, IKAch, and IKATP), and the background K+ currents (TASK-1, TWIK-1/2). Voltage-gated K+ channels consist of principal α-subunits and multiple βsubunits. The channel functional units also include the complementary proteins KV-channel associated protein, KChAP, and the KV channel interacting protein, KChIP. The major subfamilies ofα-subunits include KVN.x (n=1 to 4), the HERG channel (gene KCNH2), and KvLQT1 (gene KCNQ1). They are important in generating outward current in the heart (Augustus, 2009). Myocardial infarction causes substantial changes in K+ current expression,

 **current function in surviving border-zone cells**  A variety of K+ currents are downregulated in border-zone cells. Background K+ conductance is reduced in surviving canine subendocardial Purkinje fibers (Boyden et al., 1989), due to reduced IK1 and altered delayed-rectifier currents (Pinto & Boyden, 1998). Border-zone left ventricular cardiomyocytes show reduced Ito (Lue & Boyden, 1992). Ito decreases are most prominent within days of acute infarction and tend to resolve over the subsequent 2 months (Dun et al., 2004). The expression of subunits encoding IKr (ERG) and IKs (KvLQT1 and minK) is downregulated in 2-day postinfarction border-zone cells (Dun & Boyden, 2005; Jiang et al., 2000). Overall, the multiple forms of K+-channel dysfunction

postinfarction impair repolarization and lead to early afterdepolarizations.

in 5-day-old than in 1-day-old infarcts (Ursell et al., 1985).

 **currents** 

density, and function (Janse & Wit, 1989).

**3.4 Alterations in K+**

**3.4.1 Changed K<sup>+</sup>**

channels change their conductance in response to variations in membrane potential. Voltage-dependent gating is the commonest mechanism of gating observed in ion channels. A majority of ion channels open in response to depolarization. The pacemaker current channel (If channel) opens in response to membrane hyperpolarization. The steepness of the voltage dependence of opening or activation varies between channels (Hille, 1978).

Ion channels have two mechanisms of closure. Certain channels like the Na+ and Ca2+ channels enters a closed inactivated state during maintained depolarization. To regain their ability to open, the channel must undergo a recovery process at hyperpolarized potentials. The inactivated state may also be accessed from the closed state. Inactivation is the basis for refractoriness in cardiac muscle and is fundamental for the prevention of premature reexcitation (Hille, 1978; Leblanc & Hume, 1990).

Ligand-dependent gating is the second major gating mechanism of cardiac ion channels. The acetylcholine (Ach)-activated K+ channel, an inward-rectifying K+ channel (IKAch), is one of this class. IKAch channels are most abundant in the atria and the sinus atrial and atrioventricular nodes. The ATP-sensitive K+ channel, also termed the ADP-activated K+ channel, is a ligand-gated channel distributed abundantly in all regions of the heart. Energy depletion during ischemia increases the [ADP]/ [ATP] ratio, activates IKATP, and abbreviates the action potential. The abbreviated action potential results in less force generation and may be cardioprotective. This channel also plays a central role in ischemic preconditioning (Hille, 1978).

### **3.3 Significance and arrhythmic consequences of Ionic currents remodeling associated with myocardial infarction**

Myocardial infarction refers to the death of cardiac tissue, most often caused by critical decreases in coronary artery blood flow induced by obstructive coronary artery disease. Several mechanisms, including reentry and triggered activity due to early and delayed afterdepolarizations, contribute to ventricular tachyarrhythmia (Janse & Wit, 1989; Qin et al., 1996). Remodeling of ion-channel and transport processes cause important changes in cellular electrical activity and impulse propagation over days and weeks following acute infarction (Friedman et al., 1975; Spear et al., 1977).

Generally, by 24–48h after total coronary artery occlusion the action potentials and maximal action potential upstroke velocity (Vmax) decreased, as well as an increase in total time of repolarization. On the other hand, the cells of the epicardial border zone of the canine infarction model show a reduction in Vmax, and a shortening and triangularization of the action potential by 5 days after total artery occlusion. By 14 days post occlusion further shortening of the action potential occurs. Then by the time of the healed infarct (2 months), action potential voltage profiles have returned to nearly normal suggesting the presence of a process that might be termed 'reverse remodeling' (Figure 10) (Ursell et al., 1985). We found that the PR, QRS, QT and QTc intervals in myocardial infarction mice were significantly longer than normal mice after 4, 8, and 12 weeks (LI et al., 2009c).

These abnormalities cause severe conduction disturbances that strongly promote reentry. A particularly important arrhythmia mechanism is anisotropic reentry in the peri-infarction border zone (Dillon et al., 1988; Restivo et al., 1990). Acute myocardial infarction causes longer term (remodeling) changes over days to weeks, as well as important very early (within minutes to hours) functionally based ion-channel abnormalities. Figure 11 illustrates how different forms of ionchannel remodeling contribute to anisotropic reentry in the presence of a healed myocardial infarction (Nattel S et al., 2007).

Fig. 10. Changes in action potential duration measured to 50% (above left) and 90% repolarization (below left) of epicardial muscle fibers with increasing time after coronary artery occlusion. Asterisks denote values significantly different from control. At the right are shown representative transmembrane potential recordings; A, normal; B, 1 day; C, 5 days; D, 2 weeks; E, 2 months. Note that action potentials in the 1- and 5-day-old infarcts show loss of the plateau phase during repolarization. Action potential duration is decreased more in 5-day-old than in 1-day-old infarcts (Ursell et al., 1985).

#### **3.4 Alterations in K+ currents**

142 Advances in Electrocardiograms – Clinical Applications

channels change their conductance in response to variations in membrane potential. Voltage-dependent gating is the commonest mechanism of gating observed in ion channels. A majority of ion channels open in response to depolarization. The pacemaker current channel (If channel) opens in response to membrane hyperpolarization. The steepness of the

Ion channels have two mechanisms of closure. Certain channels like the Na+ and Ca2+ channels enters a closed inactivated state during maintained depolarization. To regain their ability to open, the channel must undergo a recovery process at hyperpolarized potentials. The inactivated state may also be accessed from the closed state. Inactivation is the basis for refractoriness in cardiac muscle and is fundamental for the prevention of premature

Ligand-dependent gating is the second major gating mechanism of cardiac ion channels. The acetylcholine (Ach)-activated K+ channel, an inward-rectifying K+ channel (IKAch), is one of this class. IKAch channels are most abundant in the atria and the sinus atrial and atrioventricular nodes. The ATP-sensitive K+ channel, also termed the ADP-activated K+ channel, is a ligand-gated channel distributed abundantly in all regions of the heart. Energy depletion during ischemia increases the [ADP]/ [ATP] ratio, activates IKATP, and abbreviates the action potential. The abbreviated action potential results in less force generation and may be cardioprotective. This channel also plays a central role in ischemic preconditioning

voltage dependence of opening or activation varies between channels (Hille, 1978).

**3.3 Significance and arrhythmic consequences of Ionic currents remodeling** 

Myocardial infarction refers to the death of cardiac tissue, most often caused by critical decreases in coronary artery blood flow induced by obstructive coronary artery disease. Several mechanisms, including reentry and triggered activity due to early and delayed afterdepolarizations, contribute to ventricular tachyarrhythmia (Janse & Wit, 1989; Qin et al., 1996). Remodeling of ion-channel and transport processes cause important changes in cellular electrical activity and impulse propagation over days and weeks following acute

Generally, by 24–48h after total coronary artery occlusion the action potentials and maximal action potential upstroke velocity (Vmax) decreased, as well as an increase in total time of repolarization. On the other hand, the cells of the epicardial border zone of the canine infarction model show a reduction in Vmax, and a shortening and triangularization of the action potential by 5 days after total artery occlusion. By 14 days post occlusion further shortening of the action potential occurs. Then by the time of the healed infarct (2 months), action potential voltage profiles have returned to nearly normal suggesting the presence of a process that might be termed 'reverse remodeling' (Figure 10) (Ursell et al., 1985). We found that the PR, QRS, QT and QTc intervals in myocardial infarction mice were significantly

These abnormalities cause severe conduction disturbances that strongly promote reentry. A particularly important arrhythmia mechanism is anisotropic reentry in the peri-infarction border zone (Dillon et al., 1988; Restivo et al., 1990). Acute myocardial infarction causes longer term (remodeling) changes over days to weeks, as well as important very early (within minutes to hours) functionally based ion-channel abnormalities. Figure 11 illustrates how different forms of ionchannel remodeling contribute to anisotropic reentry in the

reexcitation (Hille, 1978; Leblanc & Hume, 1990).

**associated with myocardial infarction** 

infarction (Friedman et al., 1975; Spear et al., 1977).

longer than normal mice after 4, 8, and 12 weeks (LI et al., 2009c).

presence of a healed myocardial infarction (Nattel S et al., 2007).

(Hille, 1978).

Cardiac K+ channels fall into three broad categories: Voltagegated (Ito, IKur, IKr, and IKs), inward rectifier channels (IK1, IKAch, and IKATP), and the background K+ currents (TASK-1, TWIK-1/2). Voltage-gated K+ channels consist of principal α-subunits and multiple βsubunits. The channel functional units also include the complementary proteins KV-channel associated protein, KChAP, and the KV channel interacting protein, KChIP. The major subfamilies ofα-subunits include KVN.x (n=1 to 4), the HERG channel (gene KCNH2), and KvLQT1 (gene KCNQ1). They are important in generating outward current in the heart (Augustus, 2009). Myocardial infarction causes substantial changes in K+ current expression, density, and function (Janse & Wit, 1989).

#### **3.4.1 Changed K<sup>+</sup> current function in surviving border-zone cells**

A variety of K+ currents are downregulated in border-zone cells. Background K+ conductance is reduced in surviving canine subendocardial Purkinje fibers (Boyden et al., 1989), due to reduced IK1 and altered delayed-rectifier currents (Pinto & Boyden, 1998). Border-zone left ventricular cardiomyocytes show reduced Ito (Lue & Boyden, 1992). Ito decreases are most prominent within days of acute infarction and tend to resolve over the subsequent 2 months (Dun et al., 2004). The expression of subunits encoding IKr (ERG) and IKs (KvLQT1 and minK) is downregulated in 2-day postinfarction border-zone cells (Dun & Boyden, 2005; Jiang et al., 2000). Overall, the multiple forms of K+-channel dysfunction postinfarction impair repolarization and lead to early afterdepolarizations.

Electrophysiological Abnormality:

postinfarction.

**3.5.1 Changes in Ca2+ current** 

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 145

involved (Liu et al., 2004). Decreases in Ito, IK1, and total delayed-rectifier current (IK) occur in rabbit hearts (Liu et al., 2004). In rats, Ito decreases correlate most closely with downregulation of Kv4.2 subunits (Perrier et al., 2004). There may be compensatory upregulation of Kv1.4 subunits (Kaprielian et al., 2002), although downregulation of Kv1.4 has also been reported (Gidh-Jain et al., 1996). Decreases in rat IK correlate with downregulation of the putative α-subunit Kv2.1 (Huang et al., 2001b). The effects of postinfarction remodeling on spatial dispersion of electrophysiological properties in noninfarcted tissues are controversial, with one study showing increases in dispersion (Huang et al., 2001b) and another decreased spatial heterogeneity (Kaprielian et al., 2002).

Calcium ions are the principal intracellular signaling ions. They regulate excitationcontraction coupling, secretion, and the activity of many enzymes and ion channels. [Ca2+]i is highly regulated despite its marked fluctuation between systole and diastole. Calcium channels are the principal portal of entry of calcium into the cells; a system of intracellular storage sites, and transporters such as the sodium-calcium exchanger, also play important roles in [Ca2+]i regulation. In cardiac muscle, two types of Ca2+ channels, the L- (low threshold type) and T-type (transient-type), transport Ca2+ into the cells. The L-type channel is found in all cardiac cell types. The T-type channel is found principally in pacemaker, atrial, and Purkinje cells. The unqualified descriptor Ca2+ channel refers to the L-type channel. Table 2 contrasts the properties of the two types of channels (Augustus, 2009). Changes in Ca2+ handling contribute importantly to arrhythmogenesis

Table 2. A comparisons of the L-type and T-type Ca2+ channels (Augustus, 2009).

subendocardial Purkinje cells, both ICaL and ICaT are reduced (Boyden & Pinto, 1994).

ICaL is diminished in border-zone cells of dogs (Dun et al., 2004), sheep (Kim et al., 2002), cats (Pinto et al., 1997), and rabbits (Litwin et al., 2000). ICaL kinetic properties also change, with slowed recovery (Dun et al., 2004) and hyperpolarizing shifts in inactivation voltage dependence (Pinto et al., 1997). The ICaL response to dihydropyridine agonists (Pu et al., 1999) and tyrosine kinase inhibitors (Yagi & Boyden, 2002) is preserved in the border zone. T-type Ca2+ current (ICaT) varies over time, being unchanged 5 days postinfarction (Aggarwal & Boyden, 1995) and increasing thereafter (Dun et al., 2004). In surviving

**3.5 Alterations in Ca2+ currents and cellular Ca2+ handling** 

Fig. 11. Contributors to anisotropic reentry in myocardial infarction. A principal mechanism underlying potentially lethal ventricular tachyarrhythmias post-myocardial infarction is anisotropic reentry, represented schematically by the black activation map in the central part of the figure (Peters et al., 1997). The numbers indicated on the map are times of electrical activation, and the curved lines (isochrones) indicate zones of tissue activated within 10 ms of each other. Crowded isochrones denote very slow conduction. Thicker black lines show lines of functional conduction block parallel to fiber orientation, due to the impaired transverse conduction (increased anisotropy) post-myocardial infarction. The impulse travels slowly in two parallel streams (thick arrows) around the lines of block, which come together to conduct through the central corridor (thinner arrows) of the reentrant pathway. The ways in which ion-channel remodeling post-myocardial infarction lead to this arrhythmia mechanism are indicated by the red points, organized into groups of dysfunction categories (blue underlined headings). Increased tissue anisotropy, which causes the unidirectional block needed for reentry initiation, arises because of connexin downregulation, reduced gap junction number and size, fewer side-to-side connections, and tissue fibrosis around muscle bundles. Unidirectional block is also favored by refractoriness heterogeneity due to spatially heterogeneous K+ channel downregulation coupled with postrepolarization refractoriness. Slowed conduction, which allows enough time for the proximal part of the central corridor to recover excitability when the reentering impulse returns, is caused by connexin downregulation, INa decreases, and reduced ICaL (ICaL is particularly important for conduction in conditions of impaired coupling). Finally, the ectopic complexes needed to engage spatially variable refractoriness and initiate reentry are provided by early afterdepolarizations promoted by K+ current downregulation and delayed afterdepolarizations caused by spontaneous diastolic Ca2+ releases (Nattel S et al., 2007).

#### **3.4.2 Changes in K<sup>+</sup> currents in normal zones of hearts with prior myocardial infarction**

Action potential duration increases and ventricular arrhythmias are features of normal-zone tissues from postinfarction rat (Perrier et al., 2004) and rabbit (Liu et al., 2004) hearts. Both reentries associated with spatial refractoriness heterogeneity and triggered activity is involved (Liu et al., 2004). Decreases in Ito, IK1, and total delayed-rectifier current (IK) occur in rabbit hearts (Liu et al., 2004). In rats, Ito decreases correlate most closely with downregulation of Kv4.2 subunits (Perrier et al., 2004). There may be compensatory upregulation of Kv1.4 subunits (Kaprielian et al., 2002), although downregulation of Kv1.4 has also been reported (Gidh-Jain et al., 1996). Decreases in rat IK correlate with downregulation of the putative α-subunit Kv2.1 (Huang et al., 2001b). The effects of postinfarction remodeling on spatial dispersion of electrophysiological properties in noninfarcted tissues are controversial, with one study showing increases in dispersion (Huang et al., 2001b) and another decreased spatial heterogeneity (Kaprielian et al., 2002).

### **3.5 Alterations in Ca2+ currents and cellular Ca2+ handling**

144 Advances in Electrocardiograms – Clinical Applications

Fig. 11. Contributors to anisotropic reentry in myocardial infarction. A principal mechanism underlying potentially lethal ventricular tachyarrhythmias post-myocardial infarction is anisotropic reentry, represented schematically by the black activation map in the central part of the figure (Peters et al., 1997). The numbers indicated on the map are times of electrical activation, and the curved lines (isochrones) indicate zones of tissue activated within 10 ms of each other. Crowded isochrones denote very slow conduction. Thicker black lines show lines of functional conduction block parallel to fiber orientation, due to the impaired transverse conduction (increased anisotropy) post-myocardial infarction. The impulse travels slowly in two parallel streams (thick arrows) around the lines of block, which come together to conduct through the central corridor (thinner arrows) of the reentrant pathway. The ways in which ion-channel remodeling post-myocardial infarction lead to this arrhythmia mechanism are indicated by the red points, organized into groups of dysfunction categories (blue underlined headings). Increased tissue anisotropy, which causes the unidirectional block needed for reentry initiation, arises because of connexin downregulation, reduced gap junction number and size, fewer side-to-side connections, and tissue fibrosis around muscle bundles. Unidirectional block is also favored by refractoriness heterogeneity due to spatially heterogeneous K+ channel downregulation coupled with postrepolarization refractoriness. Slowed conduction, which allows enough time for the proximal part of the central corridor to recover excitability when the reentering impulse returns, is caused by connexin downregulation, INa decreases, and reduced ICaL (ICaL is

particularly important for conduction in conditions of impaired coupling). Finally, the ectopic complexes needed to engage spatially variable refractoriness and initiate reentry are provided

Action potential duration increases and ventricular arrhythmias are features of normal-zone tissues from postinfarction rat (Perrier et al., 2004) and rabbit (Liu et al., 2004) hearts. Both reentries associated with spatial refractoriness heterogeneity and triggered activity is

 **currents in normal zones of hearts with prior myocardial** 

by early afterdepolarizations promoted by K+ current downregulation and delayed afterdepolarizations caused by spontaneous diastolic Ca2+ releases (Nattel S et al., 2007).

**3.4.2 Changes in K<sup>+</sup>**

**infarction** 

Calcium ions are the principal intracellular signaling ions. They regulate excitationcontraction coupling, secretion, and the activity of many enzymes and ion channels. [Ca2+]i is highly regulated despite its marked fluctuation between systole and diastole. Calcium channels are the principal portal of entry of calcium into the cells; a system of intracellular storage sites, and transporters such as the sodium-calcium exchanger, also play important roles in [Ca2+]i regulation. In cardiac muscle, two types of Ca2+ channels, the L- (low threshold type) and T-type (transient-type), transport Ca2+ into the cells. The L-type channel is found in all cardiac cell types. The T-type channel is found principally in pacemaker, atrial, and Purkinje cells. The unqualified descriptor Ca2+ channel refers to the L-type channel. Table 2 contrasts the properties of the two types of channels (Augustus, 2009). Changes in Ca2+ handling contribute importantly to arrhythmogenesis postinfarction.


Table 2. A comparisons of the L-type and T-type Ca2+ channels (Augustus, 2009).

### **3.5.1 Changes in Ca2+ current**

ICaL is diminished in border-zone cells of dogs (Dun et al., 2004), sheep (Kim et al., 2002), cats (Pinto et al., 1997), and rabbits (Litwin et al., 2000). ICaL kinetic properties also change, with slowed recovery (Dun et al., 2004) and hyperpolarizing shifts in inactivation voltage dependence (Pinto et al., 1997). The ICaL response to dihydropyridine agonists (Pu et al., 1999) and tyrosine kinase inhibitors (Yagi & Boyden, 2002) is preserved in the border zone. T-type Ca2+ current (ICaT) varies over time, being unchanged 5 days postinfarction (Aggarwal & Boyden, 1995) and increasing thereafter (Dun et al., 2004). In surviving subendocardial Purkinje cells, both ICaL and ICaT are reduced (Boyden & Pinto, 1994).

Electrophysiological Abnormality:

**3.6.2 Functional consequences** 

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 147

excitability favor unidirectional block and reentry (Janse & Wit, 1989). Isolated border-zone cardiomyocytes also have reduced dV/dtmax (Lue & Boyden, 1992) and marked abnormalities in INa, including reduced current density, accelerated inactivation, and slowed reactivation (Pu & Boyden, 1997). Computer simulations suggest that both INa and ICaL abnormalities contribute to conduction abnormalities in the reentry circuit (Baba et al., 2005), in keeping with the key role of ICaL in the context of reduced coupling (Shaw & Rudy, 1997). Protein kinase A activators partially improve INa in peri-infarct zone cells, and the response to phosphatase inhibitors suggests that INa is hyperphosphorylated (Baba et al., 2004). In late postinfarction rat cardiomyocytes, changes in INa properties and in ion-channel subunit expression suggest the appearance of atypical INa isoforms (Huang et al., 2001a); these changes may be due to generalized cardiac hypertrophy/dysfunction rather than infarction per se.

Oxidative stress in postinfarction tissues produces reactive intermediates that alter INa in a fashion similar to arrhythmogenic Nav1.5 subunit mutations and potentiate the effects of Na+ channel-blocking drugs (Fukuda et al., 2005). The INa blocker lidocaine differentially affects peri-infarct zone cardiomyocytes (Pu et al., 1998). These differential effects may contribute to the tendency of INa blockers to cause malignant ventricular tachyarrhythmias postinfarction (Ranger & Nattel, 1995). These paradoxical "proarrhythmic" effects of INablocking antiarrhythmic drugs on myocardial infarction tissues contribute to a mortalityenhancing potential (Cardiac Arrhythmia Suppression Trial (CAST) Investigators, 1989).

**3.7 Therapeutic implications of ionic current and transporter remodeling** 

remodeling likely contributes to these adverse responses.

**3.7.2 Ionic remodeling as a target for novel therapeutic approaches** 

downregulation (Yuan et al., 1999).

**3.7.1 Remodeling-induced modification of the response to therapeutic interventions**  Myocardial infarction greatly increases the risk of arrhythmic death, and associated remodeling sensitizes patients to the proarrhythmic effects of a variety of drugs. The risk of drug-induced Torsades de Pointes arrhythmias caused by early afterdepolarizations is approximately increased by myocardial infarction (Stanley et al., 2007). Drugs like βadrenergic agonists and phosphodiesterase inhibitors, which increase cardiac contractility by increasing intracellular cAMP concentrations, Ca2+ loading and Ca2+-induced Ca2+ release, have been used extensively to improve cardiac function in patients with severe cardiac dysfunction. Unfortunately, in the longer term they have arrhythmogenic actions and increase mortality (Gardner et al., 1985; Hagemeijer, 1993; Lubbe et al., 1992). Ionic

Many of the changes responsible for adverse effects of antiarrhythmic drugs are caused by postinfarction myocardial remodeling: increased action potential duration, localized conduction slowing, downregulation of K+ channels, abnormal diastolic Ca2+ handling, and impaired connexin function. Myocardial infarction predisposes to the proarrhythmic actions of Na+ channel blocking drugs (Cardiac Arrhythmia Suppression Trial (CAST) Investigators, 1989; Ranger & Nattel, 1995) and IKr blocking agents (Waldo et al., 1996). Responses to IKr blocking drugs may be reduced in postinfarction cells, perhaps because of IKr

Much less work has been done to study interventions targeting ion-handling processes postinfarction. An angiotensin-converting enzyme inhibitor attenuated increases in refractoriness heterogeneity and prevented afterdepolarization formation in normal zones of

### **3.5.2 Changes in cellular Ca2+ handling**

Ca2+ transients in border-zone cells are decreased in amplitude and show slowed recovery and decay (Kim et al., 2002). SERCA2A, the sarcoplasmic reticulum Ca2+ ATPase, is downregulated (Kim et al., 2002). The diminished and slowed Ca2+ transients are due to impaired spatial coordination of quantal Ca2+ releases, or sparks (Litwin et al., 2000). Na+- Ca2+ exchange function is unaltered, and action potential abnormalities are not responsible for Ca2+ handling abnormalities (Pu et al., 2000). Surviving subendocardial Purkinje cells show marked abnormalities in subcellular Ca2+ release events, with spontaneous and spatiotemporally nonuniform microreleases that can trigger arrhythmic episodes (Boyden et al., 2003). Drugs that suppress Ca2+ microreleases by either inhibiting sarcoplasmic reticulum Ca2+ release channels or inositol trisphosphate receptors may constitute a novel antiarrhythmic approach postinfarction (Boyden et al., 2004).

#### **3.6 Alterations in Na+ current**

The human cardiac sodium channel hNaV1.5 is a member of the family of voltage-gated sodium channels (hNaV1 to 9). The channel consists of a primary α-and multiple secondary β-subunits. The sodium channel consists of 4 homologous domains, DI through DIV (Noda et al., 1984) arranged in a 4-fold circular symmetry to form the channel (Figure 12) (Herbert & Chahine, 2006).

Fig. 12. Putative transmembrane organization of the sodium channel. The channel consists of 4 homologous domains, DI through DIV. The amino and carboxyl termini are intracellular. (Herbert & Chahine, 2006).

Each sodium channel opens very briefly (<1ms) during more than 99% of depolarizations (Patlak & Ortiz, 1985). The channel occasionally shows alternative gating modes consisting of isolated brief openings occurring after variable and prolonged latencies and bursts of openings during which the channel opens repetitively for hundreds of milliseconds. The isolated brief openings are the result of the occasional return from the inactivated state. The bursts of openings are the result of occasional failure of inactivation (Patlak & Ortiz, 1985). The cardiac sodium channel has consensus sites for phosphorylation by protein kinase, protein kinase C, and Ca-calmodulin kinase (Frohnwieser et al., 1997).

#### **3.6.1 Na+ current changes**

Surviving border-zone tissue is characterized by reduced phase 0 amplitude and upstroke velocity (dV/dtmax), suggestive of reduced INa (Spear et al., 1979). These abnormalities in excitability favor unidirectional block and reentry (Janse & Wit, 1989). Isolated border-zone cardiomyocytes also have reduced dV/dtmax (Lue & Boyden, 1992) and marked abnormalities in INa, including reduced current density, accelerated inactivation, and slowed reactivation (Pu & Boyden, 1997). Computer simulations suggest that both INa and ICaL abnormalities contribute to conduction abnormalities in the reentry circuit (Baba et al., 2005), in keeping with the key role of ICaL in the context of reduced coupling (Shaw & Rudy, 1997). Protein kinase A activators partially improve INa in peri-infarct zone cells, and the response to phosphatase inhibitors suggests that INa is hyperphosphorylated (Baba et al., 2004). In late postinfarction rat cardiomyocytes, changes in INa properties and in ion-channel subunit expression suggest the appearance of atypical INa isoforms (Huang et al., 2001a); these changes may be due to generalized cardiac hypertrophy/dysfunction rather than infarction per se.

### **3.6.2 Functional consequences**

146 Advances in Electrocardiograms – Clinical Applications

Ca2+ transients in border-zone cells are decreased in amplitude and show slowed recovery and decay (Kim et al., 2002). SERCA2A, the sarcoplasmic reticulum Ca2+ ATPase, is downregulated (Kim et al., 2002). The diminished and slowed Ca2+ transients are due to impaired spatial coordination of quantal Ca2+ releases, or sparks (Litwin et al., 2000). Na+- Ca2+ exchange function is unaltered, and action potential abnormalities are not responsible for Ca2+ handling abnormalities (Pu et al., 2000). Surviving subendocardial Purkinje cells show marked abnormalities in subcellular Ca2+ release events, with spontaneous and spatiotemporally nonuniform microreleases that can trigger arrhythmic episodes (Boyden et al., 2003). Drugs that suppress Ca2+ microreleases by either inhibiting sarcoplasmic reticulum Ca2+ release channels or inositol trisphosphate receptors may constitute a novel

The human cardiac sodium channel hNaV1.5 is a member of the family of voltage-gated sodium channels (hNaV1 to 9). The channel consists of a primary α-and multiple secondary β-subunits. The sodium channel consists of 4 homologous domains, DI through DIV (Noda et al., 1984) arranged in a 4-fold circular symmetry to form the channel (Figure 12) (Herbert

Fig. 12. Putative transmembrane organization of the sodium channel. The channel consists

Each sodium channel opens very briefly (<1ms) during more than 99% of depolarizations (Patlak & Ortiz, 1985). The channel occasionally shows alternative gating modes consisting of isolated brief openings occurring after variable and prolonged latencies and bursts of openings during which the channel opens repetitively for hundreds of milliseconds. The isolated brief openings are the result of the occasional return from the inactivated state. The bursts of openings are the result of occasional failure of inactivation (Patlak & Ortiz, 1985). The cardiac sodium channel has consensus sites for phosphorylation by protein kinase,

Surviving border-zone tissue is characterized by reduced phase 0 amplitude and upstroke velocity (dV/dtmax), suggestive of reduced INa (Spear et al., 1979). These abnormalities in

of 4 homologous domains, DI through DIV. The amino and carboxyl termini are

protein kinase C, and Ca-calmodulin kinase (Frohnwieser et al., 1997).

**3.5.2 Changes in cellular Ca2+ handling**

**3.6 Alterations in Na+**

& Chahine, 2006).

**3.6.1 Na+**

antiarrhythmic approach postinfarction (Boyden et al., 2004).

 **current** 

intracellular. (Herbert & Chahine, 2006).

 **current changes** 

Oxidative stress in postinfarction tissues produces reactive intermediates that alter INa in a fashion similar to arrhythmogenic Nav1.5 subunit mutations and potentiate the effects of Na+ channel-blocking drugs (Fukuda et al., 2005). The INa blocker lidocaine differentially affects peri-infarct zone cardiomyocytes (Pu et al., 1998). These differential effects may contribute to the tendency of INa blockers to cause malignant ventricular tachyarrhythmias postinfarction (Ranger & Nattel, 1995). These paradoxical "proarrhythmic" effects of INablocking antiarrhythmic drugs on myocardial infarction tissues contribute to a mortalityenhancing potential (Cardiac Arrhythmia Suppression Trial (CAST) Investigators, 1989).

### **3.7 Therapeutic implications of ionic current and transporter remodeling**

### **3.7.1 Remodeling-induced modification of the response to therapeutic interventions**

Myocardial infarction greatly increases the risk of arrhythmic death, and associated remodeling sensitizes patients to the proarrhythmic effects of a variety of drugs. The risk of drug-induced Torsades de Pointes arrhythmias caused by early afterdepolarizations is approximately increased by myocardial infarction (Stanley et al., 2007). Drugs like βadrenergic agonists and phosphodiesterase inhibitors, which increase cardiac contractility by increasing intracellular cAMP concentrations, Ca2+ loading and Ca2+-induced Ca2+ release, have been used extensively to improve cardiac function in patients with severe cardiac dysfunction. Unfortunately, in the longer term they have arrhythmogenic actions and increase mortality (Gardner et al., 1985; Hagemeijer, 1993; Lubbe et al., 1992). Ionic remodeling likely contributes to these adverse responses.

Many of the changes responsible for adverse effects of antiarrhythmic drugs are caused by postinfarction myocardial remodeling: increased action potential duration, localized conduction slowing, downregulation of K+ channels, abnormal diastolic Ca2+ handling, and impaired connexin function. Myocardial infarction predisposes to the proarrhythmic actions of Na+ channel blocking drugs (Cardiac Arrhythmia Suppression Trial (CAST) Investigators, 1989; Ranger & Nattel, 1995) and IKr blocking agents (Waldo et al., 1996). Responses to IKr blocking drugs may be reduced in postinfarction cells, perhaps because of IKr downregulation (Yuan et al., 1999).

### **3.7.2 Ionic remodeling as a target for novel therapeutic approaches**

Much less work has been done to study interventions targeting ion-handling processes postinfarction. An angiotensin-converting enzyme inhibitor attenuated increases in refractoriness heterogeneity and prevented afterdepolarization formation in normal zones of

Electrophysiological Abnormality:

cGMP (Figure 14) (Harris, 2001; Wong et al., 2008).

through gap junctions (Wong et al., 2008).

into changes in the number of operative gap junction channel.

**4.2 Diversity of connexin expression in the normal heart** 

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 149

(Severs et al., 1993). Gap junction are permeable to relatively large molecules (Harris, 2001). Depending on the connexins type, pore diameter ranges between approximately 6.5 and 15A °(Harris, 2001), which is wide enough to allow the passage of water, all relevant cations and anions, including Na+, K+, and Ca2+ and most second messengers as IP3, cAMP, and

Fig. 14. Organisation of connexins into gap junctions and molecules capable of diffusing

The mechanisms by which gap junction channel close or open are not fully elucidated. Gating can take place in at least two independent ways: a rapid, voltage-driven mechanism that can change the channel conformation between a fully open and a nearly completely closed state within a few ms (voltage gating), and a slower (up to 30 ms) mechanism (Harris, 2001; Moreno et al., 2002a). Phosphorylation alters the probability of the different conductance states as well as intracellular trafficking and assembly of connexins depending on the connexins type, the phosphorylation site and, possibly, the biochemical environment (Van et al., 2001). The half-life of connexins is relatively short (less than 2h for Cx43) (Laing & Beyer, 2000) so that changes in the rates of synthesis or degradation are rapidly translated

Three principal connexins are expressed in cardiac myocytes, Cx43, Cx40, and Cx45. Although Cx43 predominates in the heart as a whole, it is typically co-expressed in characteristic combinations and relative quantities with Cx40 and/or Cx45 in a chamberrelated and myocyte-type-specific manner (Vozzi et al., 1999). Although a few other connexins have been reported in cardiac tissue, these are minor components, species variants, or not been confirmed. Figure 15 gives an overview of the typical connexin

expression patterns of the normal adult mammalian heart (Severs et al., 2008).

rats with prior infarctions (Li et al., 2004). The combined α- and β-adrenoceptor antagonist carvedilol suppresses downregulation of both Na+ (Maltsev et al., 2002) and L-type Ca2+ (Li et al., 2005) currents following myocardial infarction. Protein kinase A activators can partially restore suppressed INa in the infarct border zone (Baba et al., 2004).

Advances in molecular cardiology have identified several potential targets for gene therapy. In a porcine model, focal gene transfer in the border zone of myocardial infarction to silence the KCNH2 potassium channel has been shown to abolish ventricular arrhythmias (Sasano et al., 2006). Furthermore, overexpression of the HERG potassium channel in isolated rabbit ventricular myocytes shortens action potential duration and reduces the frequency of early after-depolarizations (Nuss et al., 1999). Additional investigation is needed to confirm the safety and efficacy of targeted gene therapy in human. However, successful transition of gene therapy into clinical therapies will require the development of safe and efficient transgene vectors and delivery systems.

### **4. Cardiac gap junction remodeling**

### **4.1 Gap junction structure, permeability and regulation**

The gap junction, a type of cell-to-cell junction, is composed of low-resistance intercellular pathways and mediates electrical and metabolic coupling between adjacent cells. Each gap junction channel is formed by two end-to-end connected hemichannels (also known as connexons) contributed by each of the two adjacent cells. In chordates, hemichannels are hexameric structures formed by six connexins (Cx). Connexins have four transmembrane domains with two extracellular loops and with the N-terminal and C-terminal domains in the cytoplasm (Harris, 2001; Sosinsky, 2000). Twenty different connexin types have been identified in mouse and 21 in man (Sohl & Willecke, 2004). Cells can usually express a variety of connexins, and the six connexins forming a hemichannel may not be identical (for example Cx43 and Cx40), thus forming a heteromeric hemichannel. Although not all combinations are possible (Harris, 2001), hemichannels formed by a particular connexins can dock to hemichannels with a different composition (Harris, 2001). A principal ultrastructural feature of the intercalated disk and gap junction was illustrated in Figure 13

Fig. 13. Principal ultrastructural features of the intercalated disk and gap junction. A, Thinsection electron micrograph showing the appearance of the 3 types of cell-cell junction, the gap junction (GJ), fascia adherens (FA), and desmosome (D). B, Higher magnification thinsection of a gap junction. C, Structure of gap junction as revealed by freeze-fracture electron microscopy. The gap junction is seen as a cluster of particles. Each particle represents a connexon hemichannel. (a) ×36,250; (b) ×350,000; (c) ×98,000 (Severs et al., 1993).

rats with prior infarctions (Li et al., 2004). The combined α- and β-adrenoceptor antagonist carvedilol suppresses downregulation of both Na+ (Maltsev et al., 2002) and L-type Ca2+ (Li et al., 2005) currents following myocardial infarction. Protein kinase A activators can

Advances in molecular cardiology have identified several potential targets for gene therapy. In a porcine model, focal gene transfer in the border zone of myocardial infarction to silence the KCNH2 potassium channel has been shown to abolish ventricular arrhythmias (Sasano et al., 2006). Furthermore, overexpression of the HERG potassium channel in isolated rabbit ventricular myocytes shortens action potential duration and reduces the frequency of early after-depolarizations (Nuss et al., 1999). Additional investigation is needed to confirm the safety and efficacy of targeted gene therapy in human. However, successful transition of gene therapy into clinical therapies will require the development of safe and efficient

The gap junction, a type of cell-to-cell junction, is composed of low-resistance intercellular pathways and mediates electrical and metabolic coupling between adjacent cells. Each gap junction channel is formed by two end-to-end connected hemichannels (also known as connexons) contributed by each of the two adjacent cells. In chordates, hemichannels are hexameric structures formed by six connexins (Cx). Connexins have four transmembrane domains with two extracellular loops and with the N-terminal and C-terminal domains in the cytoplasm (Harris, 2001; Sosinsky, 2000). Twenty different connexin types have been identified in mouse and 21 in man (Sohl & Willecke, 2004). Cells can usually express a variety of connexins, and the six connexins forming a hemichannel may not be identical (for example Cx43 and Cx40), thus forming a heteromeric hemichannel. Although not all combinations are possible (Harris, 2001), hemichannels formed by a particular connexins can dock to hemichannels with a different composition (Harris, 2001). A principal ultrastructural feature of the intercalated disk and gap junction was illustrated in Figure 13

Fig. 13. Principal ultrastructural features of the intercalated disk and gap junction. A, Thinsection electron micrograph showing the appearance of the 3 types of cell-cell junction, the gap junction (GJ), fascia adherens (FA), and desmosome (D). B, Higher magnification thinsection of a gap junction. C, Structure of gap junction as revealed by freeze-fracture electron microscopy. The gap junction is seen as a cluster of particles. Each particle represents a connexon hemichannel. (a) ×36,250; (b) ×350,000; (c) ×98,000 (Severs et al., 1993).

partially restore suppressed INa in the infarct border zone (Baba et al., 2004).

transgene vectors and delivery systems.

**4. Cardiac gap junction remodeling**

**4.1 Gap junction structure, permeability and regulation** 

(Severs et al., 1993). Gap junction are permeable to relatively large molecules (Harris, 2001). Depending on the connexins type, pore diameter ranges between approximately 6.5 and 15A °(Harris, 2001), which is wide enough to allow the passage of water, all relevant cations and anions, including Na+, K+, and Ca2+ and most second messengers as IP3, cAMP, and cGMP (Figure 14) (Harris, 2001; Wong et al., 2008).

Fig. 14. Organisation of connexins into gap junctions and molecules capable of diffusing through gap junctions (Wong et al., 2008).

The mechanisms by which gap junction channel close or open are not fully elucidated. Gating can take place in at least two independent ways: a rapid, voltage-driven mechanism that can change the channel conformation between a fully open and a nearly completely closed state within a few ms (voltage gating), and a slower (up to 30 ms) mechanism (Harris, 2001; Moreno et al., 2002a). Phosphorylation alters the probability of the different conductance states as well as intracellular trafficking and assembly of connexins depending on the connexins type, the phosphorylation site and, possibly, the biochemical environment (Van et al., 2001). The half-life of connexins is relatively short (less than 2h for Cx43) (Laing & Beyer, 2000) so that changes in the rates of synthesis or degradation are rapidly translated into changes in the number of operative gap junction channel.

### **4.2 Diversity of connexin expression in the normal heart**

Three principal connexins are expressed in cardiac myocytes, Cx43, Cx40, and Cx45. Although Cx43 predominates in the heart as a whole, it is typically co-expressed in characteristic combinations and relative quantities with Cx40 and/or Cx45 in a chamberrelated and myocyte-type-specific manner (Vozzi et al., 1999). Although a few other connexins have been reported in cardiac tissue, these are minor components, species variants, or not been confirmed. Figure 15 gives an overview of the typical connexin expression patterns of the normal adult mammalian heart (Severs et al., 2008).

Electrophysiological Abnormality:

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 151

Fig. 17. Expression patterns of Cx43, Cx40 and Cx45 in different cardiomyocyte sub-types. This triple-labelled confocal montage shows part of the conduction system (common bundle and right bundle branch) which expresses Cx40 and Cx45). These two connexins are not detected in the adjacent working ventricular myocytes of the septum, which instead express

Two principal gap junction-related alterations have been reported in the diseased ventricle: disturbances in the distribution of gap junctions and reduced levels of their major component, Cx43. "Lateralization"of Cx43 gap junction label is a prominent feature of the border zone of surviving myocytes around infarct scar tissue in the human ventricle (Smith et al., 1991). Electron microscopy reveals that both laterally disposed gap junctions connecting adjacent cells, and internalized (non-functional) gap junctional membrane,

By 6–12h after ligation, the normal distribution was lost for Cx43, desmoplakin, and cadherin at the intercalated disks in the infarct zone. By 24–48 h after ligation, the expression of Cx43 markedly decreased, to 5% of the levels of shamoperated hearts (Figure 18) (Takamatsu, 2008). At 4 days post-infarction in a dog model, lateral gap junction label in the extended infarct border zone has been correlated spatially with electrophysiologically

Gap junctional changes distant from the infarct scar tissue, in particular reduction in the size and the number of gap junctions per unit length of intercalated disc, and fewer side-to-side connections between myocytes, have been described as longer term remodelling events in dog myocardium (Kostin et al., 2003). Our study showed that Cx43 mRNA and Cx43 protein reduced significantly at ischemic zone at 4, 8 and 12 weeks of myocardial infarction mice

Cx43. This example comes from rat heart (Coppen et al., 1999).

**4.3 Gap junction remodeling in myocardial infarction 4.3.1 Connexin 43 remodeling in myocardial infarction** 

contribute to this abnormal pattern (Smith et al., 1991).

(Figure 19) (LI et al., 2010; Zhao et al., 2009).

identified figure-of-eight re-entrant circuits (Peters et al., 1997).

The working cardiomyocytes of the ventricle are extensively interconnected by clusters of connexin43-containing gap junctions located at the intercalated disks (Figure 16) (Severs et al., 2004). The intercalated disks of working ventricular myocardium have a step-like configuration, with the gap junctions situated predominantly in the membrane segments that lie parallel with the long axis of the cell (Severs, 1990), with larger gap junctions typically circumscribing the disk periphery (Gourdie et al., 1991). This and other features of gap junction organization and aspects of tissue architecture such as the size and shape of the cells combine to ensure preferential propagation of the impulse in the longitudinal axis and hence the normal pattern of anisotropic spread of the impulse of healthy ventricular myocardium.

Fig. 16. An isolated ventricular myocyte labelled for Cx43 (green) illustrating localization of the gap junctions in clusters at the intercalated disks (Severs et al., 2004).

The gap junctions of atrial myocytes contain abundant Cx40 (Vozzi et al., 1999; Dupont et al., 2001), co-localized with Cx43 within the same individual gap-junctional plaques (Severs et al., 2001). Working ventricular myocytes, by contrast, normally lack detectable Cx40. In both ventricular and atrial working myocardium, Cx45 is present in very low quantities (Vozzi et al., 1999; Dupont et al., 2001). The specialized cardiomyocytes of the impulse generation and conduction system are distinct from the working ventricular and atrial cells both in terms of general morphology (Severs, 1989) and connexin expression profiles (Figure 17) (Coppen et al., 1999).

Fig. 15. Summary of the typical connexin expression patterns of the mammalian heart

The working cardiomyocytes of the ventricle are extensively interconnected by clusters of connexin43-containing gap junctions located at the intercalated disks (Figure 16) (Severs et al., 2004). The intercalated disks of working ventricular myocardium have a step-like configuration, with the gap junctions situated predominantly in the membrane segments that lie parallel with the long axis of the cell (Severs, 1990), with larger gap junctions typically circumscribing the disk periphery (Gourdie et al., 1991). This and other features of gap junction organization and aspects of tissue architecture such as the size and shape of the cells combine to ensure preferential propagation of the impulse in the longitudinal axis and hence the normal pattern of anisotropic spread of the impulse of healthy ventricular

Fig. 16. An isolated ventricular myocyte labelled for Cx43 (green) illustrating localization of

The gap junctions of atrial myocytes contain abundant Cx40 (Vozzi et al., 1999; Dupont et al., 2001), co-localized with Cx43 within the same individual gap-junctional plaques (Severs et al., 2001). Working ventricular myocytes, by contrast, normally lack detectable Cx40. In both ventricular and atrial working myocardium, Cx45 is present in very low quantities (Vozzi et al., 1999; Dupont et al., 2001). The specialized cardiomyocytes of the impulse generation and conduction system are distinct from the working ventricular and atrial cells both in terms of general morphology (Severs, 1989) and connexin expression profiles

the gap junctions in clusters at the intercalated disks (Severs et al., 2004).

(Severs et al., 2008).

myocardium.

(Figure 17) (Coppen et al., 1999).

Fig. 17. Expression patterns of Cx43, Cx40 and Cx45 in different cardiomyocyte sub-types. This triple-labelled confocal montage shows part of the conduction system (common bundle and right bundle branch) which expresses Cx40 and Cx45). These two connexins are not detected in the adjacent working ventricular myocytes of the septum, which instead express Cx43. This example comes from rat heart (Coppen et al., 1999).

### **4.3 Gap junction remodeling in myocardial infarction 4.3.1 Connexin 43 remodeling in myocardial infarction**

Two principal gap junction-related alterations have been reported in the diseased ventricle: disturbances in the distribution of gap junctions and reduced levels of their major component, Cx43. "Lateralization"of Cx43 gap junction label is a prominent feature of the border zone of surviving myocytes around infarct scar tissue in the human ventricle (Smith et al., 1991). Electron microscopy reveals that both laterally disposed gap junctions connecting adjacent cells, and internalized (non-functional) gap junctional membrane, contribute to this abnormal pattern (Smith et al., 1991).

By 6–12h after ligation, the normal distribution was lost for Cx43, desmoplakin, and cadherin at the intercalated disks in the infarct zone. By 24–48 h after ligation, the expression of Cx43 markedly decreased, to 5% of the levels of shamoperated hearts (Figure 18) (Takamatsu, 2008). At 4 days post-infarction in a dog model, lateral gap junction label in the extended infarct border zone has been correlated spatially with electrophysiologically identified figure-of-eight re-entrant circuits (Peters et al., 1997).

Gap junctional changes distant from the infarct scar tissue, in particular reduction in the size and the number of gap junctions per unit length of intercalated disc, and fewer side-to-side connections between myocytes, have been described as longer term remodelling events in dog myocardium (Kostin et al., 2003). Our study showed that Cx43 mRNA and Cx43 protein reduced significantly at ischemic zone at 4, 8 and 12 weeks of myocardial infarction mice (Figure 19) (LI et al., 2010; Zhao et al., 2009).

Electrophysiological Abnormality:

may promote arrhythmogenesis.

infarction group (Zhao et al., 2009).

**infarction** 

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 153

'hibernating myocardium' refers to regions of ventricular myocardium that do not contract properly but which recover after normal blood flow is restored following coronary artery bypass surgery. In hibernating myocardium, the large Cx43 gap junctions typically found at the periphery of the intercalated disc are smaller in size, and the overall amount of immunodetectable Cx43 per intercalated disc is reduced, compared with normally perfused myocardial regions of the same heart (Kaprielian et al., 1998). These findings were the first indication that Cx43 gap junction remodelling contributes to impaired ventricular contraction,

Because Cx45 is not expressed widely in working ventricular myocytes in the normal adult heart, there is no report on the alternation of distribution and expression of Cx45 after myocardial infarction. However, previous study showed that expression of Cx45 in the failing human ventricle was up-regulated, alongside the reduction in Cx43, thus significantly altering the Cx43: Cx45 ratio (Yamada et al., 2003). Our datas showed that Cx45 mRNA and Cx45 protein increased significantly at ischemic zone at 4, 8 and 12 weeks in myocardial infarction mice (Figure 20) (Zhao et al., 2009). Up-regulation of Cx45 in myocardial infarction hearts inplicates a novel mechanism whereby connexin remodeling

Fig. 20. Ratio change of connexin 45/β-actin for myocardial infarction mice after 4, 8 and 12 weeks MSCs, bone marrow mesenchymal stem cells; aP < 0.01, vs. the normal area in the same group; bP < 0.01, vs. the ischemic zone in the same group; cP < 0.01, vs. myocardial

**4.3.3 Possibility of formation of heteromeric gap junction channels in myocardial** 

Expression of multiple connexin isoforms induces the formation of hetero-multimeric gap junction channels with distinct gating and permeability properties than their homomultimeric counterparts. Cx43 and Cx45 can form heterotypic channels that have unitary conductances lower than those of homotypic Cx43 channels and that fail to pass the fluorescent dye Lucifer yellow (Zhong et al., 2002; Moreno et al., 2002b; Moreno et al., 1995). When Cx43-expressing ROS cells are transfected to coexpress Cx45, electrical coupling is reduced even though Cx43 abundance, phosphorylation, and localization do not change (Koval et al., 1995). Coexpression of Cx45 with Cx43 in HeLa cells reduced the number of neighboring cells that were chemically coupled. Thus, increased Cx45 expression may

in addition to arrhythmia, in human ischemic heart disease (Kaprielian et al., 1998).

**4.3.2 Connexin 45 remodeling in myocardial infarction** 

Fig. 18. Remodeling process of cell–cell and cell–extracellular matrix (cell–ECM) interactions at the stump of myocardial infarction. (a) Normal; (b) 24–48 h. At early phase after ligation (to 48 h), borderline cardiomyocytes facing the infarct lose neighboring cells and form bluntended stumps while maintaining some desmoplakin, and cadherin loses Cx43. (c) 48 h–day 3. By day 3, integrins such as b1-integrin cluster at stumps where basement membranes are partially formed. The formation of intracellular junctions composed of desmoplakin and cadherin is initiated between cell processes. (d) Days 3–4; (e) days 8–15, After day 3, stumps change into fine cytoplasmic processes toward the infarct. Cx43 expression between cell processes forms intracellular junctional complexes with desmoplakin and cadherin, similar to those of typical intercalated disks. Cx43 expression is also observed at transverse cell boundaries of borderline and vicinity cardiomyocytes. (f) In the chronic phase (days 60–90), integrin–ECM couplings at the tips of cell processes and collagen accumulation around the processes increase as wound healing proceeds. Cell processes fuse together, and the intercalated disk-like structures diminish (Takamatsu, 2008).

Fig. 19. A, Cx43 expression at normal zones in myocardial infarction mice after 4 weeks; B, Cx43 expression at ischemic zones in myocardial infarction mice after 4 weeks; C, Cx43 expression at infarcted zones in myocardial infarction mice after 4 weeks (×400) (LI et al., 2010).

Apart from alterations in connexin43 level, rapid dephosphorylation of Cx43 and translocation of Cx43 from gap junctions into the cytosol has been reported when electrical uncoupling is induced by acute ischaemia in the Langendorff-perfused rat heart (Beardslee et al., 2000). These processes are reversible upon reperfusion and substantially reduced with ischemic preconditioning (LI et al., 2009a). Dephosphorylated Cx43 is associated with the laterally distributed gap junction label, while those gap junctions that remain in the classic, polar intercalated disc orientation contain phosphorylated Cx43 (Lampe et al., 2006).

A rather different form of gap junction remodelling is associated with 'hibernating myocardium' in patients with ischemic heart disease (Kaprielian et al., 1998). The term 'hibernating myocardium' refers to regions of ventricular myocardium that do not contract properly but which recover after normal blood flow is restored following coronary artery bypass surgery. In hibernating myocardium, the large Cx43 gap junctions typically found at the periphery of the intercalated disc are smaller in size, and the overall amount of immunodetectable Cx43 per intercalated disc is reduced, compared with normally perfused myocardial regions of the same heart (Kaprielian et al., 1998). These findings were the first indication that Cx43 gap junction remodelling contributes to impaired ventricular contraction, in addition to arrhythmia, in human ischemic heart disease (Kaprielian et al., 1998).

### **4.3.2 Connexin 45 remodeling in myocardial infarction**

152 Advances in Electrocardiograms – Clinical Applications

Fig. 18. Remodeling process of cell–cell and cell–extracellular matrix (cell–ECM) interactions at the stump of myocardial infarction. (a) Normal; (b) 24–48 h. At early phase after ligation (to 48 h), borderline cardiomyocytes facing the infarct lose neighboring cells and form bluntended stumps while maintaining some desmoplakin, and cadherin loses Cx43. (c) 48 h–day 3. By day 3, integrins such as b1-integrin cluster at stumps where basement membranes are partially formed. The formation of intracellular junctions composed of desmoplakin and cadherin is initiated between cell processes. (d) Days 3–4; (e) days 8–15, After day 3, stumps change into fine cytoplasmic processes toward the infarct. Cx43 expression between cell processes forms intracellular junctional complexes with desmoplakin and cadherin, similar to those of typical intercalated disks. Cx43 expression is also observed at transverse cell boundaries of borderline and vicinity cardiomyocytes. (f) In the chronic phase (days 60–90), integrin–ECM couplings at the tips of cell processes and collagen accumulation around the processes increase as wound healing proceeds. Cell processes fuse together, and the

Fig. 19. A, Cx43 expression at normal zones in myocardial infarction mice after 4 weeks; B, Cx43 expression at ischemic zones in myocardial infarction mice after 4 weeks; C, Cx43 expression at infarcted zones in myocardial infarction mice after 4 weeks (×400) (LI et al.,

Apart from alterations in connexin43 level, rapid dephosphorylation of Cx43 and translocation of Cx43 from gap junctions into the cytosol has been reported when electrical uncoupling is induced by acute ischaemia in the Langendorff-perfused rat heart (Beardslee et al., 2000). These processes are reversible upon reperfusion and substantially reduced with ischemic preconditioning (LI et al., 2009a). Dephosphorylated Cx43 is associated with the laterally distributed gap junction label, while those gap junctions that remain in the classic,

A rather different form of gap junction remodelling is associated with 'hibernating myocardium' in patients with ischemic heart disease (Kaprielian et al., 1998). The term

polar intercalated disc orientation contain phosphorylated Cx43 (Lampe et al., 2006).

intercalated disk-like structures diminish (Takamatsu, 2008).

2010).

Because Cx45 is not expressed widely in working ventricular myocytes in the normal adult heart, there is no report on the alternation of distribution and expression of Cx45 after myocardial infarction. However, previous study showed that expression of Cx45 in the failing human ventricle was up-regulated, alongside the reduction in Cx43, thus significantly altering the Cx43: Cx45 ratio (Yamada et al., 2003). Our datas showed that Cx45 mRNA and Cx45 protein increased significantly at ischemic zone at 4, 8 and 12 weeks in myocardial infarction mice (Figure 20) (Zhao et al., 2009). Up-regulation of Cx45 in myocardial infarction hearts inplicates a novel mechanism whereby connexin remodeling may promote arrhythmogenesis.

Fig. 20. Ratio change of connexin 45/β-actin for myocardial infarction mice after 4, 8 and 12 weeks MSCs, bone marrow mesenchymal stem cells; aP < 0.01, vs. the normal area in the same group; bP < 0.01, vs. the ischemic zone in the same group; cP < 0.01, vs. myocardial infarction group (Zhao et al., 2009).

### **4.3.3 Possibility of formation of heteromeric gap junction channels in myocardial infarction**

Expression of multiple connexin isoforms induces the formation of hetero-multimeric gap junction channels with distinct gating and permeability properties than their homomultimeric counterparts. Cx43 and Cx45 can form heterotypic channels that have unitary conductances lower than those of homotypic Cx43 channels and that fail to pass the fluorescent dye Lucifer yellow (Zhong et al., 2002; Moreno et al., 2002b; Moreno et al., 1995). When Cx43-expressing ROS cells are transfected to coexpress Cx45, electrical coupling is reduced even though Cx43 abundance, phosphorylation, and localization do not change (Koval et al., 1995). Coexpression of Cx45 with Cx43 in HeLa cells reduced the number of neighboring cells that were chemically coupled. Thus, increased Cx45 expression may

Electrophysiological Abnormality:

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 155

Fig. 22. Cultured mice atrial HL-1 cardiomyocytes were stained with antibodies against Cx43 (red) and mitochondria (Ox-Phos Com plex II, green) and analyzed by confocal laser scan microscopy. Merged image demonstrates colocalization as yellow po ints (arrows).

Fig. 23. Cx43 and translocase of the outer membrane 20 (Tom20) protein level in mitochondria isolated from left ventricular mycardium from young and aged mice. A: Western blot analysis was performed on mitochondrial protein extracts from young (< 3 mo) and aged (> 13mo) mice and on total right ventricular proteins (< 3 mo) for Na-K-ATPase and Cx43. Ponceau S staining demonstrates equal protein loading. B: Western blot analysis was performed for Tom20 on mitochondrial protein extracts from young and aged mice and on total right ventricular proteins. Ponceau S staining demonstrates equal protein loading. C: quantification of the Cx43 and Tom20 immunoreactivity in mitochondria of young (<3 mo, n = 14) and aged (> 13 mo, n = 10) mice normalized to Ponceau S staining. \*P<0.05 vs. <3 mo (Boengler et al., 2007). In Antonio et al (Rodriguez-Sinovas et al., 2006) study, treatment of isolated mitochondria with digitonin resulted in a marked reduction of voltage dependent anion channel immunoreactivity (VDA), whereas an adenine nucleotide transporter (ANT) specific signal was detected by confocal laser scan microscopy. An observations, by using western blotting

Additional ly, the phase contrast image was shown (Boengler et al., 2005).

reduce coupling and slow conduction or create microheterogeneities in coupling without slowing macroscopic conduction (Yamada et al., 2003).

Previos studies showed that heterotypic combination of Cx45 and Cx43 in mammalian cell lines impairs voltage gating of Cx43 and alters residual conductances, suggesting that connexon interactions may play a distinct role in modulation of intercellular communication (Elenes et al., 2001; Thomas et al., 2004). Cx43 and Cx45 formed heteromeric channels that exhibit reduced single channel conductance compared with Cx43 homomeric channels (Martinez et al., 2002). Our datas demonstrated colocalization of Cx45 and Cx43 in both control and ischemic ventricular myocardium (Figure 21).

Not only do Cx45 and Cx43 form hybrid channels in vitro, colocalization of these connexins in ventricular myocyte gap junctions provides and opportunity for hybrid channel formation in vivo, where interaction between Cx45 and Cx43 in hybrid channels could alter conductance states and gating responsiveness relative to the biophysical properties of homotypic channels (Yamada et al., 2003; Moreno et al., 2004). Our dates further suggested that enhanced expression of Cx45 in the ischemic heart occurs in junctions containing reduced levels of Cx43, raising the intriguing possibility that the relative stoichiometries of Cx45 and Cx43 within gap junctional plaques may be dramatically altered in the ischemic heart (Zhao et al., 2009). The limitation for our study is that the colocalization data are qualitative. In the absence of technological support of electrophysiologic confirmation of hybrid gap junction channels connecting ventricular myocytes in vivo, we can only speculate on the functional effects of increased Cx45 in the myocardial infarction heart.

Fig. 21. Colocalizations (white arrows) of Cx45 and Cx43 in ischemic ventricular myocardium after 12 weeks in myocardial infarction mice by confocal microscopy examination (Scale bar = 20μm).

### **4.3.4 Mitochondrial Cx43 in myocardial infarction**

Although it is generally assumed that Cx43 is exclusively localized at the sarcolemma, Cx43 has been found recently at cardiomyocyte mitochondria linner and outer membrane (Boengler et al., 2005; Rodriguez-Sinovas et al., 2006; Goubaeva et al., 2007). Boengler et al (Boengler et al., 2005) presented evidence at first for the presence of Cx43 in mitochondria from mouse, rat, pig and human left ventricular myocardium obtained from fluorescence-activated cell sorting and western blot analyses, as well as confocal and immunoelectron microscopy (Figure 22).

reduce coupling and slow conduction or create microheterogeneities in coupling without

Previos studies showed that heterotypic combination of Cx45 and Cx43 in mammalian cell lines impairs voltage gating of Cx43 and alters residual conductances, suggesting that connexon interactions may play a distinct role in modulation of intercellular communication (Elenes et al., 2001; Thomas et al., 2004). Cx43 and Cx45 formed heteromeric channels that exhibit reduced single channel conductance compared with Cx43 homomeric channels (Martinez et al., 2002). Our datas demonstrated colocalization of Cx45 and Cx43 in both

Not only do Cx45 and Cx43 form hybrid channels in vitro, colocalization of these connexins in ventricular myocyte gap junctions provides and opportunity for hybrid channel formation in vivo, where interaction between Cx45 and Cx43 in hybrid channels could alter conductance states and gating responsiveness relative to the biophysical properties of homotypic channels (Yamada et al., 2003; Moreno et al., 2004). Our dates further suggested that enhanced expression of Cx45 in the ischemic heart occurs in junctions containing reduced levels of Cx43, raising the intriguing possibility that the relative stoichiometries of Cx45 and Cx43 within gap junctional plaques may be dramatically altered in the ischemic heart (Zhao et al., 2009). The limitation for our study is that the colocalization data are qualitative. In the absence of technological support of electrophysiologic confirmation of hybrid gap junction channels connecting ventricular myocytes in vivo, we can only speculate on the functional effects of increased Cx45 in the myocardial infarction heart.

Fig. 21. Colocalizations (white arrows) of Cx45 and Cx43 in ischemic ventricular myocardium after 12 weeks in myocardial infarction mice by confocal microscopy

Although it is generally assumed that Cx43 is exclusively localized at the sarcolemma, Cx43 has been found recently at cardiomyocyte mitochondria linner and outer membrane (Boengler et al., 2005; Rodriguez-Sinovas et al., 2006; Goubaeva et al., 2007). Boengler et al (Boengler et al., 2005) presented evidence at first for the presence of Cx43 in mitochondria from mouse, rat, pig and human left ventricular myocardium obtained from fluorescence-activated cell sorting and western blot analyses, as well as confocal and immunoelectron microscopy (Figure 22).

examination (Scale bar = 20μm).

**4.3.4 Mitochondrial Cx43 in myocardial infarction** 

slowing macroscopic conduction (Yamada et al., 2003).

control and ischemic ventricular myocardium (Figure 21).

Fig. 22. Cultured mice atrial HL-1 cardiomyocytes were stained with antibodies against Cx43 (red) and mitochondria (Ox-Phos Com plex II, green) and analyzed by confocal laser scan microscopy. Merged image demonstrates colocalization as yellow po ints (arrows). Additional ly, the phase contrast image was shown (Boengler et al., 2005).

Fig. 23. Cx43 and translocase of the outer membrane 20 (Tom20) protein level in mitochondria isolated from left ventricular mycardium from young and aged mice. A: Western blot analysis was performed on mitochondrial protein extracts from young (< 3 mo) and aged (> 13mo) mice and on total right ventricular proteins (< 3 mo) for Na-K-ATPase and Cx43. Ponceau S staining demonstrates equal protein loading. B: Western blot analysis was performed for Tom20 on mitochondrial protein extracts from young and aged mice and on total right ventricular proteins. Ponceau S staining demonstrates equal protein loading. C: quantification of the Cx43 and Tom20 immunoreactivity in mitochondria of young (<3 mo, n = 14) and aged (> 13 mo, n = 10) mice normalized to Ponceau S staining. \*P<0.05 vs. <3 mo (Boengler et al., 2007).

In Antonio et al (Rodriguez-Sinovas et al., 2006) study, treatment of isolated mitochondria with digitonin resulted in a marked reduction of voltage dependent anion channel immunoreactivity (VDA), whereas an adenine nucleotide transporter (ANT) specific signal was detected by confocal laser scan microscopy. An observations, by using western blotting

Electrophysiological Abnormality:

markedly diminished (Yao et al., 2003).

pathologic pro-arrhythmic"hits" (Fishman, 2005).

**4.5.1 Pharmacological therapeutic implications** 

specific GJ blockers (Dhein, 1998, 2005).

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 157

(Fishman, 2005). In the healing canine infarct model, areas of GJ remodeling characterized by redistribution of Cx43 to lateral cell borders, correlates with the location of the central common pathway of figure-of-eight reentrant circuits (Peters et al., 1997). Additionally, junctional conductance of side-to-side coupled cells from the epicardial border zone is

What needs to be noted is that dysregulated connexin expression occurs as but one element of a widespread remodeling process - one that in volves many of the ion-handling proteins that regulate cardiac excitability (Fishman, 2005). Cardiac-specific Cx43 gene-targeted mutant mice showed significant slowing of impulse propagation and despite the relatively small mass of the murine ventricle, they all succumb to spontaneous or easily inducible sustained ventricular tachyarrhythmias (Fishman, 2005). Moreover, optical mapping studies suggest that most, if not all of these arrhythmias are due to reentry (Gutstein et al., 2001). On the other hand, studies of several additional genetic models in which Cx43 expression is modified are instructive, including the heteryzygous (Cx43+/-) germline knockout mice, chimeric (Cx43-/-/Cx43+/+) mice, as well as the recently described Cx43 O-CKO mice (Guerrero et al., 1997). Taken together, data from these murine models suggest that in the absence of additional pathologic stimuli, less widespread or more modest reductions in cellcell coupling are insufficient to support sustained ventricular arrhythmias (Fishman, 2005). For example, Cx43 germline heterozygous mice subjected to myocardial infarction reportedly have no increase in the frequency of spontaneous or inducible arrhythmias compared to wildtype mice with infarcts (Betsuyaku, 2004). Moreover, the imposition of ischemia in isolated-perfused hearts from Cx43+/- mice provokes only non-sustained ventricular tachycardia (Lerner et al., 2000). Cx43 chimeric mice, which have relatively large foci of uncoupled myocytes within the ventricle, display a substantial increase in spontaneous PVCs and short runs of VT (Gutstein et al., 2005). The O-CKO mice only develop inducible or spontaneous sustained VT when the anatomic extent of knockout is marked, encompassing more than 60% of the myocardium (Danik et al., 2004). Thus, the spatial extent and magnitude of uncoupling of Cx43 is likely to be insufficient to support the development of sustained ventricular tachyarrhythmias in the absence of additional

**4.5 Therapeutic Implications of gap junction remodeling after myocardial infarction** 

Pharmacology of GJ intercellular communication is still at its beginnings. Figure 26 shows a schematic view of a gap junction channel as a target of pharmacology (Salameh & Dhein, 2005). Potential mechanisms controlling the level of intercellular communication in the heart include regulation of connexin turnover (synthesis and degradation), cellular distribution and phosphorylation. There are numerous data showing that some compounds can upregulate Cx43 via modulation either synthesis or degradation and enhance gap junctional communication (Salameh & Dhein, 2005). Although many substances have effects on Gap junction-mediated intercellular communication (GJMIC), there is a lack of powerful and

Substances, such as AAP10 (Weng et al., 2002) or its derivative ZP123 (Xing et al., 2003), that increase intercellular coupling have presented themselves as valuable new tools to investigate the role of intercellular coupling in physiology and pathophysiology. It has been shown that uncoupling correlates with the development of some types of arrhythmias, and in several cases application of drugs that increase coupling has confirmed this role (Axelsen

and immunofluorescence with anti-VDAC as a marker for the outer mitochondrial membrane and with anti-ANT as a marker for the inner mitochondrial membrane, confirmed that Cx43 was not only present in the inner and outer mitochondrial membrane, but the mitochondrial Cx43 is phosphorylated (Goubaeva et al., 2007). Furthermore, with progressing age, GJ and mitochondrial levels of Cx43 in ventricular and atrial tissue homogenates are reduced. In mice hearts, sarcolemmal Cx43 content was reduced in aged (> 13 month) com pared with young (< 3 month). Also in mitochondria isolated from aged mice left ventricular myocardium, western blot analysis indicated a 40% decrease in Cx43 content compared with mitochondria isolated from young mice hearts. The reduced levels of Cx43 may contribute to the age-related loss of cardioprotection (Figure 23) (Boengler et al., 2007).

There is very little research on the relationship between mitochondria connexin and myocardial infarction until now, and Cx43 is the only connexin that has been found at cardiomyocyte mitochondria. Recentlly, our data showed that Heptanol (a GJ blocker) preconditioning protects myocardium from ischemia-reperfusion injury. The mechanism may be related to increasing in mitochondrial membrane potential and attenuating the decrease in mitochondria Cx43 expression induced by isehemia-reperfusion (Figure 24) (HE, 2009, 2010).

Fig. 24. Western blotting (30 μg total protein / lane) shows the increase of Cx43 protein in myoca rdial mitochondria in heptanol preconditioning on myocardial isehemia/reperfusion injury of rabbits. A: expression of Cx43 protein detected by Western blotting; B: results of the relative density of Cx43 protein; n = 3. P<0.05 vs IR; \*P< 0.05 vs sham (HE, 2009, 2010).

### **4.4 Electrophysiological consequences of gap junction remodeling**

Reductions in intercellular coupling lead to slowing of impulse propagation and amplification of the normal electrical heterogeneity within the ventricular myocardium. Individually, each of these electrophysiological effects of uncoupling are predicted to favor reentry, and together they may very well synergistically enhance arrhythmic risk (Kleber & Rudy, 2004).

Anatomic evidence of GJ remodeling, typically documented by immunohistochemical means, has been correlated with functional measures of aberrant cell-cell coupling such as slowing of impulse propagation and changes in anisotropy, as well as arrhythmogenicity

and immunofluorescence with anti-VDAC as a marker for the outer mitochondrial membrane and with anti-ANT as a marker for the inner mitochondrial membrane, confirmed that Cx43 was not only present in the inner and outer mitochondrial membrane, but the mitochondrial Cx43 is phosphorylated (Goubaeva et al., 2007). Furthermore, with progressing age, GJ and mitochondrial levels of Cx43 in ventricular and atrial tissue homogenates are reduced. In mice hearts, sarcolemmal Cx43 content was reduced in aged (> 13 month) com pared with young (< 3 month). Also in mitochondria isolated from aged mice left ventricular myocardium, western blot analysis indicated a 40% decrease in Cx43 content compared with mitochondria isolated from young mice hearts. The reduced levels of Cx43 may contribute to the age-related loss of cardioprotection (Figure 23) (Boengler et

There is very little research on the relationship between mitochondria connexin and myocardial infarction until now, and Cx43 is the only connexin that has been found at cardiomyocyte mitochondria. Recentlly, our data showed that Heptanol (a GJ blocker) preconditioning protects myocardium from ischemia-reperfusion injury. The mechanism may be related to increasing in mitochondrial membrane potential and attenuating the decrease in mitochondria Cx43 expression induced by isehemia-reperfusion (Figure 24) (HE,

Fig. 24. Western blotting (30 μg total protein / lane) shows the increase of Cx43 protein in myoca rdial mitochondria in heptanol preconditioning on myocardial isehemia/reperfusion injury of rabbits. A: expression of Cx43 protein detected by Western blotting; B: results of the relative density of Cx43 protein; n = 3. P<0.05 vs IR; \*P< 0.05 vs sham (HE, 2009, 2010).

Reductions in intercellular coupling lead to slowing of impulse propagation and amplification of the normal electrical heterogeneity within the ventricular myocardium. Individually, each of these electrophysiological effects of uncoupling are predicted to favor reentry, and together they may very well synergistically enhance arrhythmic risk (Kleber &

Anatomic evidence of GJ remodeling, typically documented by immunohistochemical means, has been correlated with functional measures of aberrant cell-cell coupling such as slowing of impulse propagation and changes in anisotropy, as well as arrhythmogenicity

**4.4 Electrophysiological consequences of gap junction remodeling** 

al., 2007).

2009, 2010).

Rudy, 2004).

(Fishman, 2005). In the healing canine infarct model, areas of GJ remodeling characterized by redistribution of Cx43 to lateral cell borders, correlates with the location of the central common pathway of figure-of-eight reentrant circuits (Peters et al., 1997). Additionally, junctional conductance of side-to-side coupled cells from the epicardial border zone is markedly diminished (Yao et al., 2003).

What needs to be noted is that dysregulated connexin expression occurs as but one element of a widespread remodeling process - one that in volves many of the ion-handling proteins that regulate cardiac excitability (Fishman, 2005). Cardiac-specific Cx43 gene-targeted mutant mice showed significant slowing of impulse propagation and despite the relatively small mass of the murine ventricle, they all succumb to spontaneous or easily inducible sustained ventricular tachyarrhythmias (Fishman, 2005). Moreover, optical mapping studies suggest that most, if not all of these arrhythmias are due to reentry (Gutstein et al., 2001).

On the other hand, studies of several additional genetic models in which Cx43 expression is modified are instructive, including the heteryzygous (Cx43+/-) germline knockout mice, chimeric (Cx43-/-/Cx43+/+) mice, as well as the recently described Cx43 O-CKO mice (Guerrero et al., 1997). Taken together, data from these murine models suggest that in the absence of additional pathologic stimuli, less widespread or more modest reductions in cellcell coupling are insufficient to support sustained ventricular arrhythmias (Fishman, 2005).

For example, Cx43 germline heterozygous mice subjected to myocardial infarction reportedly have no increase in the frequency of spontaneous or inducible arrhythmias compared to wildtype mice with infarcts (Betsuyaku, 2004). Moreover, the imposition of ischemia in isolated-perfused hearts from Cx43+/- mice provokes only non-sustained ventricular tachycardia (Lerner et al., 2000). Cx43 chimeric mice, which have relatively large foci of uncoupled myocytes within the ventricle, display a substantial increase in spontaneous PVCs and short runs of VT (Gutstein et al., 2005). The O-CKO mice only develop inducible or spontaneous sustained VT when the anatomic extent of knockout is marked, encompassing more than 60% of the myocardium (Danik et al., 2004). Thus, the spatial extent and magnitude of uncoupling of Cx43 is likely to be insufficient to support the development of sustained ventricular tachyarrhythmias in the absence of additional pathologic pro-arrhythmic"hits" (Fishman, 2005).

### **4.5 Therapeutic Implications of gap junction remodeling after myocardial infarction 4.5.1 Pharmacological therapeutic implications**

Pharmacology of GJ intercellular communication is still at its beginnings. Figure 26 shows a schematic view of a gap junction channel as a target of pharmacology (Salameh & Dhein, 2005). Potential mechanisms controlling the level of intercellular communication in the heart include regulation of connexin turnover (synthesis and degradation), cellular distribution and phosphorylation. There are numerous data showing that some compounds can upregulate Cx43 via modulation either synthesis or degradation and enhance gap junctional communication (Salameh & Dhein, 2005). Although many substances have effects on Gap junction-mediated intercellular communication (GJMIC), there is a lack of powerful and specific GJ blockers (Dhein, 1998, 2005).

Substances, such as AAP10 (Weng et al., 2002) or its derivative ZP123 (Xing et al., 2003), that increase intercellular coupling have presented themselves as valuable new tools to investigate the role of intercellular coupling in physiology and pathophysiology. It has been shown that uncoupling correlates with the development of some types of arrhythmias, and in several cases application of drugs that increase coupling has confirmed this role (Axelsen

Electrophysiological Abnormality:

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 159

2005), seem to provide only modest improvement of contractile heart function (Janssens et al., 2006) because neither BM cells (Murry et al., 2004) nor SMs (Reinecke et al., 2002) adopt a cardiac cell fate or couple electrically with the host myocardium (Leobon et al., 2003; Murry et al., 2004). Moreover, VT has been reported in several of the patients transplanted with SMs (Hagege et al., 2006), raising concerns as to whether engraftment of cells enhances the risk of VT, the most frequent cause of sudden death after myocardial infarction (Solomon et al., 2005; Henkel et al., 2006). So it is very important to fully answer these two questions before cell or stem cell therapy was proposed widely in clinic: whether cell or stem cell can differenciate into cardiomyocytes? Whether cell or stem cell can form functional coupling with the host, which is

A study (Virginijus et al., 2004) showed that human mesenchymal stem cells (hMSCs) can express connexins and couple to one another via Cx43 and Cx40. In addition, they formed functional gap junction channels with cells tranfected with Cx43, Cx40 or Cx45 as well as canine ventricular cardiomyocytes. Those are the foundermantal conditions for mesenchymal

Besides, another study (Wilhelm et al., 2007) showed that cardiomyocyte transplantation has the potential to impart electrical stability to the injured heart, thereby markedly reducing the major factor leading to sudden death. This protective effect was independent of the documented modest augmentation of left ventricular function and was associated with improved electrical coupling within the infarct by the engraftment of Cx43-expressing embryonic cardiomyocytes (eCMs). This enhanced coupling reduced vulnerability to VT by decreasing the incidence of conduction block within the infarct and/or by a modulatory effect on border-zone cardiomyocytes. This study also found that expression of the cardiac gap-junction protein Cx43 is the critical factor underlying augmented intercellular electrical conduction and protection from arrhythmia, because engraftment of Cx43-expressing SMs

Fig. 27. Identification of connexins in gap junctions of hMSCs Immunostaining of Cx43 (A), Cx40 (B) and Cx45 (C). D, immunoblot analysis of Cx43 in canine ventricular myocytes and hMSCs. Whole cell lysates (120μg) from ventricular cells or hMSCs were resolved by SDS, transferred to membrans, and blotted with Cx43 antibodies. Migration of molecular weight

markers is indicated to the right of the blot (Virginijus et al., 2004).

important for the physiological communication between cardiac myocytes?

stem cells therapy for heart disease including myocardial infarction (see Figure 27).

Fig. 26. Schematic view of a gap junction channel and its constituent component, the connexin, as a target of pharmacology (Salameh & Dhein, 2005).

 et al., 2007). Two recent studies showed (Haugan et al. 2005, Axelsen et al. 2007 (Haugan et al., 2005; Axelsen et al., 2007) that an increase of gap junctional conductance by specific peptide, rotigaptide, can prevent atrial conduction slowing or re-entrant ventricular tachycardia in ischemic heart. Suppression of ischemia-induced dephosphorylation of connexin seems to be one of the mechanisms involved. This mechanism was thought to be involved in the antiarrhythmic effects of ischemic preconditioning (Schulz et al., 2007). On the other hand, the reduction in gap junction communication that takes place during ischemia has been regarded as a beneficial and protective measure (Garcia-Dorado et al., 2004). Several investigators have examined the role of uncoupling GJ communication on myocardial infarct size. Thus, pharmacological uncoupling of GJ communication with nonselective uncouplers of GJ communication has been reported to reduce infarct size in animal experiments. Addition of the GJ blocker heptanol at concentration has a marked protective effect when applied at the time of reperfusion (Garcia-Dorado et al., 1997). Recent studies analyzing the effect of GJ uncouplers when administered during reoxygenation or reperfusion have consistently documented a reduction of necrosis (enzyme release or infarct size) (Saltman et al., 2002; Sebbag et al., 2003). Besides, electrical uncoupling by heptanol significantly lowers defibrillation threshold and dispersion of ventricular fibrillation cycle length without altering refractoriness (Qi et al., 2003). Although the beneficial and protective effort of GJ uncouplers delighted our researches, an important limitation is the lack of specific, rapid, reversible, and safe inhibitors of GJ intercellular communication.

Modulation of GJ intercellular coupling may open interesting new therapeutic approaches for a large number of diseases including myocardial infarction. However, in many respects, we have not completely understood the complex networking and its long-term regulation. This again underlines the need for in vivo and organbased models to evaluate the possibilities of pharmacological approaches towards an acute or chronic modulation of gap junctions. Moreover, the development of connexin- or organ specific gap junction modulator drugs is a challenging task for pharmacologists.

#### **4.5.2 Stem cell therapy**

Stem cell transplantation has emerged as a potential treatment strategy for heart failure secondary to acute or chronic ischaemic heart disease (Laflamme & Murry, 2005). But currently two autologous cell types, namely bone marrow (BM) cells and skeletal myoblasts (SMs), which were used in clinical trials in patients after myocardial infarction (Murry et al.,

Fig. 26. Schematic view of a gap junction channel and its constituent component, the

specific, rapid, reversible, and safe inhibitors of GJ intercellular communication.

drugs is a challenging task for pharmacologists.

**4.5.2 Stem cell therapy** 

Modulation of GJ intercellular coupling may open interesting new therapeutic approaches for a large number of diseases including myocardial infarction. However, in many respects, we have not completely understood the complex networking and its long-term regulation. This again underlines the need for in vivo and organbased models to evaluate the possibilities of pharmacological approaches towards an acute or chronic modulation of gap junctions. Moreover, the development of connexin- or organ specific gap junction modulator

Stem cell transplantation has emerged as a potential treatment strategy for heart failure secondary to acute or chronic ischaemic heart disease (Laflamme & Murry, 2005). But currently two autologous cell types, namely bone marrow (BM) cells and skeletal myoblasts (SMs), which were used in clinical trials in patients after myocardial infarction (Murry et al.,

 et al., 2007). Two recent studies showed (Haugan et al. 2005, Axelsen et al. 2007 (Haugan et al., 2005; Axelsen et al., 2007) that an increase of gap junctional conductance by specific peptide, rotigaptide, can prevent atrial conduction slowing or re-entrant ventricular tachycardia in ischemic heart. Suppression of ischemia-induced dephosphorylation of connexin seems to be one of the mechanisms involved. This mechanism was thought to be involved in the antiarrhythmic effects of ischemic preconditioning (Schulz et al., 2007). On the other hand, the reduction in gap junction communication that takes place during ischemia has been regarded as a beneficial and protective measure (Garcia-Dorado et al., 2004). Several investigators have examined the role of uncoupling GJ communication on myocardial infarct size. Thus, pharmacological uncoupling of GJ communication with nonselective uncouplers of GJ communication has been reported to reduce infarct size in animal experiments. Addition of the GJ blocker heptanol at concentration has a marked protective effect when applied at the time of reperfusion (Garcia-Dorado et al., 1997). Recent studies analyzing the effect of GJ uncouplers when administered during reoxygenation or reperfusion have consistently documented a reduction of necrosis (enzyme release or infarct size) (Saltman et al., 2002; Sebbag et al., 2003). Besides, electrical uncoupling by heptanol significantly lowers defibrillation threshold and dispersion of ventricular fibrillation cycle length without altering refractoriness (Qi et al., 2003). Although the beneficial and protective effort of GJ uncouplers delighted our researches, an important limitation is the lack of

connexin, as a target of pharmacology (Salameh & Dhein, 2005).

2005), seem to provide only modest improvement of contractile heart function (Janssens et al., 2006) because neither BM cells (Murry et al., 2004) nor SMs (Reinecke et al., 2002) adopt a cardiac cell fate or couple electrically with the host myocardium (Leobon et al., 2003; Murry et al., 2004). Moreover, VT has been reported in several of the patients transplanted with SMs (Hagege et al., 2006), raising concerns as to whether engraftment of cells enhances the risk of VT, the most frequent cause of sudden death after myocardial infarction (Solomon et al., 2005; Henkel et al., 2006). So it is very important to fully answer these two questions before cell or stem cell therapy was proposed widely in clinic: whether cell or stem cell can differenciate into cardiomyocytes? Whether cell or stem cell can form functional coupling with the host, which is important for the physiological communication between cardiac myocytes?

A study (Virginijus et al., 2004) showed that human mesenchymal stem cells (hMSCs) can express connexins and couple to one another via Cx43 and Cx40. In addition, they formed functional gap junction channels with cells tranfected with Cx43, Cx40 or Cx45 as well as canine ventricular cardiomyocytes. Those are the foundermantal conditions for mesenchymal stem cells therapy for heart disease including myocardial infarction (see Figure 27).

Besides, another study (Wilhelm et al., 2007) showed that cardiomyocyte transplantation has the potential to impart electrical stability to the injured heart, thereby markedly reducing the major factor leading to sudden death. This protective effect was independent of the documented modest augmentation of left ventricular function and was associated with improved electrical coupling within the infarct by the engraftment of Cx43-expressing embryonic cardiomyocytes (eCMs). This enhanced coupling reduced vulnerability to VT by decreasing the incidence of conduction block within the infarct and/or by a modulatory effect on border-zone cardiomyocytes. This study also found that expression of the cardiac gap-junction protein Cx43 is the critical factor underlying augmented intercellular electrical conduction and protection from arrhythmia, because engraftment of Cx43-expressing SMs

Fig. 27. Identification of connexins in gap junctions of hMSCs Immunostaining of Cx43 (A), Cx40 (B) and Cx45 (C). D, immunoblot analysis of Cx43 in canine ventricular myocytes and hMSCs. Whole cell lysates (120μg) from ventricular cells or hMSCs were resolved by SDS, transferred to membrans, and blotted with Cx43 antibodies. Migration of molecular weight markers is indicated to the right of the blot (Virginijus et al., 2004).

Electrophysiological Abnormality:

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 161

Fig. 29. A, Cx43 staining by FITC (green) at normal zones of ventricular myocardium. B, Cx43 staining by FITC at ischemic zones of ventricular myocardium. C, Cx43 staining by FITC at ischemic zones of ventricular myocardium after MSCs transplantation. D, Cx45 staining by TR (red) at normal zones of ventricular myocardium. E, Cx45 staining by TR at ischemic zones of ventricular myocardium. F, Cx45 staining by TR at ischemic zones of

In this chapter, we have presented mechanisms of sympathetic neural remodeling, electrical remodeling and gap Junction remodeling, which closely relate to the electrophysiological abnormality after myocardial infarction. Pathophysiological remodeling in myocardial infarction is multifactorial and complex, which involves changes in the structure and function of the myocardium. Although we anthropogenically divided the arrhythogenic mechamism of myocardial infarction into three parts, we have emphasized the importance of interrelationship among them which occurs at the cellular and molecular levels. Elucidating the role of myocardial remodeling and its molecular underpinnings present opportunities for the development of novel gene- and cell-based strategies as well as more pharmacotherapies. Besides, the localization of Cx43 at mitochondria opens a new door for us to reveal the cardioprotection against myocardial infarction. Stem cells, especially MSCs, give us a new hope on the potential treatment strategy for reducing life-threatening post-infarct arrhythmias

This work was supported by grants from the National Natural Science Foundation of China (No. 30560051); the Science Foundation for Returnees of Guangxi Zhuang Autonomous

and for heart failure secondary to acute or chronic ischaemic heart disease.

ventricular myocardium after MSCs transplantation.

**5. Conclusions and perspective** 

**6. Acknowledgment** 

Fig. 28. d, Ventricular burst stimulation does not induce VT. Upper trace, surface ECG; lower trace, His-level electrogram. e, EGFP-positive eCMs integrate into the infarct. f, EGFP positive eCMs are striated (α-actinin staining, red) and are in direct contact with EGFPnegative host cardiomyocytes. g, Cx43 staining (red) illustrates gap-junction formation between engrafted EGFP-positive eCMs (arrows) and with native cardiomyocytes (arrowheads). h, Summary of VT inducibility. Note strongly elevated susceptibility to VT induction in shaminjected group (Sh) compared with control group; eCM engraftment reduces VT inducibility compared with sham injection. Conversely, VT remains frequent after transplantation of SMs, BM and cardiac myofibroblasts (cFib). i, Improvement of left ventricular ejection fraction after eCM or SM transplantation. Numbers above bars indicate n; error bars show s.d. Scale bar, 60 mm (e), 5 mm (f), and 11mm (g).

protected against VT induction (see Figure 28). Thus, cell therapy including stem cell therapy in combination with Cx43 gene transfer represents a promising therapeutic strategy to decrease the risk of potentially fatal arrhythmias in injured heart.

In our recently study on mice, Laser Scanning Confocal Microscope examination found that DAPI-labeled MSCs distributed widely at the host ischemic zone and expressed Cx43. Fluorescence PCR, Immunohistochemistry, Immunofluorescence and immuno-electron microscope studies showed that Cx43 mRNA and Cx43 protein exprssions were statistically higher in MSCs group compared to MI group; Cx45 mRNA and Cx45 protein expressions were statistically lower in MSCs group compared to MI group (see Figure 29). We concluded that MSCs transplantation, which could survive at the host ischemic zone and express Cx43, can modulate the electrophysiological abnormality of myocardial infarction by up-regulating Cx43 and down-regulating Cx45 both at Protein and mRNA levels. In a word, MSCs have the plasticity of differentiating into cardiac muscle cell-like cells, which can modulate the electrophysiological abnormality via regulation of Cx43 and Cx45 remodeling following with myocardial infarction (Li et al., 2009b, 2010; Zhao et al., 2009, 2010).

Fig. 29. A, Cx43 staining by FITC (green) at normal zones of ventricular myocardium. B, Cx43 staining by FITC at ischemic zones of ventricular myocardium. C, Cx43 staining by FITC at ischemic zones of ventricular myocardium after MSCs transplantation. D, Cx45 staining by TR (red) at normal zones of ventricular myocardium. E, Cx45 staining by TR at ischemic zones of ventricular myocardium. F, Cx45 staining by TR at ischemic zones of ventricular myocardium after MSCs transplantation.

### **5. Conclusions and perspective**

160 Advances in Electrocardiograms – Clinical Applications

Fig. 28. d, Ventricular burst stimulation does not induce VT. Upper trace, surface ECG; lower trace, His-level electrogram. e, EGFP-positive eCMs integrate into the infarct. f, EGFP positive eCMs are striated (α-actinin staining, red) and are in direct contact with EGFPnegative host cardiomyocytes. g, Cx43 staining (red) illustrates gap-junction formation between engrafted EGFP-positive eCMs (arrows) and with native cardiomyocytes (arrowheads). h, Summary of VT inducibility. Note strongly elevated susceptibility to VT induction in shaminjected group (Sh) compared with control group; eCM engraftment reduces VT inducibility compared with sham injection. Conversely, VT remains frequent after transplantation of SMs, BM and cardiac myofibroblasts (cFib). i, Improvement of left ventricular ejection fraction after eCM or SM transplantation. Numbers above bars indicate

protected against VT induction (see Figure 28). Thus, cell therapy including stem cell therapy in combination with Cx43 gene transfer represents a promising therapeutic strategy

In our recently study on mice, Laser Scanning Confocal Microscope examination found that DAPI-labeled MSCs distributed widely at the host ischemic zone and expressed Cx43. Fluorescence PCR, Immunohistochemistry, Immunofluorescence and immuno-electron microscope studies showed that Cx43 mRNA and Cx43 protein exprssions were statistically higher in MSCs group compared to MI group; Cx45 mRNA and Cx45 protein expressions were statistically lower in MSCs group compared to MI group (see Figure 29). We concluded that MSCs transplantation, which could survive at the host ischemic zone and express Cx43, can modulate the electrophysiological abnormality of myocardial infarction by up-regulating Cx43 and down-regulating Cx45 both at Protein and mRNA levels. In a word, MSCs have the plasticity of differentiating into cardiac muscle cell-like cells, which can modulate the electrophysiological abnormality via regulation of Cx43 and Cx45 remodeling following with

n; error bars show s.d. Scale bar, 60 mm (e), 5 mm (f), and 11mm (g).

to decrease the risk of potentially fatal arrhythmias in injured heart.

myocardial infarction (Li et al., 2009b, 2010; Zhao et al., 2009, 2010).

In this chapter, we have presented mechanisms of sympathetic neural remodeling, electrical remodeling and gap Junction remodeling, which closely relate to the electrophysiological abnormality after myocardial infarction. Pathophysiological remodeling in myocardial infarction is multifactorial and complex, which involves changes in the structure and function of the myocardium. Although we anthropogenically divided the arrhythogenic mechamism of myocardial infarction into three parts, we have emphasized the importance of interrelationship among them which occurs at the cellular and molecular levels. Elucidating the role of myocardial remodeling and its molecular underpinnings present opportunities for the development of novel gene- and cell-based strategies as well as more pharmacotherapies. Besides, the localization of Cx43 at mitochondria opens a new door for us to reveal the cardioprotection against myocardial infarction. Stem cells, especially MSCs, give us a new hope on the potential treatment strategy for reducing life-threatening post-infarct arrhythmias and for heart failure secondary to acute or chronic ischaemic heart disease.

### **6. Acknowledgment**

This work was supported by grants from the National Natural Science Foundation of China (No. 30560051); the Science Foundation for Returnees of Guangxi Zhuang Autonomous

Electrophysiological Abnormality:

1969. ISSN 0009-7322

ISSN 0028-4793

ISSN 0008-6363

6147

Vol.24, No.1-2, pp. 82-90. ISSN 0192-253X

*Res,* Vol.95, No.10, pp. 1035-1041. ISSN 0009-7330

*Pfluqers Arch,* Vol.448, No.4, pp. 363–375. ISSN 0031-6768

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 163

Boyden PA, Dun W, Barbhaiya C & ter Keurs HE. (2004). 2APB- and JTV519(K201)-sensitive

Canty JM Jr, Suzuki G, Banas MD, Verheyen F, Borgers M & Fallavollita JA. (2004).

Cao JM, Chen LS, KenKnight BH, Ohara T, Lee MH, Tsai J, Lai WW, Karagueuzian HS, Wolf

Cao JM, Fishbein MC, Han JB, Lai WW, Lai AC, Wu TJ, Czer L, Wolf PL, Denton TA,

Cardiac Arrhythmia Suppression Trial (CAST) Investigators. (1989). Preliminary report:

Chow LT, Chow WH, Lee JC, Chow SS, Anderson RH & Gosling JA. (1998). Postmortem

ventricular myocardium. *J Anat,* Vol.192, No.1, pp. 73-80. ISSN 0021-8782 Cittadini A, Monti MG, Isgaard J, Casaburi C, Strömer H, Di Gianni A, Serpico R,

Coppen SR, Severs NJ & Gourdie RG. (1999). Connexin45 (a6) expression delineates an

Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armanini MP, Ling LH,

Desplantez T, Halliday D, Dupont E & Weingart R. (2004). Cardiac connexins Cx43 and

Dhein S. (1998). Gap junction channels in the cardiovascular system: pharmacological and

Dhein S, Polontchouk L, Salameh A & Haefliger J.-A. (2002). Pharmacological modulation

connexin 40. *Biol Cell,* Vol.94, No.7-8, pp. 409-22. ISSN 0248-4900

heart. *Heart Rhythm,* Vol.1, No.2, pp. 218–226. ISSN 1547-5271

*Circ Res,* Vol.86, No.7, pp. 816–821. ISSN 0009-7330

sudden death. *Circ Res,* Vol.94, No.8, pp. 1142-1149. ISSN 0009-7330

micro Ca2+ waves in arrhythmogenic Purkinje cells that survive in infarcted canine

Hibernating myocardium: chronically adapted to ischemia but vulnerable to

PL, Fishbein MC & Chen PS. (2000a). Nerve sprouting and sudden cardiac death.

Shintaku IP, Chen PS & Chen LS. (2000b). Relationship between regional cardiac hyperinnervation and ventricular arrhythmia. *Circulation,* Vol.101, No.16, pp. 1960–

effect of encainide and flecainide on mortality in a randomized trial of arrhythmia suppression after myocardial infarction. *N Engl J Med,* Vol.321, No.6, pp. 406–412.

changes in the immunohistochemical demonstration of nerves in human

Saldamarco L, Vanasia M & Saccà L. (2003). Aldosterone receptor blockade improves left ventricular remodeling and increases ventricular fibrillation threshold in experimental heart failure. *Cardiovasc Res,* Vol.58, No.3, pp. 555–564.

extended conduction system in the embryonic and mature rodent heart. *Dev Genet,* 

McMahon SB, Shelton DL & Levinson AD. (1994). Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. *Cell,* Vol.76, No.6, pp. 1001–1011. ISSN 0092-8674 Danik SB, Liu F, Zhang J, Suk HJ, Morley GE, Fishman GI & Gutstein DE. (2004).

Modulation of cardiac gap junction expression and arrhythmic susceptibility. *Circ* 

Cx45: formation of diverse gap junction channels with diverse electrical properties.

physiological modulation. *Trends Pharmacol Sci,* Vol.19, No.6, pp. 229-41. ISSN 0165-

and differential regulation of the cardiac gap junction proteins connexin 43 and

Region of China (No.0448014) and the Key Scientific Research Subject of Medical Treatment and Public Health of Guangxi Zhuang Autonomous Region of China (No.200301).

### **7. References**


Region of China (No.0448014) and the Key Scientific Research Subject of Medical Treatment

Aggarwal R & Boyden PA. (1995). Diminished Ca2+ and Ba2+ currents in myocytes surviving

Armour, JA. (2010). Functional anatomy of intrathoracic neurons innervating the atria and

Augustus O. Grant. (2009). Cardiac Ion Channels. *Circ Arrhythm Electrophysiol.* 2, 185-194.

Axelsen LN, Haugan K, Stahlhut M, Kjølbye AL, Hennan JK, Holstein-Rathlou NH, Petersen

Baba S, Dun W & Boyden PA. (2004). Can PKA activators rescue Na+ channel function in

Baba S, Dun W, Cabo C & Boyden PA. (2005). Remodeling in cells from different regions of

Beardslee MA, Lerner DL, Tadros PN, Laing JG, Beyer EC, Yamada KA, Kléber AG,

Betsuyaku T, Kanno S, Lerner DL, Schuessler RB, Saffitz JE & Yamada KA. (2004).

Boengler K, Dodoni G, Rodriguez-Sinovas A, Cabestrero A, Ruiz-Meana M, Gres P,

preconditioning. *Cardiovasc Res,* Vol.67, No.2, pp. 234-244. ISSN 0008-6363 Boengler K, Konietzka I, Buechert A, Heinen Y, Garcia-Dorado D, Heusch G & Schulz R.

Boyden PA & Pinto JM. (1994). Reduced calcium currents in subendocardial Purkinje

Boyden PA, Barbhaiya C, Lee T & ter Keurs HE. (2003). Nonuniform Ca2+ transients in

ischemia. *Circ Res,* Vol.87, No.8, pp. 656-62. ISSN 0009-7330

hearts. *Circ Res,* Vol.65, No.4, pp. 955–970. ISSN 0009-7330

No.6, pp. 2747–2759. ISSN 0009-7322

*Res,* Vol.57, No.3, pp. 681–693. ISSN 0008-6363

mice. *Cardiovasc Pathol,* Vol.13, No.3, pp. 156-164. ISSN 1054-8807

in the epicardial border zone of the 5-day infarcted canine heart. *Circ Res,* Vol.77,

JS & Nielsen MS. (2007). Increasing Gap Junctional Coupling: A Tool for Dissecting the Role of Gap Junctions. *J Membrane Biol,* Vol.216, No.1, pp. 23-35. ISSN 0022-2631

epicardial border zone cells that survive in the infarcted canine heart? *Cardiovasc* 

the reentrant circuit during ventricular tachycardia. *Circulation,* Vol.112, No.16, pp.

Schuessler RB & Saffitz JE. (2000). Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by

Spontaneous and inducible ventricular arrhythmias after myocardial infarction in

Konietzka I, Lopez-Iglesias C, Garcia-Dorado D, Di Lisa F, Heusch G & Schulz R. (2005). Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic

(2007). Loss of ischemic preconditionings cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. *Am J Physiol Heart Circ Physiol,* Vol.292, No.4, pp. H1764-H1769. ISSN 0363-6135 Boyden PA, Albala A & Dresdner KP Jr. (1989). Electrophysiology and ultrastructure of

canine subendocardial Purkinje cells isolated from control and 24-hour infarcted

myocytes that survive in the 24- and 48-hour infarcted heart. *Circulation,* Vol.89,

arrhythmogenic Purkinje cells that survive in the infarcted canine heart. *Cardiovasc* 

and Public Health of Guangxi Zhuang Autonomous Region of China (No.200301).

ventricles. *Heart Rhythm,* Vol.7, No.7, pp. 994-996. ISSN 1547-5271

No.6, pp. 1180–1191. ISSN 0009-7330

*Res,* Vol.64, No.2, pp. 260–267. ISSN 0008-6363

**7. References** 

ISSN 1941-3149

2386–2396. ISSN 0009-7322


Electrophysiological Abnormality:

97-103. ISSN 0006-291X

No.3, pp. 333-339. ISSN 0009-7330

0363-6135

ISSN 0009-7322

545. ISSN 1045-3873

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 165

Gardner PI, Ursell PC, Fenoglio JJ Jr & Wit AL. (1985). Electrophysiologic and anatomic

Gidh-Jain M, Huang B, Jain P & el-Sherif N. (1996). Differential expression of voltage-gated

myocardial infarction. *Circ Res,* Vol.79, No.4, pp. 669–675. ISSN 0009-7330 Goubaeva F, Mikami M, Giardina S, Ding B, Abe J & Yang J. (2007). Cardiac mitochondrial

Gourdie RG, Green CR & Severs NJ. (1991). Gap junction distribution in adult mammalian

Guerrero PA, Schuessler RB, Davis LM, Beyer EC, Johnson CM, Yamada KA & Saffitz JE.

Gulbenkian S, Saetrum Opgaard O, Ekman R, Costa Andrade N, Wharton J, Polak JM,

Gutstein DE, Danik SB, Lewitton S, France D, Liu F, Chen FL, Zhang J, Ghodsi N, Morley

Gwechenberger M, Mendoza LH, Youker KA, Frangogiannis NG, Smith CW, Michael LH &

Hagège AA, Marolleau JP, Vilquin JT, Alhéritière A, Peyrard S, Duboc D, Abergel E, Messas

Harris AL. (2001). Emerging issues of connexin channels: biophysics fills the gap. *Q Rev* 

Haugan K, Olsen KB, Hartvig L, Petersen JS, Holstein-Rathlou NH, Hennan JK & NlelsenI

HE Yan, ZENG Zhi-yu, ZHONG Guo-qiang, LI Jin-yi, LI Wei-ke & LI Wei. (2009). Cx43 in

heart failure. *Eur Heart J,* Vol.14, No.4, pp. 551–566. ISSN 0195-668X

*Biophys,* Vol.34, No.3, pp. 325-472. ISSN 0033-5835

microscopy. *J Cell Sci,* Vol.99, No.1, pp. 41–55. ISSN 0021-9533

mutation. *J Clin Invest,* Vol.99, No.8, pp. 1991-1998. ISSN 0021-9738

*Circulation,* Vol.72, No.3, pp. 596–611. ISSN 0009-7322

basis for fractionated electrograms recorded from healed myocardial infarcts.

K+ channel genes in left ventricular remodeled myocardium after experimental

connexin 43 regulates apoptosis. *Biochem Biophys Res Commun,* Vol.352, No.1, pp.

myocardium revealed by an antipeptide antibody and laser scanning confocal

(1997). Slow ventricular conduction in mice heterozygous for a connexin43 null

Queiroz e Melo J & Edvinsson L. (1993). Peptidergic innervation of human epicardial coronary arteries. *Circ Res,* Vol.73, No.3, pp. 579–588. ISSN 0009-7330 Gutstein DE, Morley GE, Tamaddon H, Vaidya D, Schneider MD, Chen J, Chien KR,

Stuhlmann H & Fishman GI. (2001). Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. *Circ Res,* Vol.88,

GE & Fishman GI. (2005). Focal gap junction uncoupling and spontaneous ventricular ectopy. *Am J Physiol Heart Circ Physiol,* Vol.289, No.3, pp. H1091-8. ISSN

Entman ML. (1999). Cardiac myocytes produce interleukin-6 in culture and in viable border zone of reperfused infarctions. *Circulation,* Vol.99, No.4, pp. 546-551.

E, Mousseaux E, Schwartz K, Desnos M & Menasché P (2006). Skeletal myoblast transplantation in ischemic heart failure: long-term follow-up of the first phase I cohort of patients. *Circulation,* Vol.114, No. (1 Suppl), pp. 1108–13. ISSN 0009-7322 Hagemeijer F. (1993). Calcium sensitization with pimobendan: pharmacology,

haemodynamic improvement, sudden death in patients with chronic congestive

MS. (2005). The antiarrhythmic peptide analog ZP123 prevents atrial conduction slowing during metabolic stress. *J Cardiovasc Electrophysiol,* Vol.16, No.5, pp. 537-

mitochondria participates in the protection for heptanol preconditioning on


Dillon SM, Allessie MA, Ursell PC & Wit AL. (1988). Influences of anisotropic tissue

Dun W, Baba S, Yagi T & Boyden PA. (2004). Dynamic remodeling of K+ and Ca2+ currents

Dupont E, Ko Y, Rothery S, Coppen SR, Baghai M, Haw M & Severs NJ (2001). The gap-

Esler M, Jennings G, Lambert G, Meredith I, Horne M & Eisenhofer G (1990). Overflow of

Fishman GI. (2005). Gap junction remodeling and ventricular arrhythmias. *Heart Rhythm,* 

Friedman PL, Fenoglio JJ & Wit AL. (1975). Time course for reversal of electrophysiological

Frohnwieser B, Chen, L-Q, Schreibmayer W & Kallen RG. (1997). Modulation of the human

Fukuda K, Davies SS, Nakajima T, Ong BH, Kupershmidt S, Fessel J, Amarnath V, Anderson

Gao L, Wang W, Li YL, Schultz HD, Liu D, Cornish KG & Zucker IH. (2005). Simvastatin

Garcia-Dorado D, Inserte J, Ruiz-Meana M, González MA, Solares J, Juliá M, Barrabés JA &

Garcia-Dorado D, Rogriguez-Sinovas A & Ruiz-Meana M. (2004). Gap junction-mediated

channels. *Circ Res,* Vol.97, No.12, pp. 1262–1269. ISSN 0009-7330

*Circulation,* Vol.96, No.10, pp. 3579-3586. ISSN 0009-7322

*Res,* Vol.61, No.3 pp. 386-410. ISSN 0008-6363

atrial fibrillation. *Circulation,* Vol.103, No.6, pp. 842-9. ISSN 0009-7322 Elenes S, Martinez AD, Delmar M, Beyer EC & Moreno AP (2001). Heterotypic docking of

infarcts. *Circ Res,* Vol.63, No.1, pp. 182–206. ISSN 0009-7330

pp. H667–H673. ISSN 0363-6135

No.3, pp. 1406-1418. ISSN 0006-3495

Vol.2, No.8, pp. 887-9. ISSN 1547-5271

No.4, pp. 2118-2120. ISSN 0892-6638

No.12, pp. 1763–1770. ISSN 0009-7322

0009-7330

*Physiol Rev,* Vol.70, No.4, pp. 963–985. ISSN 0031-9333

structure on reentrant circuits in the epicardial border zone of subacute canine

in cells that survived in the epicardial border zone of canine healed infarcted heart. *Am J Physiol Heart Circ Physiol,* Vol.287, No.3, pp. H1046–H1054. ISSN 0363-6135 Dun W & Boyden PA. (2005). Diverse phenotypes of outward currents in cells that have

survived in the 5-day-infarcted heart. *Am J Physiol Heart Circ Physiol,* Vol.289, No.2,

junctional protein, connexin40, is elevated in patients susceptible to post-operative

Cx43 and Cx45 connexons blocks fast voltage gating of Cx43. *Biophys J,* Vol.81,

catecholamine neurotransmitters to the circulation: source, fate, and functions.

and ultrastructural abnormalities in subendocardial Purkinje fibers surviving extensive myocardial infarction in dogs. *Circ Res,* Vol.36, No.1, pp. 127–144. ISSN

cardiac sodium channel a-subunit by cAMP-dependent protein kinase and the responsible sequence domain*. J Physiol,* Vol.498, No.2, pp. 309-318. ISSN 0022-3751 Fuchs M, Hilfiker A, Kaminski K, Hilfiker-Kleiner D, Guener Z, Klein G, Podewski E,

Schieffer B, Rose-John S & Drexler H. (2003). Role of interleukin-6 for LV remodeling and survival after experimental myocardial infarction. *FASEB J,* Vol.17,

ME, Boyden PA, Viswanathan PC, Roberts LJ 2nd & Balser JR. (2005). Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium

therapy normalizes sympathetic neural control in experimental heart failure: roles of angiotensin II type 1 receptors and NAD(P)H oxidase. *Circulation,* Vol.112,

Soler-Soler J. (1997). Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion.

spread of cell injury and death during myocardial ischemia-reperfusion. *Cardiovas* 


Electrophysiological Abnormality:

0363-6135

660. ISSN 0009-7322

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 167

Kaprielian RR, Gunning M, Dupont E, Sheppard MN, Rothery SM, Underwood R, Pennell

Kim YK, Kim SJ, Kramer CM, Yatani A, Takagi G, Mankad S, Szigeti GP, Singh D, Bishop

human heart. *Mol Cell Biochem,* Vol.242, No.1, pp. 135–144. ISSN 0300-8177 Koval M, Geist ST, Westphale EM, Kemendy EM, Civitelli R, Beyer EC & Steinberg TH

Laflamme, M. A. & Murry, C. E. (2005). Regenerating the heart. *Nature Biotechnol,* Vol.23,

Laing JG, Beyer EC. (2000). Degradation of gap junctions and connexins. In: *Gap junctions.* 

Lampe PD, Cooper CD, King TJ & Burt JM. (2006). Analysis of Connexin43 phosphorylated

Leblanc N & Hume JR. (1990). Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum. *Science,* Vol.248, No.4953, pp. 372-376. ISSN 0036-8075 Leobon B, Garcin I, Menasche P, Vilquin JT, Audinat E & Charpak S. (2003). Myoblasts

host. *Proc. Natl Acad. Sci,* Vol.100, No.13, pp. 7808–7811. ISSN 0027-8424 Lerner DL, Yamada KA, Schuessler RB & Saffitz JE. (2000). Accelerated onset and increased

LI Jinyi, ZHONG Guoqiang, HE Yan & LING Yun. (2009a). Cardiac Connexin 43 and

Li Jin-yi, Zhong Guo-qiang, He Yan, Wen Li-na, Ke Hong-hong, Wei Zhuo, Deng Yan & Wu

infarction. *J Mol Cell Cardiol,* Vol.34, No.1, pp. 63–73. ISSN 0022-2828 Kleber AG & Rudy Y. (2004). Basic mechanisms of cardiac impulse propagation and associated arrhythmias. *Physiol Rev,* Vol.84, No.2, pp. 431-488. ISSN 0031-9333 Kostin S, Rieger M, Dammer S, Hein S, Richter M, Klövekorn WP, Bauer EP & Schaper J.

Vol.118, No.2, pp. 243–255. ISSN 0092-8674

Academic Press, ISBN 0-12-153349-2, San Diego

*Circulation,* Vol.101, No.5, pp. 547-552. ISSN 0009-7322

No.7, pp. 845–856. ISSN 1087-0156

3435–3442. ISSN 0021-9533

No.2, pp. 97-105. ISSN 1007-9688

transients. *Am J Physiol Heart Circ Physiol,* Vol.283, No.3, pp. H1157–H1168. ISSN

DJ, Fox K, Pepper J, Poole-Wilson PA & Severs NJ. (1998). Down-regulation of immunodetectable connexin43 and decreased gap junction size in the pathogenesis of chronic hibernation in the human left ventricle. *Circulation,* Vol.97, No.7, pp. 651–

SP, Shannon RP, Vatner DE & Vatner SF. (2002). Altered excitation-contraction coupling in myocytes from remodeled myocardium after chronic myocardial

(2003). Gap junction remodeling and altered connexin43 expression in the failing

(1995). Transfected connexin 45 alters gap junction permeability in cells expressing endogenous connexin 43. *J Cell Biol,* Vol.130, No.4, pp. 987-995. ISSN 0021-9525 Kuruvilla R, Zweifel LS, Glebova NO, Lonze BE, Valdez G, Ye H & Ginty DD. (2004). A

neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling. *Cell,* 

*Molecular basis of cell communication in health and disease.* Peracchia C, pp. 23-41,

at S325, S328 and S330 in normoxic and ischemic heart. *J Cell Sci,* Vol.119, No16, pp.

transplanted into rat infarcted myocardium are functionally isolated from their

incidence of ventricular arrhythmias induced by ischemia in Cx43-deficient mice.

Ischemic Cardioprotection. *South China Journal of Cardiovascular Diseases,* Vol.10,

Zhi-fu. (2009b). Effects of allogenic bone marrow mesenchymal stem cell transplantation on electrophysiological abnormality and left ventricular

myocardial ischemia/reperfusion injury of rabbits. *Chinese Pharmacological Bulletin,* Vol.25, No.12, pp. 1660-1665. ISSN 1001-1978


HE Yan, ZHONG Guo-qiang, ZENG Zhi-yu, LI Wei-ke, LI Wei & LI Jin-yi. (2010). Effects of

Henkel DM, Witt BJ, Gersh BJ, Jacobsen SJ, Weston SA, Meverden RA & Roger VL. (2006).

Herbert E & Chahine M. (2006). Clinical aspects and physiopathology of Brugada syndrome:

Hille B. (1978). Ionic channels in excitable membranes. Current problems and biophysical

Huang B, El-Sherif T, Gidh-Jain M, Qin D & El-Sherif N. (2001a). Alterations of sodium

Ieda M, Kanazawa H, Kimura K, Hattori F, Ieda Y, Taniguchi M, Lee JK, Matsumura K,

Issa ZF, Ujhelyi MR, Hildebrand KR, Zhou X, Rosenberger J, Groh WJ, Miller JM & Zipes

Janse MJ & Wit AL. (1989). Electrophysiological mechanisms of ventricular arrhythmias

Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, Kalantzi M, Herbots

Jiang M, Cabo C, Yao J, Boyden PA & Tseng G. (2000). Delayed rectifier K+ currents have

Jin H, Lyon AR & Akar FG. (2008). Arrhythmia mechanisms in the failing heart. *Pacing Clin* 

Kaplan DR & Miller FD. (2003). Axon growth inhibition: signals from the p75 neurotrophin

Kaprielian R, Sah R, Nguyen T, Wickenden AD & Backx PH. (2002). Myocardial infarction in

Vol.25, No.12, pp. 1660-1665. ISSN 1001-1978

Vol.52, No.2, pp. 246–254. ISSN 0008-6363

1049–1069. ISSN 0031-9333

ISSN 0008-4212

*Pathophysiology,* Vol.26, No.3, pp. 461-465. ISSN 1000-4718

study. *Am. Heart J,* Vol.151, No.4, pp. 806–812. ISSN 0002-8703

approaches. *Biophys J.* Vol.22, No.2, pp. 283-94. ISSN 0006-3495

patterning. *Nat Med,* Vol.13, No.5, pp. 604–612. ISSN 1078-8956

*Rhythm,* Vol.2, No.10, pp. 1122–1127. ISSN 1547-5271

trial. *Lancet,* Vol.367, No.9505, pp. 113–121. ISSN 0140-6736

*Electrophysiol,* Vol.31, No.8, pp. 1048-56. ISSN 0147-8389

ventricle. *Cardiovasc Res,* Vol.48, No.1, pp. 34–43. ISSN 0008-6363

receptor. *Nat Neurosci,* Vol.6, No.5 pp. 435–436. ISSN 1097-6256

*J Cardiovasc Electrophysiol,* Vol.12, No.2, pp. 218–225. ISSN 1045-3873 Huang B, Qin D & El-Sherif N. (2001b). Spatial alterations of Kv channels expression and

myocardial ischemia/reperfusion injury of rabbits. *Chinese Pharmacological Bulletin,*

heptanol preconditioning on structure,function and Cx43 content of mitochondria in rabbit model of myocardial ischemia/reperfusion injury. *Chinese Journal of* 

Ventricular arrhythmias after acute myocardial infarction: a 20-year community

review of current concepts. *Can J Physiol Pharmacol,* Vol.84, No.8-9, pp. 795-802.

channel kinetics and gene expression in the postinfarction remodeled myocardium.

K(+) currents in post-myocardial infarction remodeled rat heart. *Cardiovasc Res,* 

Tomita Y, Miyoshi S, Shimoda K, Makino S, Sano M, Kodama I, Ogawa S & Fukuda K. (2007). Sema3a maintains normal heart rhythm through sympathetic innervation

DP. (2005). Intrathecal clonidine reduces the incidence of ischemiaprovoked ventricular arrhythmias in a canine postinfarction heart failure model. *Heart* 

resulting from myocardial ischemia and infarction. *Physiol Rev,* Vol.69, No.4, pp.

L, Sinnaeve P, Dens J, Maertens J, Rademakers F, Dymarkowski S, Gheysens O, Van Cleemput J, Bormans G, Nuyts J, Belmans A, Mortelmans L, Boogaerts M & Van de Werf F. (2006). Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled

reduced amplitudes and altered kinetics in myocytes from infarcted canine

rat eliminates regional heterogeneity of AP profiles, Ito K+ currents, [Ca2+]i

transients. *Am J Physiol Heart Circ Physiol,* Vol.283, No.3, pp. H1157–H1168. ISSN 0363-6135


Electrophysiological Abnormality:

ISSN 0033-0620

Norwell.

0836

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 169

Maltsev VA, Sabbab HN & Undrovinas AI. (2002). Down-regulation of sodium current in

Marmar Vaseghi & Kalyanam Shivkumar. (2008). The Role of the Autonomic Nervous

Martinez AD, Hayrapetyan V, Moreno AP & Beyer EC: (2002). Connexin 43 and connexin 45

Michael G, Xiao L, Qi XY, Dobrev D & Nattel S. (2009). Remodelling of cardiac

Moreno AP, Chanson M, Elenes S, Anumonwo J, Scerri I, Gu H, Taffet SM & Delmar M.

Moreno AP, Zhong G, Hayrapetyan V. (2002b). Heteromultimeric gap junction channels: a

Moreno AP, Hayrapetyan V, Zhong G. (2004). Homomeric and Heteromeric Gap Junctions.

Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO, Rubart M, Pasumarthi

Murry CE, Field LJ & Menasche P. (2005). Cell-based cardiac repair: reflections at the 10 year point. *Circulation,* Vol.112, No.20, pp. 3174–3183. ISSN 0009-7322 Nademanee K, Taylor R, Bailey WE, Rieders DE & Kosar EM. (2000). Treating electrical

Nattel S & Carlsson L. (2006). Innovative approaches to anti-arrhythmic drug therapy. *Nat* 

Nattel S, Maguy A, Le Bouter S, Yeh YH. (2007). Arrhythmogenic ion-channel remodeling in

Nerbonne JM & Kass RS. (2005). Molecular physiology of cardiac repolarization. *Physiol Rev,* 

Nian M, Lee P, Khaper N & Liu P. (2004). Inflammatory cytokines and postmyocardial infarction remodeling. *Circ Res,* Vol.94, No.12, pp. 1543–1553. ISSN 0009-7330 Noda M, Shimizu S, Tanabe T, Takai T, Kayano T, Ikeda T, Takahashi H, Nakayama H,

120-126, W.B Saunders Company, ISBN 0-7216-0323-8, Philadelphia

Vol.59, No.9, pp. 1561–1568. ISSN 1420-682X

*Cardiovasc Res*, Vol.81, No.3, pp. 491-9. ISSN 0008-6363

gating. *Circ Res,* Vol.90, No.4, pp. 450-7. ISSN 0009-7330

*Circulation,* Vol.102, No.7, pp. 742–747. ISSN 0009-7322

Vol.87, No.2, pp. 425-56. ISSN 0031-9333

Vol.85, No.4, pp. 1205–1253. ISSN 0031-9333

*Rev Drug Discov,* Vol.5, No.12, pp. 1034-1049. ISSN 1474-1776

chronic heart failure: effect of long-term therapy with carvedilol. *Cell Mol Life Sci,* 

System in Sudden Cardiac Death. *Prog Cardiovasc Dis,* Vol.50, No.6, pp. 404–419.

form heteromeric gap junction channels in which individual components determine permeability and regulation. *Circ Res,* Vol.90, No.10, pp. 1100-1107. ISSN 0009-7330

repolarization: how homeostatic responses can lead to arrhythmogenesis.

(2002a). Role of the carboxyl terminal of connexin43 in transjunctional fast voltage

connection with cardiac physiology and pathology. In: *Heart Cell Coupling and Impulse Propagation in Health and Disease-Basic Science for the Cardiologist,* WC. De Mello, M. Janse, pp. 89-108. Kluwer Academic Publishers. ISBN 1-4020-7182-5,

In: *Cardiac electrophysiololgy from cell to bedside.* Douglas P.Zipes and Jose Jalife. pp.

KB, Virag JI, Bartelmez SH, Poppa V, Bradford G, Dowell JD, Williams DA & Field LJ. (2004). Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. *Nature,* Vol.428, No.6983, pp. 664–668. ISSN 0028-

storm: sympathetic blockade versus advanced cardiac life support-guided therapy.

the heart: heart failure, myocardial infarction, and atrial fibrillation. *Physiol Rev.*

Kanaoke Y, Minamino N, Kangawa R, Matsuo H, Raftery MA, Hirose T, Inayama S, Hayashida H, Miyata T & Numa S. (1984). Primary structure of electrophorus

remodeling in rats with myocardial infarction. *Journal of Clinical Rehabilitative Tissue Engineering Research,* Vol.13, No.27, pp. 5211-5216. ISSN 1671-5926


LI Jin-yi, ZHONG Guo-qiang, WEI Zhuo, XU Wei-yan, KE Hong-hong, NING Zong & WU

*laboratorium animalis scientia sinica,* Vol.17, No.6, pp. 419-423. ISSN 1005-4847 LI Jinyi, ZHONG Guoqiang, KE Honghong, HE Yan, WEN Lina, WEI Zhuo & ZHAO

Li W, Knowlton D, Van Winkle DM & Habecker BA. (2004). Infarction alters both the

*Physiol Heart Circ Physiol,* Vol.286, No.6, pp. H2229-H2236. ISSN 0363-6135 Li X, Huang CX, Jiang H, Cao F & Wang T. (2005). The beta-adrenergic blocker carvedilol

Li Y, Xue Q, Ma J, Zhang CT, Qiu P, Wang L, Gao W, Cheng R, Lu ZY & Wang SW. (2004).

Litwin SE, Zhang D & Bridge JH. (2000). Dyssynchronous Ca(2+) sparks in myocytes from infarcted hearts. *Circ Res,* Vol.87, No.11, pp. 1040–1047. ISSN 0009-7330 Liu N, Niu H, Li Y, Zhang C, Zhou Q, Ruan Y, Pu J & Lu Z. (2004). The changes of

Lorentz CU, Alston EN, Belcik T, Lindner JR, Giraud GD & Habecker BA. (2010).

Lue WM & Boyden PA. (1992). Abnormal electrical properties of myocytes from chronically

Luisi AJ Jr, Fallavollita JA, Suzuki G & Canty JM Jr. (2002). Spatial inhomogeneity of

Mahajan A, Moore J, Cesario DA & Shivkumar K. (2005). Use of thoracic epidural anesthesia

*Circulation,* Vol.85, No.3, pp. 1175–1188. ISSN 0009-7322

*Engineering Research,* Vol.13, No.27, pp. 5211-5216. ISSN 1671-5926

Vol.30, No.4, pp. 337-342. ISSN 1001-6325

1458–1463. ISSN 1671-4083

1633. ISSN 0735-1097

pp. 779–781. ISSN 0009-7322

1359–1362. ISSN 1547-5271

1606

*Med J,* Vol.118, No.5, pp. 377–382. ISSN 0366-6999

remodeling in rats with myocardial infarction. *Journal of Clinical Rehabilitative Tissue* 

ZHI-fu. (2009c). Establishment of a rat model of myocardial infarction and the postinfarction changes in electrophysiology and lef ventricular function. *Acta* 

Yanmei. (2010). Transplantation of allogenic mesenchymal stem cells up-regulates connexin 43 expression in rats with myocardial infarction. *Basic & Clinical Medicine,*

distribution and noradrenergic properties of cardiac sympathetic neurons. *Am J* 

restores L-type calcium current in a myocardial infarction model of rabbit. *Chin* 

Effects of imidapril on heterogeneity of action potential and calcium current of ventricular myocytes in infarcted rabbits. *Acta Pharmacol Sin,* Vol.25, No.11, pp.

potassium currents in rabbit ventricle with healed myocardial infarction. *J Huazhong Univ Sci Technolog Med Sci,* Vol.24, No.2, pp. 128–131. ISSN 1672-0733 Long JB, Jay SM, Segal SS & Madri JA. (2009). VEGF-A and semaphorin3A: modulators of

vascular sympathetic innervation. *Dev Biol,* Vol.334, No.1, pp. 119–132. ISSN 0012-

Heterogeneous ventricular sympathetic innervation, altered b-adrenergic receptor expression, and rhythm instability in mice lacking the p75 neurotrophin receptor. *Am J Physiol Heart Circ Physiol,* Vol.298, No.6, pp. H1652-H1660. ISSN 0363-6135 Lubbe WF, Podzuweit T & Opie LH. (1992). Potential arrhythmogenic role of cyclic

adenosine monophosphate (AMP) and cytosolic calcium overload: implications for prophylactic effects of beta-blockers in myocardial infarction and proarrhythmic effects of phosphodiesterase inhibitors. *J Am Coll Cardiol,* Vol.19, No.7, pp. 1622–

infarcted canine heart. Alterations in Vmax and the transient outward current.

sympathetic nerve function in hibernating myocardium. *Circulation,* Vol.106, No.7,

for management of electrical storm: a case report. *Heart Rhythm,* Vol.2, No.12, pp.


Electrophysiological Abnormality:

1311. ISSN 0009-7322

*Res*,

7330

1078-8956

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 171

Qi X, Varma P, Newman D & Dorian P. (2001). Gap junction blockers decrease defibrillation

Qin D, Zhang ZH, Caref EB, Boutjdir M, Jain P & el-Sherif N. (1996). Cellular and ionic basis

Ranger S & Nattel S. (1995). Determinants and mechanisms of flecainideinduced promotion

Rautaharju PM, Zhou SH, Wong S, Calhoun HP, Berenson GS, Prineas R & Davignon A.

Reinecke H, Poppa V & Murry CE. (2002). Skeletal muscle stem cells do not

Ren C, Wang F, Li G, Jiao Q, Bai J, Yu D, Hao W, Wang R & Cao JM. (2008). Nerve sprouting

fibrillation in rat. *Auton Neurosci,* Vol.144, No.1-2, pp. 22–9. ISSN 1566-0702 Restivo M, Gough WB & el-Sherif N. (1990). Ventricular arrhythmias in the subacute

reentrant rhythms. *Circ Res,* Vol.66, No.5, pp. 1310–1327. ISSN 0009-7330 Rodriguez-Sinovas A, Boengler K, Cabestrero A, Gres P, Morente M, Ruiz-Meana M,

Roell W, Lewalter T, Sasse P, Tallini YN, Choi BR, Breitbach M, Doran R, Becher UM,

infarct arrhythmia. *Nature,* Vol.450, No.7171, pp. 819-824. ISSN 0028-0836 Rubart M & Zipes DP. (2005). Mechanisms of sudden cardiac death. *J Clin Invest,* Vol.115,

Saltman AE, Aksehirli TO, Valiunas V, Gaudette GR, Matsuyama N, Brink P & Krukenkamp

Salameh A & Dhein S. (2005). Pharmacology of gap junctions. New pharmacological targets

Sasano T, McDonald AD, Kikuchi K & Donahue JK. (2006). Molecular ablation of ventricular

*Cardiovasc Surg,* Vol.124, No.2, pp. 371-376. ISSN 0022-5223

*Circulation,* Vol.104, No.13, pp. 1544-1549. ISSN 0009-7322

age. *Can J Cardiol,* Vol.8, No.7, pp. 690-695. ISSN 0828-282X

Vol.34, No.2, pp. 241–249. ISSN 0022-2828

No.9, pp. 2305–2315. ISSN 0021-9738

No.1-2, pp. 36-58. ISSN 0005-2736

Vol.79, No.3, pp. 461–473. ISSN 0009-7330

thresholds with out changes in ventricular refractoriness in isolated rabbit hearts.

of arrhythmias in postinfarction remodeled ventricular myocardium. *Circ* 

of ventricular tachycardia in anesthetized dogs. *Circulation,* Vol.92, No.5, pp. 1300–

(1992). Sex differences in the evolution of the electrocardiographic QT interval with

transdifferentiate into cardiomyocytes after cardiac grafting. *J. Mol. Cell. Cardiol,* 

suppresses myocardial I(to) and I(K1) channels and increases severity to ventricular

myocardial infarction period. High-resolution activation and refractory patterns of

Konietzka I, Miró E, Totzeck A, Heusch G, Schulz R & Garcia-Dorado D. (2006). Translocation of Connexin 43 to the Inner Mitochondrial Membrane of Cardiomyocytes Through the Heat Shock Protein 90-Dependent TOM Pathway and Its Importance for Card ioprotection. *Circ Res,* Vol.99, No.1, pp. 93-101. ISSN 0009-

Hwang SM, Bostani T, von Maltzahn J, Hofmann A, Reining S, Eiberger B, Gabris B, Pfeifer A, Welz A, Willecke K, Salama G, Schrickel JW, Kotlikoff MI & Fleischmann BK. (2007). Engraftment of connexin 43-expressing cells prevents post-

IB. (2002). Gap junction uncoupling protects the heart against ischemia. *J Thorac* 

for treatment of arrhythmia, seizure and cancer? *Biochim Biophys Acta,* Vol.1719,

tachycardia after myocardial infarction. *Nat Med,* Vol.12, No.11, pp. 1256. ISSN

electricus sodium channel deduced from cDNA sequence. *Nature,* Vol.312, No.5990, pp. 121-127. ISSN 0028-0836


Nori SL, Gaudino M, Alessandrini F, Bronzetti E & Santarelli P. (1995).

Nuss HB, Marbán E & Johns DC. (1999). Overexpression of a human potassium channel

Oh YS, Jong AY, Kim DT, Li H, Wang C, Zemljic-Harpf A, Ross RS, Fishbein MC, Chen PS &

Oki T, Fukuda N, Kawano T, Iuchi A, Tabata T, Manabe K, Kageji Y, Sasaki M, Yamada H &

Perrier E, Kerfant BG, Lalevee N, Bideaux P, Rossier MF, Richard S, Gomez AM & Benitah

Peters NS, Coromilas J, Severs NJ & Wit AL. (1997). Disturbed connexin43 gap junction

Pinto JM, Yuan F, Wasserlauf BJ, Bassett AL & Myerburg RJ. (1997). Regional gradation of L-

Pinto JM & Boyden PA. (1998). Reduced inward rectifying and increased E-4031-sensitive

Pu J & Boyden PA. (1997). Alterations of Na+ currents in myocytes from epicardial border

Pu J, Balser JR & Boyden PA. (1998). Lidocaine action on Na+ currents in ventricular

Pu J, Ruffy F & Boyden PA. (1999). Effects of Bay Y 5959 on Ca2+ currents and intracellular

heart. *J Cardiovasc Pharmacol,* Vol.33, No.6, pp. 929–937. ISSN 0160-2446 Pu J, Robinson RB & Boyden PA. (2000). Abnormalities in Ca(i) handling in myocytes that

*Cell Cardiol,* Vol.32, No.8, pp. 1509–1523. ISSN 0022-2828

*Cardiovasc Electrophysiol,* Vol.8, No.5, pp. 548–560. ISSN 1045-3873

in mice. *Heart Rhythm,* Vol.3, No.6, pp. 728-736. ISSN 1547-5271

*J Gen Physiol,* Vol.86, No.1, pp. 89-104. ISSN 0022-1295

*Circulation,* Vol.110, No.7, pp. 776–783. ISSN 0009-7322

Vol.95, No.4, pp. 988–996. ISSN 0009-7322

No.4, pp. 431–440. ISSN 0009-7330

pp. 121-127. ISSN 0028-0836

Vol.103, No.6, pp. 889. ISSN 0021-9738

ISSN 0145-5680

7330

electricus sodium channel deduced from cDNA sequence. *Nature,* Vol.312, No.5990,

Immunohistochemical evidence for sympathetic denervation and reinnervation after necrotic injury in rat myocardium. *Cell Mol Biol,* Vol.41, No.6, pp. 799–807.

suppresses cardiac hyperexcitability in rabbit ventricular myocytes. *J Clin Invest,* 

Chen LS. (2006). Spatial distribution of nerve sprouting after myocardial infarction

Ito S. (1995). Histopathologic studies of innervation of normal and prolapsed human mitral valves. *J Heart Valve Dis,* Vol.4, No.5, pp. 496–502. ISSN 0966-8519 Patlak JB & Ortiz M. (1985). Slow currents through single sodium channels of adult rat heart.

JP. (2004). Mineralocorticoid receptor antagonism prevents the electrical remodeling that precedes cellular hypertrophy after myocardial infarction.

distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. *Circulation,* 

type calcium currents in the feline heart with a healed myocardial infarct. *J* 

K\_ current density in arrhythmogenic subendocardial Purkinje myocytes from the infarcted heart. *J Cardiovasc Electrophysiol,* Vol.9, No.3, pp. 299–311. ISSN 1045-3873

zone of the infarcted heart. A possible ionic mechanism for reduced excitability and postrepolarization refractoriness. *Circ Res,* Vol.81, No.1, pp. 110–119. ISSN 0009-

myocytes from the epicardial border zone of the infarcted heart. *Circ Res*, Vol. 83,

Ca2+ in cells that have survived in the epicardial border of the infarcted canine

survive in the infarcted heart are not just due to alterations in repolarization. *J Mol* 


Electrophysiological Abnormality:

Sympathetic Neural Remodeling, Electrical Remodeling and Gap Junction Remodeling 173

Sosinsky G. (2000). Gap junction structure: new structures and new insights. In: *Gap* 

Spear JF, Michelson EL, Spielman SR & Moore EN. (1977). The origin of ventricular

Spear JF, Horowitz LN, Hodess AB, MacVaugh H 3rd & Moore EN. (1979). Cellular

Takamatsu T. (2008). Arrhythmogenic substrates in myocardial infarct. *Pathol Int,* Vol.58,

Ursell PC, Gardner PI, Albala A, Fenoglio Jr. JJ & Wit AL. (1985). Structural and

Van Veen AA, van Rijen HV & Opthof T. (2001). Cardiac gap junction channels: modulation

Vozzi C, Dupont E, Coppen SR, Yeh HI & Severs NJ. (1999). Chamber-related differences in

Vracko R, Thorning D & Frederickson RG. (1990). Fate of nerve fibers in necrotic, healing, and healed rat myocardium. *Lab Invest,* Vol.63, No.4, pp. 490–501. ISSN 0023-6837 Vracko R, Thorning D & Frederickson RG. (1991). Nerve fibers in human myocardial scars.

Waldo AL, Camm AJ, deRuyter H, Friedman PL, MacNeil DJ, Pauls JF, Pitt B, Pratt CM,

Wen H, Jiang H, Lu Z, Hu X, He B, Tang Q & Huang C. (2010). Carvedilol ameliorates

kinase C activation. *FASEB J,* Vol.16, No.9, pp. 1114–6. ISSN 0892-6638 Wong RC, Pera MF & Pébay A. (2008). Role of Gap Junctions in Embryonic and Somatic

Xin P, Pan Y, Zhu W, Huang S, Wei M & Chen C. (2010). Favorable effects of resveratrol on

Stem Cells. *Stem Cell Rev,* Vol.4, No.4, pp. 283-292. ISSN 1550-8943

*Journal of Pharmacology,* Vol.649, No.1-3, pp. 293-300. ISSN 0014-2999 Xing D, Kjølbye AL, Nielsen MS, Petersen JS, Harlow KW, Holstein-Rathlou NH & Martins

*Hum Pathol,* Vol.22, No.2, pp. 138–146. ISSN 0046-8177

occlusion. *Circulation,* Vol.55, No.6, pp. 844–852. ISSN 0009-7322

activation. *Circulation,* Vol.59, No.2, pp. 247–256. ISSN 0009-7322

22, Academic Press,: ISBN 0-12-153349-2, San Diego

No.9, pp. 533-43. ISSN 1320-5463

617–626. ISSN 0022-3751

1003. ISSN 0022-2828

0008-6363

0140-6736

*junctions. Molecular basis of cell communication in health and disease.* Peracchia C, pp. 1-

arrhythmias 24 hours following experimental anterior septal coronary artery

electrophysiology of human myocardial infarction. 1. Abnormalities of cellular

electrophysiological changes in the epicardial border zone of canine myocardial infarcts during infarct healing. *Circ Res,* Vol.56, No.3, pp. 436–451. ISSN 0009-7330 Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB,

Rosen MR, Brink PR & Cohen IS. (2004). Human mesenchymal stem cells make cardiac connexins and form functional gap junctions*.J Physiol,* Vol.555, No.3, pp.

of expression and channel properties. *Cardiovasc Res,* Vol.51, No2, pp. 217–29. ISSN

connexin expression in the human heart. *J Mol Cell Cardiol,* Vol.31, No5, pp. 991–

Schwartz PJ & Veltri EP. (1996). Effect of D-sotalol on mortality in patients with left ventricular dysfunction after recent and remote myocardial infarction. The SWORD Investigators survival with oral D-sotalol. *Lancet,* Vol.348, No.9019, pp. 7–12. ISSN

sympathetic nerve sprouting and electrical remodelling after myocardial infarction in rats. *Biomedicine & Pharmacotherapy,* Vol.64, No.7, pp. 446–450. ISSN 0753-3322 Weng S, Lauven M, Schaefer T, Polontchouk L, Grover R & Dhein S. (2002). Pharmacological

modification of gap junction coupling by an antiarrhythmic peptide via protein

sympathetic neural remodeling in rats following myocardial infarction. *European* 

JB. (2003). ZP123 increases gap junctional conductance and prevents reentrant


Schulz R, Boengler K, Totzeck A, Luo Y, Garcia-Dorado D & Heusch G. (2007). Connexin 43

Schwartz PJ & Stone HL. (1980). Left stellectomy in the prevention of ventricular fibrillation

Schwartz PJ, Motolese M, Pollavini G, Lotto A, Ruberti U, Trazzi R, Bartorelli C & Zanchetti

Sebbag L, Verbinski SG, Reimer KA & Jennings RB. (2003). Protection of ischemic

Severs NJ, (1989). Constituent cells of the heart and isolated cell models in cardiovascular

Severs NJ. (1990). The cardiac gap junction and intercalated disc. *Int J Cardiol,* Vol.26, No.2,

Severs NJ, Gourdie RG, Harfst E, Peters NS & Green CR. (1993). Intercellular junctions and

Severs NJ, Rothery S, Dupont E, Coppen SR, Yeh HI, Ko YS, Matsushita T, Kaba R &

Severs NJ, Coppen SR, Dupont E, Yeh HI, Ko YS, Matsushita T. (2004). Gap junction

Severs NJ, Bruce AF, Dupont E & Rothery S. (2008). Remodelling of gap junctions and

Shaw RM & Rudy Y. (1997). Ionic mechanisms of propagation in cardiac tissue. Roles of the

Sohl G & Willecke K. (2004). Gap junctions and the connexin protein family. *Cardiovasc Res,* 

Solomon SD, Zelenkofske S, McMurray JJ, Finn PV, Velazquez E, Ertl G, Harsanyi A,

junction coupling. *Circ Res,* Vol.81, No.5, pp. 727–741. ISSN 0009-7330 Smith JH, Green CR, Peters NS, Rothery S & Severs NJ. (1991). Altered patterns of gap

infarction. *Circulation,* Vol.62, No.6, pp. 1256-1265. ISSN 0009-7322

*Cell Cardiol,* Vol.35, No.2, pp. 165-176. ISSN 0022-2828

model. *J Microsc,* Vol.169, No.3, pp. 299–328. ISSN 0022-2720

Isenberg G, pp. 3-41, CRC Press, Boca Raton.

Vol.139, No.4, pp. 801–821. ISSN 0002-9440

Vol.62, No.2, pp. 228–232. ISSN 0008-6363

2581–2588. ISSN 0028-4793

pp. 137-73. ISSN 0167-5273

301–22. ISSN 1059-910X

0008-6363

ISSN 0008-6363

ISSN 1382-4147

1045-3873

in ischemic pre- and postconditioning. *Heart Fail Rev,* Vol.12, No.3-4, pp. 261-266.

caused by acute myocardial ischemia in conscious dogs with anterior myocardial

A. (1992). Italian Sudden Death Prevention Group. Prevention of sudden cardiac death after a first myocardial infarction by pharmacologic or surgical antiadrenergic interventions. *J Cardiovasc Electrophysiol,* Vol.3, No.1, pp. 2–16. ISSN

myocardium in dogs using intracoronary 2, 3-butanedione monoxime (BDM). *J Mol* 

research. In: *Isolated Adult Cardiomyocytes: Structure and metabolism,* Piper HM,

the application of microscopical techniques: the cardiac gap junction as a case

Halliday D. (2001). Immunocytochemical analysis of connexin expression in the healthy and diseased cardiovascular system. *Microsc Res Tech,* Vol.52, No.3, pp.

alterations in human cardiac disease. *Cardiovasc Res.* Vol.62, No.2, pp. 368-77. ISSN

connexin expression in diseased myocardium. *Cardiovasc Res,* Vol.80, No.1, pp. 9-19.

sodium and L-type calcium currents during reduced excitability and decreased gap

junction distribution in ischemic heart disease. An immunohistochemical study of human myocardium using laser scanning confocal microscopy. *Am J Pathol,* 

Rouleau JL, Maggioni A, Kober L, White H, Van de Werf F, Pieper K, Califf RM & Pfeffer MA. (2005). Sudden death in patients with myocardial infarction and left ventricular dysfunction, heart failure, or both. *N Eng J Med,* Vol.352, No.25, pp.


**10** 

*China* 

**Novel Porcine Models of Myocardial** 

**and Future Application** 

*Department of Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing* 

Jianxun Liu and Xinzhi Li

**Ischemia/Infarction – Technical Progress,** 

Cardiovascular diseases, the first killer for human being, constitute the global economic burden. The updated data from World Health Organization (WHO) addresses that the mortality rank of myocardial infarction will increase from No.5 in 2000 to Top One till 2020. And 30 percent of overall mortality is due to the cardiovascular diseases. Their prevalence has severely affected patients' healthy conditions. Therefore, it is the priority for us to enforce the

Heart attacks are most commonly caused by a fatty "plaque" dislodging from the blood vessel wall. Inflammation in vessels and heart also plays an important role in this process. This provokes tiny cells in the bloodstream known as platelets to clump together and block the blood circulation to important organs. To elucidate the exact mechanism of the mentioned process, researchers need to develop various pre-clinical approaches to simulate the clinical presentation in animal models, cell strains, or even in tube reactions. Among those methodologies, animal model are more acceptable than the others due to its easily "from bench to bed" translational property. The use of appropriate large-animal models is essential if some new therapeutic strategies are to be critically evaluated in a preclinical

Over the past decade, swine have been increasingly used in studies of myocardial ischemia because of their numerous similarities to humans, including minimal preexisting coronary collaterals as well as similar coronary anatomy and physiology. In this chapter, the author will consequently review the most commonly used swine models of myocardial ischemia with special attention to regional myocardial blood flow and function and critically evaluates the strengths and weaknesses of each model in terms of utility for preclinical

Also in this part, several porcine models of myocardial ischemia/infarction developed in our department will be summarized briefly regarding with the technical progress. We would also like to share our experiences about new adventures in the field of

research on the mechanisms of pathogenesis and treatment of cardiovascular diseases.

**1. Introduction**

setting before their use in humans.

electrocardiogram mapping using multiple electrodes.

trials.

**Modified Electrocardiograms Validating,** 

ventricular tachycardia during myocardial ischemia in open chest dogs. *J Cardiovasc Electrophysiol,* Vol.14, No.5, pp. 510-20. ISSN 1045-3873


### **Novel Porcine Models of Myocardial Ischemia/Infarction – Technical Progress, Modified Electrocardiograms Validating, and Future Application**

Jianxun Liu and Xinzhi Li *Department of Pharmacology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing* 

*China* 

### **1. Introduction**

174 Advances in Electrocardiograms – Clinical Applications

Yagi T & Boyden PA. (2002). Protein tyrosine kinases and L-type Ca2+ currents in cells that

Yamada KA, Rogers JG, Sundset R, Steinberg TH & Saffitz JE. (2003). Up-regulation of

Yao JA, Hussain W, Patel P, Peters NS, Boyden PA & Wit AL. (2003). Remodeling of gap

Yuan F, Pinto JM, Li Q, Wasserlauf BJ, Yang X, Bassett AL & Myerburg RJ. (1999).

Yuan MJ, Huang CX, Tang YH, Wang X, Huang H, Chen YJ & Wang T. (2009). A novel

*European Journal of Pharmacology,* Vol.618, No.1-3, pp. 52-57. ISSN 0014-2999 Zhao Yan-mei, Zhong Guo-qiang, Li Jin-yi, He Yan, Ke Hong-hong & Wang Dong-xu.

Zhao Yan-mei, Zhong Guo-qiang, Ke Hong-hong & Li Jin-yi. (2010). Effects of different

Zhong G, Hayrapetyan V & Moreno AP. (2002). The Formation of mono-heteromeric Cx43-

Zhou S, Chen LS, Miyauchi Y, Miyauchi M, Kar S, Kangavari S, Fishbein MC, Sharifi B &

Zhou S, Jung BC, Tan AY, Trang VQ, Gholmieh G, Han SW, Lin SF, Fishbein MC, Chen PS,

Zipes DP. (1990). Influence of myocardial ischemia and infarction on autonomic innervation

*Electrophysiol,* Vol.14, No.5, pp. 510-20. ISSN 1045-3873

*Pharmacol,* Vol.40, No.5, pp. 669–677. ISSN 0160-2446

*Res,* Vol.92, No.4, pp. 437-443. ISSN 0009-7330

*Research,* Vol.13, No.45, pp. 8895-8900. ISSN 1671-5926

*Biophysical Journal,* Vol.82, No.1, pp. 633b. ISSN 0006-3495

in dogs. *Circ Res,* Vol.95, No.1, pp. 76–83. ISSN 0009-7330

of heart. *Circulation,* Vol.82, No.4, pp. 95–105. ISSN 0009-7322.

ISSN 1045-3873

1045-3873

1001-6325

9. ISSN 1547-5271

ventricular tachycardia during myocardial ischemia in open chest dogs. *J Cardiovasc* 

have survived in epicardial border zone of canine infarcted heart. *J Cardiovasc* 

connexin45 in heart failure. *J Cardiovasc Electrophysiol,* Vol.14, No.11, pp. 1205–1212.

junctional channel function in epicardial border zone of healing canine infarcts. *Circ* 

Characteristics of I(K) and its response to quinidine in experimental healed myocardial infarction. *J Cardiovasc Electrophysiol,* Vol.10, No.6, pp. 844–854. ISSN

peptide ghrelin inhibits neural remodeling after myocardial infarction in rats.

(2009). mRNA expression of connectin 43 and connectin 45 following transplantation of allogenic bone marrow mesenchymal stem cells in rats with acute myocardial infarction. *Journal of Clinical Rehabilitative Tissue Engineering* 

conditions on rat bone marrow mesenchymal stem cells differentiating into cardiomyocytes in vitro. *Basic & Clinical Medicine,* Vol.30, No.6, pp. 561-565. ISSN

Cx45/43 gap junctions uncovers gating and selectivity properties of their channels.

Chen PS. (2004). Mechanisms of cardiac nerve sprouting after myocardial infarction

Chen LS. (2008). Spontaneous stellate ganglion nerve activity and ventricular arrhythmia in a canine model of sudden death. *Heart Rhythm*. Vol.5, No.1, pp. 131Cardiovascular diseases, the first killer for human being, constitute the global economic burden. The updated data from World Health Organization (WHO) addresses that the mortality rank of myocardial infarction will increase from No.5 in 2000 to Top One till 2020. And 30 percent of overall mortality is due to the cardiovascular diseases. Their prevalence has severely affected patients' healthy conditions. Therefore, it is the priority for us to enforce the research on the mechanisms of pathogenesis and treatment of cardiovascular diseases.

Heart attacks are most commonly caused by a fatty "plaque" dislodging from the blood vessel wall. Inflammation in vessels and heart also plays an important role in this process. This provokes tiny cells in the bloodstream known as platelets to clump together and block the blood circulation to important organs. To elucidate the exact mechanism of the mentioned process, researchers need to develop various pre-clinical approaches to simulate the clinical presentation in animal models, cell strains, or even in tube reactions. Among those methodologies, animal model are more acceptable than the others due to its easily "from bench to bed" translational property. The use of appropriate large-animal models is essential if some new therapeutic strategies are to be critically evaluated in a preclinical setting before their use in humans.

Over the past decade, swine have been increasingly used in studies of myocardial ischemia because of their numerous similarities to humans, including minimal preexisting coronary collaterals as well as similar coronary anatomy and physiology. In this chapter, the author will consequently review the most commonly used swine models of myocardial ischemia with special attention to regional myocardial blood flow and function and critically evaluates the strengths and weaknesses of each model in terms of utility for preclinical trials.

Also in this part, several porcine models of myocardial ischemia/infarction developed in our department will be summarized briefly regarding with the technical progress. We would also like to share our experiences about new adventures in the field of electrocardiogram mapping using multiple electrodes.

Novel Porcine Models of Myocardial Ischemia/Infarction

difficult, compared with closed chest procedures.

**2.1.2 Mechanically chronic ischemic animal models** 

incomplete) of coronary occlusion (Inou et al., 1980).

**2.1.3 Swine models with thrombosis formation in coronary artery** 

feasible.

2006).

regional perfusion, and wall motion in different treatment group.

– Technical Progress, Modified Electrocardiograms Validating, and Future Application 177

emission computed tomographic scans were performed to compare the stroke volume,

Most of these myocardial infarction animal models using dogs or pigs is created by coronary artery ligation after surgical opening of the chest and exposure of the heart. Hereby, numerous disadvantages and experimental limitations may be associated with this technique: There is evidence suggesting that normal cardiac mechanics are disturbed in acute models after thoracotomy. Opening the chest and pericardium have both been suspected and reported to influence the pattern of left ventricular remodeling in chronic models (Kraitchman et al., 2000; Ludemann et al., 2007). Furthermore, the surgical trauma may cause a high rate of complications, resulting in high mortality. In addition, the trauma may render successful recovery from anesthesia in models of myocardial infarction more

Besides, to date, there are various myocardial ischemic animal models, such as ligating the coronary artery (Takahashi et al., 2005), placing constrictors on the coronary artery (Laham et al., 2000), putting microembolus into the coronary artery (Huang et al., 2004), or injecting ferric chloride via the vein (Dogne et al., 2005). However, operations in these methods lead to much damage in animals, and channelization can not be restored in the obstructed coronary artery. More considerably, the mimic pathophysiological alterations deviate from clinical data, especially autopsy. Nowadays, cardiovascular medicine research requires the availability of appropriate experimental animal models that are as close to humans as

The most widely used porcine model of chronic ischemia has been the ameroid constrictor. Originally described by Litvak and colleagues in 1957, these constrictors are constructed of the hygroscopic material casein encased within a steel sleeve. When the device is implanted around an artery, the constrictor absorbs water and swells, compressing the artery and producing total coronary occlusion over a period of 14-30 or more days (Elzinga, 1969). The major advantage of the ameroid constrictor model is its simplicity. Inherent limitations to the use of these occluders include an inability to control the rate or degree (sometimes

Another large-animal model of chronic ischemia has involved placing an adjustable hydraulic occluder around an epicardial coronary artery to produce a fixed degree of coronary stenosis (Bolukoglu et al., 1992). This model is similar to the fixed stenosis model in that the coronary artery is reduced in diameter by an external device, although with this latter model the occluder is typically placed either proximal or distal to a myocardial flow probe. Another famous study using this animal is about the relationship between hibernating myocardium and cell autophage (Yan et al., 2005). Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by coronary hydraulic stenosis. Autophagy, triggered by ischemia, could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited (Yan et al.,

Coronary artery thrombosis is widely accepted as a major cause of myocardial infarction (MI). The abrupt transition from a stable, often clinically silent, disease to a symptomatic

### **2. Animal models of myocardial ischemia/infarction**

### **2.1 Myocardial ischemic or infarction models**

High mortality and morbidity associated with myocardial ischemia or even the infarction necessitates modelling the process in animal. In a hospital setting, because the primary concern is resuscitation and maintaining of the patient's life, but not research in the underlying mechanism of the diseases. To study the pathophysiological changes associated with myocardial ischemic injury and how they lead to the establishment of cardiac cell death and regional infarction, animal models of clinical pathophysiology should gain our serious concern to overcome the limitations of clinical studies and to play an invaluable role in advancing mechanistic insight. Animal model studies will considerably provided new ideas and hypotheses that have been tested in clinical studies and have thus increased the level of knowledge regarding cardioprotection in the ischemic milieu.

Moreover, the use of experimental models of heart diseases in animals is an obligatory step for the understanding of mechanisms involved in pathologies consecutive to cardiac metabolic or functional disorders. As for any kind of therapeutic approach, to demonstrate its efficacy and safety there is also a need for animal models. Among factors of importance, there are animal species, coronary thrombosis induction method, thrombus composition and site of formation.

### **2.1.1 Acute ischemic and reperfusion animal models**

Based on the clinical profiles of the sudden heart attach, people firstly sought for the mimic method to study myocardial ischemic condition by ligation of one of the three main arteries in heart. Back to 1975, myocardial ischemia was induced in swine by ligation of the anterior descending coronary artery for periods of 30 minutes to six hours. Electron microscopy was used to monitor normal and ischemic myocardium (Lichtig et al., 1975). Scientists from our institute did contribute quite a lot to this model, not in swine, but in dogs. The dog's heart was exposed by opening the chest. The acute ischemia models were prepared by ligating left anterior descending (LAD) artery. Meanwhile, timed monitoring hemodynamics and blood flow were determined by physiological polygraph and electromagnetic flow meter, respectively (Li, 1978). In another research group, the diagonal branches of the left anterior descending coronary artery in pigs were ligated for 5, 15, 30, 60 and 120 minutes. By using transmission electron microscopy, morphological observations indicated that initial leukocyte infiltration and inflammation during myocardial ischemia (Park et al., 1985).

These kinds of open-chest model were widely accepted during 1990s and were still prevalent in 2000s. With development of the diagnostic methods, more and more advanced technologies were employed in the research of heart diseases and animal modelling. In one of representative works, nine open-chest swine undergoing myocardial ischemia were instrumented for measurement of regional myocardial blood flow (microsphere method), contractile function (sonomicrometry), and hemodynamics. L-[1-14C] Lactate or L-[U-13C] lactate was infused intravenously using a primed continuous infusion technique to quantify regional myocardial lactate release. A close inverse relation between regional myocardial lactate release and regional subendocardial blood flow during graded ischemia was revealed (Guth et al., 1990). When researcher evaluated the utility of transplanting bone marrow stromal cells in a porcine myocardial infarction model, a myocardial infarction was created by occluding the distal left anterior descending artery in pigs with coils and Gelfoam sponge (Tomita et al., 2002). In this study, sestamibi technetium single-photon

High mortality and morbidity associated with myocardial ischemia or even the infarction necessitates modelling the process in animal. In a hospital setting, because the primary concern is resuscitation and maintaining of the patient's life, but not research in the underlying mechanism of the diseases. To study the pathophysiological changes associated with myocardial ischemic injury and how they lead to the establishment of cardiac cell death and regional infarction, animal models of clinical pathophysiology should gain our serious concern to overcome the limitations of clinical studies and to play an invaluable role in advancing mechanistic insight. Animal model studies will considerably provided new ideas and hypotheses that have been tested in clinical studies and have thus increased the

Moreover, the use of experimental models of heart diseases in animals is an obligatory step for the understanding of mechanisms involved in pathologies consecutive to cardiac metabolic or functional disorders. As for any kind of therapeutic approach, to demonstrate its efficacy and safety there is also a need for animal models. Among factors of importance, there are animal species, coronary thrombosis induction method, thrombus composition and

Based on the clinical profiles of the sudden heart attach, people firstly sought for the mimic method to study myocardial ischemic condition by ligation of one of the three main arteries in heart. Back to 1975, myocardial ischemia was induced in swine by ligation of the anterior descending coronary artery for periods of 30 minutes to six hours. Electron microscopy was used to monitor normal and ischemic myocardium (Lichtig et al., 1975). Scientists from our institute did contribute quite a lot to this model, not in swine, but in dogs. The dog's heart was exposed by opening the chest. The acute ischemia models were prepared by ligating left anterior descending (LAD) artery. Meanwhile, timed monitoring hemodynamics and blood flow were determined by physiological polygraph and electromagnetic flow meter, respectively (Li, 1978). In another research group, the diagonal branches of the left anterior descending coronary artery in pigs were ligated for 5, 15, 30, 60 and 120 minutes. By using transmission electron microscopy, morphological observations indicated that initial leukocyte infiltration and inflammation during myocardial ischemia (Park et al., 1985). These kinds of open-chest model were widely accepted during 1990s and were still prevalent in 2000s. With development of the diagnostic methods, more and more advanced technologies were employed in the research of heart diseases and animal modelling. In one of representative works, nine open-chest swine undergoing myocardial ischemia were instrumented for measurement of regional myocardial blood flow (microsphere method), contractile function (sonomicrometry), and hemodynamics. L-[1-14C] Lactate or L-[U-13C] lactate was infused intravenously using a primed continuous infusion technique to quantify regional myocardial lactate release. A close inverse relation between regional myocardial lactate release and regional subendocardial blood flow during graded ischemia was revealed (Guth et al., 1990). When researcher evaluated the utility of transplanting bone marrow stromal cells in a porcine myocardial infarction model, a myocardial infarction was created by occluding the distal left anterior descending artery in pigs with coils and Gelfoam sponge (Tomita et al., 2002). In this study, sestamibi technetium single-photon

**2. Animal models of myocardial ischemia/infarction** 

level of knowledge regarding cardioprotection in the ischemic milieu.

**2.1.1 Acute ischemic and reperfusion animal models** 

**2.1 Myocardial ischemic or infarction models** 

site of formation.

emission computed tomographic scans were performed to compare the stroke volume, regional perfusion, and wall motion in different treatment group.

Most of these myocardial infarction animal models using dogs or pigs is created by coronary artery ligation after surgical opening of the chest and exposure of the heart. Hereby, numerous disadvantages and experimental limitations may be associated with this technique: There is evidence suggesting that normal cardiac mechanics are disturbed in acute models after thoracotomy. Opening the chest and pericardium have both been suspected and reported to influence the pattern of left ventricular remodeling in chronic models (Kraitchman et al., 2000; Ludemann et al., 2007). Furthermore, the surgical trauma may cause a high rate of complications, resulting in high mortality. In addition, the trauma may render successful recovery from anesthesia in models of myocardial infarction more difficult, compared with closed chest procedures.

Besides, to date, there are various myocardial ischemic animal models, such as ligating the coronary artery (Takahashi et al., 2005), placing constrictors on the coronary artery (Laham et al., 2000), putting microembolus into the coronary artery (Huang et al., 2004), or injecting ferric chloride via the vein (Dogne et al., 2005). However, operations in these methods lead to much damage in animals, and channelization can not be restored in the obstructed coronary artery. More considerably, the mimic pathophysiological alterations deviate from clinical data, especially autopsy. Nowadays, cardiovascular medicine research requires the availability of appropriate experimental animal models that are as close to humans as feasible.

### **2.1.2 Mechanically chronic ischemic animal models**

The most widely used porcine model of chronic ischemia has been the ameroid constrictor. Originally described by Litvak and colleagues in 1957, these constrictors are constructed of the hygroscopic material casein encased within a steel sleeve. When the device is implanted around an artery, the constrictor absorbs water and swells, compressing the artery and producing total coronary occlusion over a period of 14-30 or more days (Elzinga, 1969). The major advantage of the ameroid constrictor model is its simplicity. Inherent limitations to the use of these occluders include an inability to control the rate or degree (sometimes incomplete) of coronary occlusion (Inou et al., 1980).

Another large-animal model of chronic ischemia has involved placing an adjustable hydraulic occluder around an epicardial coronary artery to produce a fixed degree of coronary stenosis (Bolukoglu et al., 1992). This model is similar to the fixed stenosis model in that the coronary artery is reduced in diameter by an external device, although with this latter model the occluder is typically placed either proximal or distal to a myocardial flow probe. Another famous study using this animal is about the relationship between hibernating myocardium and cell autophage (Yan et al., 2005). Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by coronary hydraulic stenosis. Autophagy, triggered by ischemia, could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited (Yan et al., 2006).

### **2.1.3 Swine models with thrombosis formation in coronary artery**

Coronary artery thrombosis is widely accepted as a major cause of myocardial infarction (MI). The abrupt transition from a stable, often clinically silent, disease to a symptomatic

Novel Porcine Models of Myocardial Ischemia/Infarction

completely and instantly embolized, post-operation.

the duration of the occlusion.

– Technical Progress, Modified Electrocardiograms Validating, and Future Application 179

Pathohistological analysis revealed myocardial degeneration, necrosis, fibrosis, inflammatory cell infiltration and granulation tissue hyperblastosis. Our data showed that myocardial ischemia induced by injecting self-embolus into the LAD coronary artery in Chinese miniature swine is quite close to clinical pathophysiological conditions. (Fig. 1.)

Fig. 1. Changes of coronary embolism were disclosed by the coronary angiography. (A) the LAD of the model group was normal at pre-operation; (B) the LAD of the model group was

In other lab, injection of thrombogenic material, such as microcoils or thrombin fibrinogen mixtures, have also been proposed and explored (Koning et al., 1993; Naslund et al., 1992). In these studies, after injection into a coronary artery, the blood flow transports these materials downstream until their size matches the diameter of the coronary artery. Standardization of the infarction size is not possible because there is physiologic variation of the diameter of the coronary arteries between individual animals of similar body weight. Furthermore, with these approaches, either reperfusion is not possible or occurs after spontaneous lysis of the occluding material. This approach does not allow for controlling

On the other hand, X-ray-guided placement of a balloon catheter in the coronary artery and inflation of the balloon for coronary artery occlusion avoids extensive surgery and allows the control of both the location and the time of occlusion. This approach has been used in several studies. Balloon occlusion of the LAD can successfully be performed in pigs to create reperfused or occlusive myocardial infarction. The technique allows for a relatively short preparation time compared with open-chest surgery, control over the duration of ischemia, and standardization the location of the occlusion and infarcts size. The resulting changes of the myocardial function are solely due to the ischemic injury. This renders results obtained

life-threatening condition results from endothelial injury or plaque disruption followed by thrombosis (Falk et al., 1995). Because of this acute intracoronary thrombotic occlusion, the primary goal of therapy is rapid, complete, and sustained restoration of infarct-related artery blood flow. In this case, the choice of the animal model of thrombosis to evaluate the efficiency of antiplatelet, antithrombotic or thrombolytic drugs in preclinical studies is crucial. Numerous animal models of thrombosis-induced MI have been proposed in the last decades (Leadley et al., 2000). Another example was from porcine model of myocardial infarction induced by topical application of ferric chloride to the LAD coronary artery, which was validated to quite close to clinical pathophysiological conditions, such as thrombus formation occurring after atherosclerotic plaque rupture (Dogne et al., 2005).

In dogs, left circumflex coronary artery (LCx) thrombosis was induced by vascular electrolytic injury (Hennan et al., 2001; Romson et al., 1980). After a flow probe and stenosis had been placed around the LCx, an intracoronary electrode was inserted through the LCx arterial wall, with the uninsulated portion positioned against the endothelial surface. An anodal current of 150 µA was applied to the endothelial surface to initiate the experiment. This operation is relatively difficult to master or needs much more practice, since the electrode is introduced into coronary artery. Another shortcoming is bleeding due to artery injury during procedure, especially the introduction of electrode.

In our department, to evaluate the thrombolytic effects of heparin-like drugs, swine's endoarterium was injuried and coronary thrombi were formed gradually through direct electrical stimulation on the coronary artery of animals (Liu et al., 2002). However, some limitations of this model should be pointed out. First, we are aware of the fact that the maintenance of pigs is expensive and difficult, and requires special facilities that are beyond the capabilities of most laboratories. Secondly, the pig presents a high myocardial sensitivity to hypoxia. Acute reduction of coronary blood flow can easily induce paroxystic ventricular fibrillation which may then requires the use of prophylactic antiarrythmic agents.

### **2.1.4 Making myocardial ischemic animal models via interventional techniques**

Most models listed above in the pig, with or without subsequent reperfusion need thoracotomy. The majority of these employ surgical techniques of coronary occlusion following thoracotomy. During these complex operations, the subtle enviorment inside of the chest will be somewhat disturbed. The endovascular and minimal invasion techniques offer a series of advantages over the surgical technique such as a lower incidence of infections and of related complications which can compromise the survival of the animal and the validity of the results.

These disadvantages raise the demand for closed-chest models for myocardial infarction in large animals. To facilitate the surgical operation, we introduced the intervention technique and selective coronary angiography into our study. One ischemic model we used was developed in Chinese miniature swine, where myocardial ischemia was performed by injecting self-embolus into the middle segment of the left anterior descending (LAD) without thoracotomy (Liu et al., 2007b; Yu et al., 2007b). Embolization occurred in the LAD coronary artery of the Chinese miniature swine injected by self-embolus. There were significant myocardial ischemia and large cardiac muscle infarction in the Chinese miniature swine, which were accompanied with increased Body surface ECG (BS-ECG) (Liu et al., 2007b), decreased hemodynamic indexes of the cardiac output, cardiac index, left cardiac work and left cardiac work index, and increased systemic vascular resistance index.

life-threatening condition results from endothelial injury or plaque disruption followed by thrombosis (Falk et al., 1995). Because of this acute intracoronary thrombotic occlusion, the primary goal of therapy is rapid, complete, and sustained restoration of infarct-related artery blood flow. In this case, the choice of the animal model of thrombosis to evaluate the efficiency of antiplatelet, antithrombotic or thrombolytic drugs in preclinical studies is crucial. Numerous animal models of thrombosis-induced MI have been proposed in the last decades (Leadley et al., 2000). Another example was from porcine model of myocardial infarction induced by topical application of ferric chloride to the LAD coronary artery, which was validated to quite close to clinical pathophysiological conditions, such as thrombus formation occurring after atherosclerotic plaque rupture (Dogne et al., 2005). In dogs, left circumflex coronary artery (LCx) thrombosis was induced by vascular electrolytic injury (Hennan et al., 2001; Romson et al., 1980). After a flow probe and stenosis had been placed around the LCx, an intracoronary electrode was inserted through the LCx arterial wall, with the uninsulated portion positioned against the endothelial surface. An anodal current of 150 µA was applied to the endothelial surface to initiate the experiment. This operation is relatively difficult to master or needs much more practice, since the electrode is introduced into coronary artery. Another shortcoming is bleeding due to artery

In our department, to evaluate the thrombolytic effects of heparin-like drugs, swine's endoarterium was injuried and coronary thrombi were formed gradually through direct electrical stimulation on the coronary artery of animals (Liu et al., 2002). However, some limitations of this model should be pointed out. First, we are aware of the fact that the maintenance of pigs is expensive and difficult, and requires special facilities that are beyond the capabilities of most laboratories. Secondly, the pig presents a high myocardial sensitivity to hypoxia. Acute reduction of coronary blood flow can easily induce paroxystic ventricular

fibrillation which may then requires the use of prophylactic antiarrythmic agents.

**2.1.4 Making myocardial ischemic animal models via interventional techniques** 

Most models listed above in the pig, with or without subsequent reperfusion need thoracotomy. The majority of these employ surgical techniques of coronary occlusion following thoracotomy. During these complex operations, the subtle enviorment inside of the chest will be somewhat disturbed. The endovascular and minimal invasion techniques offer a series of advantages over the surgical technique such as a lower incidence of infections and of related complications which can compromise the survival of the animal

These disadvantages raise the demand for closed-chest models for myocardial infarction in large animals. To facilitate the surgical operation, we introduced the intervention technique and selective coronary angiography into our study. One ischemic model we used was developed in Chinese miniature swine, where myocardial ischemia was performed by injecting self-embolus into the middle segment of the left anterior descending (LAD) without thoracotomy (Liu et al., 2007b; Yu et al., 2007b). Embolization occurred in the LAD coronary artery of the Chinese miniature swine injected by self-embolus. There were significant myocardial ischemia and large cardiac muscle infarction in the Chinese miniature swine, which were accompanied with increased Body surface ECG (BS-ECG) (Liu et al., 2007b), decreased hemodynamic indexes of the cardiac output, cardiac index, left cardiac work and left cardiac work index, and increased systemic vascular resistance index.

injury during procedure, especially the introduction of electrode.

and the validity of the results.

Pathohistological analysis revealed myocardial degeneration, necrosis, fibrosis, inflammatory cell infiltration and granulation tissue hyperblastosis. Our data showed that myocardial ischemia induced by injecting self-embolus into the LAD coronary artery in Chinese miniature swine is quite close to clinical pathophysiological conditions. (Fig. 1.)

Fig. 1. Changes of coronary embolism were disclosed by the coronary angiography. (A) the LAD of the model group was normal at pre-operation; (B) the LAD of the model group was completely and instantly embolized, post-operation.

In other lab, injection of thrombogenic material, such as microcoils or thrombin fibrinogen mixtures, have also been proposed and explored (Koning et al., 1993; Naslund et al., 1992). In these studies, after injection into a coronary artery, the blood flow transports these materials downstream until their size matches the diameter of the coronary artery. Standardization of the infarction size is not possible because there is physiologic variation of the diameter of the coronary arteries between individual animals of similar body weight. Furthermore, with these approaches, either reperfusion is not possible or occurs after spontaneous lysis of the occluding material. This approach does not allow for controlling the duration of the occlusion.

On the other hand, X-ray-guided placement of a balloon catheter in the coronary artery and inflation of the balloon for coronary artery occlusion avoids extensive surgery and allows the control of both the location and the time of occlusion. This approach has been used in several studies. Balloon occlusion of the LAD can successfully be performed in pigs to create reperfused or occlusive myocardial infarction. The technique allows for a relatively short preparation time compared with open-chest surgery, control over the duration of ischemia, and standardization the location of the occlusion and infarcts size. The resulting changes of the myocardial function are solely due to the ischemic injury. This renders results obtained

Novel Porcine Models of Myocardial Ischemia/Infarction

think over the necessity and specific characters in each animal model.

cardiovascular disease on any genetic background (Moreno et al., 2011).

**2.1.7 Computer-based simulation of ischemia and reperfusion model** 

others (Rodriguez et al., 2006).

**2.2 Evaluation of myocardial ischemia and infarction** 

– Technical Progress, Modified Electrocardiograms Validating, and Future Application 181

performed in mice and rats; on the assumption that the results can be translated to humans. Transgenic mice are widely accepted and used in many labs. To delve the roles of prostaglandin H synthase-1 and prostaglandin H-synthase-2, known colloquially as COX-1 (target of aspirin: first level heart disease prevention drug) and COX-2 (target of many antiinflammatory drugs), in cardiovascular systems, Four different strains of mice were successfully generated and characterized including: (i) genetic knockdown of COX-1 (80- 97% reduction) (Yu et al., 2005); (ii) genetic knockdown of COX-2 (80-95 % reduction) (Seta et al., 2009); (iii) knock-in of a point mutation at the COX-2 active site to abolish cyclooxygenase activity and leave peroxidase activity intact Ptgs2Y385F (Yu et al., 2006); and (iv) exchange of COX-1 into the COX-2 locus (Yu et al., 2007a). But before we head to set up more transgenic knock-in and/or knock-out mice, we should keep our own brain cool to

Tiny mouse's heart muscle mass (<100 mg) as compared to about 500 g in the human heart presents problems of magnitude and architectural complexity. This may partially explain why so many effective investigational new drugs withdraw market after phase 3 clinical trials even they have show promising pharmacological effects in experimental animal models. Scientist resorted to another popular rodent animal: rat. But hampered by lacking specific techniques to manipulate the rat stem cells which have been working very well in mice, rats were kicked out of the transgenic game for a long time. Zinc-Finger Nucleases (ZFN) technology to generate knock-in rats in which foreign genes have been inserted, or 'knocked-in', into the rat genome in a precisely targeted manner (Geurts et al., 2009). This breakthrough achievement represents a major step forward in the creation of a transgenic animal, which may serve as more predictive models of human disease, especially in cardiovascular diseases. For example, first rat knockout in the renin-angiotensin system has demonstrated the efficacy of the ZFN technology for creating knockout rats for

Experimental models, however, have their own set of limitations that hamper the comprehensive evaluation of heart functional mechanisms. At the end of the last century, some scientists pursued to develop a biochemically and biophysically detailed model that could provide a novel approach to studying myocardial ischaemia and reperfusion (Ch'en et al., 1998). Over the last decade, analysis of electrophysiological phenomena following coronary occlusion has been significantly augmented and advanced by the use of mathematical modeling and computer simulations, from the ionic channel to the whole organ. One of the major contributions of computational research in electrophysiology has been the ability of mathematical models to dissect various effects and to tease out important relations between electrophysiological parameters. This part has been well reviewed by

Conventional 12-lead electrocardiography (ECG) as well as analysis of serum markers still play a non-replaceable role clinically and even in the animal model research. In addition, a number of special techniques such as echocardiography, nuclear magnetic resonance imaging (MRI) (Wright et al., 2009), computed tomography (CT) (Baks et al., 2006; Buecker et al., 2005; Mahnken et al., 2005), single photon emission computed tomography (SPECT),

with this model more easily interpretable. Furhtermore, this model is advantageous for MRI studies because the heart is not exposed, so that susceptibility artifacts that arise from the myocardium-air border in open chest-models are avoided (Krombach et al., 2005).

### **2.1.5 Coronary atherosclerosis in swine**

Recently, advanced coronary atherosclerosis in swine was produced by combination of balloon-catheter injury and cholesterol feeding (Li et al., 2009). In this study, twelve Chinese experimental miniature swine (CEMS) were randomly divided into Control and Model groups. In the model, fed with high fat diet for 2 weeks, the pigs underwent left coronary angiography and balloon over dilation in left anterior descending artery (LAD) followed by continuous feeding with high fat diet for 8 weeks. At the end of 10 weeks, an IVUS catheter was guided to the LAD artery. Intravascular ultrasound and virtual histology (IVUS-VH) data analysis was based on border contour calculation from gray scale. Coronary angiography with (IVUS) and virtual histology (VH) will help us to detect the process of coronary atherosclerosis during coronary heart disease (CHD). IVUS classified the plaque as concentric, with fibrous 62.8%, fibro-lipidic 12.7%, necrotic 15.5%, and calcific 8.9% tissue. In vitro histopathology correctly identified the different presence of fibrous tissue, and different extent of stenosis, which was consistently correlative to the result of IVUS-VH (Fig. 2.). These models provide essential help for researchers to better understand the pathophysiological implications of the heart diseases, from endothelium injury to plaque stability, from marker protein expressions to cell death pattern. All these findings will not only present one of the scientific answers to the pathogenesis of model animals with coronary atherosclerotic heart diseases (coronary heart disease, CHD), but also will facilitate to interpret the scientific insights of prevention and therapeutic strategy.

Fig. 2. LAD angiography and IVUS-VH after balloon injury in swine fed with high-fat diet for different time (1, 2, 8w). (A, C, E) LAD angiography; (B, D, F) IVUS-VH.

### **2.1.6 Transgenic animal models for cardiovascular diseases**

For economic reasons and with the help of genetically targeted mouse manipulated in the laboratory, considerable basic and applied cardiovascular experimentation has been

with this model more easily interpretable. Furhtermore, this model is advantageous for MRI studies because the heart is not exposed, so that susceptibility artifacts that arise from the

Recently, advanced coronary atherosclerosis in swine was produced by combination of balloon-catheter injury and cholesterol feeding (Li et al., 2009). In this study, twelve Chinese experimental miniature swine (CEMS) were randomly divided into Control and Model groups. In the model, fed with high fat diet for 2 weeks, the pigs underwent left coronary angiography and balloon over dilation in left anterior descending artery (LAD) followed by continuous feeding with high fat diet for 8 weeks. At the end of 10 weeks, an IVUS catheter was guided to the LAD artery. Intravascular ultrasound and virtual histology (IVUS-VH) data analysis was based on border contour calculation from gray scale. Coronary angiography with (IVUS) and virtual histology (VH) will help us to detect the process of coronary atherosclerosis during coronary heart disease (CHD). IVUS classified the plaque as concentric, with fibrous 62.8%, fibro-lipidic 12.7%, necrotic 15.5%, and calcific 8.9% tissue. In vitro histopathology correctly identified the different presence of fibrous tissue, and different extent of stenosis, which was consistently correlative to the result of IVUS-VH (Fig. 2.). These models provide essential help for researchers to better understand the pathophysiological implications of the heart diseases, from endothelium injury to plaque stability, from marker protein expressions to cell death pattern. All these findings will not only present one of the scientific answers to the pathogenesis of model animals with coronary atherosclerotic heart diseases (coronary heart disease, CHD), but also will facilitate

myocardium-air border in open chest-models are avoided (Krombach et al., 2005).

to interpret the scientific insights of prevention and therapeutic strategy.

Fig. 2. LAD angiography and IVUS-VH after balloon injury in swine fed with high-fat diet

For economic reasons and with the help of genetically targeted mouse manipulated in the laboratory, considerable basic and applied cardiovascular experimentation has been

for different time (1, 2, 8w). (A, C, E) LAD angiography; (B, D, F) IVUS-VH.

**2.1.6 Transgenic animal models for cardiovascular diseases** 

**2.1.5 Coronary atherosclerosis in swine**

performed in mice and rats; on the assumption that the results can be translated to humans. Transgenic mice are widely accepted and used in many labs. To delve the roles of prostaglandin H synthase-1 and prostaglandin H-synthase-2, known colloquially as COX-1 (target of aspirin: first level heart disease prevention drug) and COX-2 (target of many antiinflammatory drugs), in cardiovascular systems, Four different strains of mice were successfully generated and characterized including: (i) genetic knockdown of COX-1 (80- 97% reduction) (Yu et al., 2005); (ii) genetic knockdown of COX-2 (80-95 % reduction) (Seta et al., 2009); (iii) knock-in of a point mutation at the COX-2 active site to abolish cyclooxygenase activity and leave peroxidase activity intact Ptgs2Y385F (Yu et al., 2006); and (iv) exchange of COX-1 into the COX-2 locus (Yu et al., 2007a). But before we head to set up more transgenic knock-in and/or knock-out mice, we should keep our own brain cool to think over the necessity and specific characters in each animal model.

Tiny mouse's heart muscle mass (<100 mg) as compared to about 500 g in the human heart presents problems of magnitude and architectural complexity. This may partially explain why so many effective investigational new drugs withdraw market after phase 3 clinical trials even they have show promising pharmacological effects in experimental animal models. Scientist resorted to another popular rodent animal: rat. But hampered by lacking specific techniques to manipulate the rat stem cells which have been working very well in mice, rats were kicked out of the transgenic game for a long time. Zinc-Finger Nucleases (ZFN) technology to generate knock-in rats in which foreign genes have been inserted, or 'knocked-in', into the rat genome in a precisely targeted manner (Geurts et al., 2009). This breakthrough achievement represents a major step forward in the creation of a transgenic animal, which may serve as more predictive models of human disease, especially in cardiovascular diseases. For example, first rat knockout in the renin-angiotensin system has demonstrated the efficacy of the ZFN technology for creating knockout rats for cardiovascular disease on any genetic background (Moreno et al., 2011).

### **2.1.7 Computer-based simulation of ischemia and reperfusion model**

Experimental models, however, have their own set of limitations that hamper the comprehensive evaluation of heart functional mechanisms. At the end of the last century, some scientists pursued to develop a biochemically and biophysically detailed model that could provide a novel approach to studying myocardial ischaemia and reperfusion (Ch'en et al., 1998). Over the last decade, analysis of electrophysiological phenomena following coronary occlusion has been significantly augmented and advanced by the use of mathematical modeling and computer simulations, from the ionic channel to the whole organ. One of the major contributions of computational research in electrophysiology has been the ability of mathematical models to dissect various effects and to tease out important relations between electrophysiological parameters. This part has been well reviewed by others (Rodriguez et al., 2006).

### **2.2 Evaluation of myocardial ischemia and infarction**

Conventional 12-lead electrocardiography (ECG) as well as analysis of serum markers still play a non-replaceable role clinically and even in the animal model research. In addition, a number of special techniques such as echocardiography, nuclear magnetic resonance imaging (MRI) (Wright et al., 2009), computed tomography (CT) (Baks et al., 2006; Buecker et al., 2005; Mahnken et al., 2005), single photon emission computed tomography (SPECT),

Novel Porcine Models of Myocardial Ischemia/Infarction

**2.2.2 Advanced imaging systems** 

(Higuchi et al., 2007).

(Skrzypiec-Spring et al., 2007).

**2.2.4 Other novel methodologies** 

2009).

**2.2.3 Histological analysis of infarct size** 

2003).

– Technical Progress, Modified Electrocardiograms Validating, and Future Application 183

myocardium was so promising that would result in potential clinical uses (Odenstedt et al.,

Conventional methods to quantify infarct size after myocardial infarction in mice are not ideal. Cardiologists therefore implemented a fast, high-resolution method to directly measure infarct size in vivo using three-dimensional (3D) late gadolinium enhancement MRI (3D-LGE). They had validated an improved 3D MRI method to noninvasively quantify infarct size in mice with unsurpassed spatial resolution and tissue contrast. This method is particularly suited to studies requiring early quantification of initial infarct size, for example, to measure damage before intervention with stem cells (Bohl et al., 2009). Another study group combined the small-animal PET and MRI data to acquire quantitative in vivo insights into cardiac pathophysiology, and sought to determine the feasibility of PET and MRI for the quantification of ischemic injury in the rat model. Successful integrating information from small-animal PET and clinical MRI instrumentation allows for the quantitative assessment of cardiac function and infarct size in the rat model. The MRI measurements of scar can be complemented by metabolic imaging, addressing the extent and severity of ischemic injury and providing endpoints for therapeutic interventions

In preclinical studies, still the postmortem histological analysis is considered to be the gold standard for measuring infarct size. However, there are a number of disadvantages of this technique, making a reliable non-invasive alternative highly desirable. First, histological methods leave no residual tissue for further analysis. Second, visual interpretation and planimetry of heart sections may be subjective in cases with poor viable/non-viable contrast due to hemoglobin residues within the necrotic regions or with TTC-induced geometric distortion of the sample. Finally, animals must be euthanized to measure injury, meaning longitudinal studies require separate groups of animals for each time point (Bohl et al.,

Conventional TTC staining allows the quantification of infarct size much sooner than standard histological techniques, and has been shown to be equally sensitive and specific. Therefore, the direct post-sacrifice techniques such as e.g. the TTC staining, is still the most widely used, low-cost and high throughput method to assess infarct size in animal models

Many new methods were employed in cardiovascular functional measurement after ischemia or infarction. By using a three-axis accelerometer, researchers developed a novel technique for continuous real-time assessment of myocardial ischaemia in 14 anaesthetized open-chest pigs. Two accelerometers sutured on the left ventricle (LV) surface in the perfusion areas of the left anterior descending (LAD) and circumflex (CX) arteries, measured acceleration in the longitudinal, circumferential, and radial directions, and the corresponding epicardial velocities were calculated. The accelerometer had the ability to distinguish ischaemia from interventions altering global myocardial function, by which the myocardial ischaemia can be monitored in a continuous real-time mode (Halvorsen et al.,

and positron emission tomography (PET) have been devised to support diagnosis in the patients who show ambiguous symptoms and ECG findings. Major advantage of these latter methods is that infarct size can be non-invasively and repeatedly measured in vivo. Major limitation of the non-invasive imaging techniques in humans is the lack of quantification of the risk zone.

In preclinical small animal models, the use of above mentioned novel non-invasive imaging techniques is also limited due to several technical problems (e.g. lack of area at risk determination, insufficient temporal and spatial resolution, irradiation, etc.) and their high cost. Therefore, direct post-sacrifice techniques such as e.g. the triphenyltetrazolium (TTC) staining as well as nitroblue tetrazolium (NBT) staining, is still the most widely used, lowcost method to assess infarct size in animal models.

### **2.2.1 Modified ECG and multiple-electrode mapping**

Identifying patients at risk of ST segment elevations (most frequently observed in heart attack) by use of body surface electrical measures is controversial. To probe the principal variations during myocardial ischemia, scientists have pursued to measure the electrophysiological changes of heart by modified ECG (s). In our lab, to circumvent some of these problems and to further our knowledge of the cardiovascular disorders we have undertook a series of investigations that consisted of designing novel modified ECG since 1970s. Two different modified ECGs were successfully developed and characterized including: epicardial electrograms (Li, 1978; Liu et al., 2002) and body surface ECG (BS-ECG) (Liu et al., 2007b). See the section "EECG and BS-ECG" below for the details.

Other lab worldwide besides our lab should also be reviewed here as to design very sensitive, multiple-electrode mapping technology. Mimicking the body 12-lead ECG, scientists from University of Oxford recorded ventricular epicardial electrograms from 5 anesthetized pigs with a 127-electrode sock and simultaneously investigated torso ECG using a specifically designed vest with 256 ECG electrodes. One of breakthroughs they have made in this study is that with chest reclosed, simultaneous arrays of epicardial electrograms and torso ECGs can be recorded during LAD occlusion and reperfusion (Nash et al., 2003).

The earlier identification of coronary occlusion with ST-segment elevation, the better outcomes patients undergoing heart attack may have due to timing of revascularization. Using an implantable, high-fidelity, intracardiac electrogram monitoring system with longrange telemetry in porcine subjected to acute coronary occlusion precipitated by stent thrombosis, researchers from Michigan State University real-timely detected acute STsegment elevation and analyzed its correlations with thrombotic coronary occlusion. High sensitivity and specificity (100% and 100%, respectively) in such a system make it possible to advance the time frame of reperfusion therapy and potentially prevent, rather than interrupt, acute myocardial infarction in patients with coronary artery disease (Fischell et al., 2006).

In a porcine acute infarct and reperfusion model induced by balloon occlusion of the left anterior descending coronary artery for 45 minutes, endocardial electromechanical mapping (EMM) was performed to evaluat the extent of myocardial ischemia. Even though there was significant intersegmental threshold variability at baseline and after infarction, the electromechanical activity thresholds, for infarct detection, could be established. In this study, the capacity of separating myocardium with evolving necrosis from viable

myocardium was so promising that would result in potential clinical uses (Odenstedt et al., 2003).

### **2.2.2 Advanced imaging systems**

182 Advances in Electrocardiograms – Clinical Applications

and positron emission tomography (PET) have been devised to support diagnosis in the patients who show ambiguous symptoms and ECG findings. Major advantage of these latter methods is that infarct size can be non-invasively and repeatedly measured in vivo. Major limitation of the non-invasive imaging techniques in humans is the lack of quantification of

In preclinical small animal models, the use of above mentioned novel non-invasive imaging techniques is also limited due to several technical problems (e.g. lack of area at risk determination, insufficient temporal and spatial resolution, irradiation, etc.) and their high cost. Therefore, direct post-sacrifice techniques such as e.g. the triphenyltetrazolium (TTC) staining as well as nitroblue tetrazolium (NBT) staining, is still the most widely used, low-

Identifying patients at risk of ST segment elevations (most frequently observed in heart attack) by use of body surface electrical measures is controversial. To probe the principal variations during myocardial ischemia, scientists have pursued to measure the electrophysiological changes of heart by modified ECG (s). In our lab, to circumvent some of these problems and to further our knowledge of the cardiovascular disorders we have undertook a series of investigations that consisted of designing novel modified ECG since 1970s. Two different modified ECGs were successfully developed and characterized including: epicardial electrograms (Li, 1978; Liu et al., 2002) and body surface ECG (BS-

Other lab worldwide besides our lab should also be reviewed here as to design very sensitive, multiple-electrode mapping technology. Mimicking the body 12-lead ECG, scientists from University of Oxford recorded ventricular epicardial electrograms from 5 anesthetized pigs with a 127-electrode sock and simultaneously investigated torso ECG using a specifically designed vest with 256 ECG electrodes. One of breakthroughs they have made in this study is that with chest reclosed, simultaneous arrays of epicardial electrograms and torso ECGs can be recorded during LAD occlusion and reperfusion (Nash

The earlier identification of coronary occlusion with ST-segment elevation, the better outcomes patients undergoing heart attack may have due to timing of revascularization. Using an implantable, high-fidelity, intracardiac electrogram monitoring system with longrange telemetry in porcine subjected to acute coronary occlusion precipitated by stent thrombosis, researchers from Michigan State University real-timely detected acute STsegment elevation and analyzed its correlations with thrombotic coronary occlusion. High sensitivity and specificity (100% and 100%, respectively) in such a system make it possible to advance the time frame of reperfusion therapy and potentially prevent, rather than interrupt, acute myocardial infarction in patients with coronary artery disease (Fischell et

In a porcine acute infarct and reperfusion model induced by balloon occlusion of the left anterior descending coronary artery for 45 minutes, endocardial electromechanical mapping (EMM) was performed to evaluat the extent of myocardial ischemia. Even though there was significant intersegmental threshold variability at baseline and after infarction, the electromechanical activity thresholds, for infarct detection, could be established. In this study, the capacity of separating myocardium with evolving necrosis from viable

ECG) (Liu et al., 2007b). See the section "EECG and BS-ECG" below for the details.

the risk zone.

et al., 2003).

al., 2006).

cost method to assess infarct size in animal models.

**2.2.1 Modified ECG and multiple-electrode mapping** 

Conventional methods to quantify infarct size after myocardial infarction in mice are not ideal. Cardiologists therefore implemented a fast, high-resolution method to directly measure infarct size in vivo using three-dimensional (3D) late gadolinium enhancement MRI (3D-LGE). They had validated an improved 3D MRI method to noninvasively quantify infarct size in mice with unsurpassed spatial resolution and tissue contrast. This method is particularly suited to studies requiring early quantification of initial infarct size, for example, to measure damage before intervention with stem cells (Bohl et al., 2009). Another study group combined the small-animal PET and MRI data to acquire quantitative in vivo insights into cardiac pathophysiology, and sought to determine the feasibility of PET and MRI for the quantification of ischemic injury in the rat model. Successful integrating information from small-animal PET and clinical MRI instrumentation allows for the quantitative assessment of cardiac function and infarct size in the rat model. The MRI measurements of scar can be complemented by metabolic imaging, addressing the extent and severity of ischemic injury and providing endpoints for therapeutic interventions (Higuchi et al., 2007).

### **2.2.3 Histological analysis of infarct size**

In preclinical studies, still the postmortem histological analysis is considered to be the gold standard for measuring infarct size. However, there are a number of disadvantages of this technique, making a reliable non-invasive alternative highly desirable. First, histological methods leave no residual tissue for further analysis. Second, visual interpretation and planimetry of heart sections may be subjective in cases with poor viable/non-viable contrast due to hemoglobin residues within the necrotic regions or with TTC-induced geometric distortion of the sample. Finally, animals must be euthanized to measure injury, meaning longitudinal studies require separate groups of animals for each time point (Bohl et al., 2009).

Conventional TTC staining allows the quantification of infarct size much sooner than standard histological techniques, and has been shown to be equally sensitive and specific. Therefore, the direct post-sacrifice techniques such as e.g. the TTC staining, is still the most widely used, low-cost and high throughput method to assess infarct size in animal models (Skrzypiec-Spring et al., 2007).

### **2.2.4 Other novel methodologies**

Many new methods were employed in cardiovascular functional measurement after ischemia or infarction. By using a three-axis accelerometer, researchers developed a novel technique for continuous real-time assessment of myocardial ischaemia in 14 anaesthetized open-chest pigs. Two accelerometers sutured on the left ventricle (LV) surface in the perfusion areas of the left anterior descending (LAD) and circumflex (CX) arteries, measured acceleration in the longitudinal, circumferential, and radial directions, and the corresponding epicardial velocities were calculated. The accelerometer had the ability to distinguish ischaemia from interventions altering global myocardial function, by which the myocardial ischaemia can be monitored in a continuous real-time mode (Halvorsen et al.,

Novel Porcine Models of Myocardial Ischemia/Infarction

\*\*P<0.01 vs Control.

vs Control.

– Technical Progress, Modified Electrocardiograms Validating, and Future Application 185

Fig. 4. The degree of myocardial ischemia (Σ-ST) determined by the epicardiogram mapping. We proposed that the percent of ischemic condition (before treatment) was 100%,Σ-ST % was obtained by the comparison with ischmemic condition. DIL, diltiazem, a classical calcium channel blocker. Pre-Tre: pre-treatment, Post-Tre: post-treatment. \*P<0.05,

Fig. 5. The scope of myocardial ischemia (N-ST) determined by the epicardiogram mapping. We proposed that the percent of ischemic condition (before treatment) was 100%, N-ST % was obtained by the comparison with ischmemic condition. DIL, diltiazem, a classical calcium channel blocker. Pre-Tre: pre-treatment, Post-Tre: post-treatment. \*P<0.05, \*\*P<0.01

2009). With the support of interventional techniques, intravascular ultrasound probe or intravascular Doppler velocimetry can be introduced into coronary artery and epicardial cross-sectional area and coronary flow velocity can be detected (Hutchison et al., 2005). Some study indicated that endocardial voltage amplitudes were closely related with sustaining myocardial ischemia or infarction. LV endocardial unipolar voltage (UpV) mapping was performed using the Biosense 3D navigation system 4 weeks after ameroid constrictor placement around the left circumflex coronary artery. Meanwhile, echocardiography was used to assess regional changes in myocardial wall thickening (MT) and fluorescent microspheres (4 x 10/injection) were used to quantify rest regional myocardial blood flow (MBF) in ischemic (left circumflex) and remote non-ischemic (left anterior descending) regions (Fuchs and Kornowski, 2005).

### **2.3 EECG and BS-ECG**

Once a new myocardial ischemic/infarction model was to be established, validating it should be placed on the first priority. Without any doubt, traditional electrocardiogram is of first choice. Since 1970 last century, we have made much effort in developing practically modified electrocardiograms. The first one is so-called 30-point epicardial electrocardiogram (EECG), which was introduced in exposed heart. The multi-point epicardial electrodes were sutured on the ventricular surface and physiological monitor was connected to record the electrocardiogram (EECG). (Fig. 3. ) ST segment elevated more than 2 mV was regarded as ischemic criterion to calculate the degree of myocardial ischemia (total mV of ST segment elevating, Σ-ST) and myocardial ischemic scope (total point number of ST segment elevating, N-ST) (Liu et al., 2007a; Liu et al., 2002; Yu et al., 2007b). (Fig. 4. and 5.)

Fig. 3. Schematic components of EECG mapping system (A) and representative EECG in swine (B).

Body surface electrocardiogram (BS-ECG) is another well designed method, especially for closed chest and repeated measurement during a long period. The 30 point electrode was placed on the chest surface in the cardiac projective area and then connected to recorder

2009). With the support of interventional techniques, intravascular ultrasound probe or intravascular Doppler velocimetry can be introduced into coronary artery and epicardial cross-sectional area and coronary flow velocity can be detected (Hutchison et al., 2005). Some study indicated that endocardial voltage amplitudes were closely related with sustaining myocardial ischemia or infarction. LV endocardial unipolar voltage (UpV) mapping was performed using the Biosense 3D navigation system 4 weeks after ameroid constrictor placement around the left circumflex coronary artery. Meanwhile, echocardiography was used to assess regional changes in myocardial wall thickening (MT) and fluorescent microspheres (4 x 10/injection) were used to quantify rest regional myocardial blood flow (MBF) in ischemic (left circumflex) and remote non-ischemic (left

Once a new myocardial ischemic/infarction model was to be established, validating it should be placed on the first priority. Without any doubt, traditional electrocardiogram is of first choice. Since 1970 last century, we have made much effort in developing practically modified electrocardiograms. The first one is so-called 30-point epicardial electrocardiogram (EECG), which was introduced in exposed heart. The multi-point epicardial electrodes were sutured on the ventricular surface and physiological monitor was connected to record the electrocardiogram (EECG). (Fig. 3. ) ST segment elevated more than 2 mV was regarded as ischemic criterion to calculate the degree of myocardial ischemia (total mV of ST segment elevating, Σ-ST) and myocardial ischemic scope (total point number of ST segment elevating, N-ST) (Liu et al., 2007a; Liu et al., 2002; Yu et al.,

Fig. 3. Schematic components of EECG mapping system (A) and representative EECG in

Body surface electrocardiogram (BS-ECG) is another well designed method, especially for closed chest and repeated measurement during a long period. The 30 point electrode was placed on the chest surface in the cardiac projective area and then connected to recorder

anterior descending) regions (Fuchs and Kornowski, 2005).

**2.3 EECG and BS-ECG** 

2007b). (Fig. 4. and 5.)

swine (B).

Fig. 4. The degree of myocardial ischemia (Σ-ST) determined by the epicardiogram mapping. We proposed that the percent of ischemic condition (before treatment) was 100%,Σ-ST % was obtained by the comparison with ischmemic condition. DIL, diltiazem, a classical calcium channel blocker. Pre-Tre: pre-treatment, Post-Tre: post-treatment. \*P<0.05, \*\*P<0.01 vs Control.

Fig. 5. The scope of myocardial ischemia (N-ST) determined by the epicardiogram mapping. We proposed that the percent of ischemic condition (before treatment) was 100%, N-ST % was obtained by the comparison with ischmemic condition. DIL, diltiazem, a classical calcium channel blocker. Pre-Tre: pre-treatment, Post-Tre: post-treatment. \*P<0.05, \*\*P<0.01 vs Control.

Novel Porcine Models of Myocardial Ischemia/Infarction

disease and the key molecular processes involved.

*Circ Physiol,* Vol. 296, No. 4, pp.H1200-1208

*Biol,* Vol. 69, No. 2-3, pp.515-538

*Res,* Vol. 116, No. 5, pp.431-442

procedure. *J Appl Physiol,* Vol. 27, No. 3, pp.419-421

*Am Coll Cardiol,* Vol. 48, No. 11, pp.2306-2314

*Coron Artery Dis,* Vol. 16, No. 3, pp.163-167

*Science,* Vol. 325, No. 5939, pp.433

**4. Acknowledgment** 

No. 1, pp.144-152

pp.700-704

**5. References** 

– Technical Progress, Modified Electrocardiograms Validating, and Future Application 187

heart diseases, one of the primary impediments to successful drug R&D is the frequent failure of successfully translating positive results obtained in animal models to human disease. To a large degree, this discrepancy is secondary to the substantial biological differences between species. We hope the information contained in this chapter will be helpful for researchers to consider prospectively their research plan regarding with myocardial ischemia/infarction animal study, which will someday bridge the gap between

We thank Drs. Zhen Yu and Lei Li for their hard work with animal study. This work was supported by National Natural Science Foundation of China (30830118), National Key

Baks, T., et al. (2006). Multislice computed tomography and magnetic resonance imaging for

Bohl, S., et al. (2009). Advanced methods for quantification of infarct size in mice using

Bolukoglu, H., et al. (1992). An animal model of chronic coronary stenosis resulting in hibernating myocardium. *Am J Physiol,* Vol. 263, No. 1 Pt 2, pp.H20-29 Buecker, A., et al. (2005). A feasibility study of contrast enhancement of acute myocardial

Ch'en, F. F., et al. (1998). Modelling myocardial ischaemia and reperfusion. *Prog Biophys Mol* 

Dogne, J. M., et al. (2005). Characterization of an original model of myocardial infarction

Elzinga, W. E. (1969). Ameroid constrictor: uniform closure rates and a calibration

Fuchs, S., Kornowski, R. (2005). Correlation between endocardial voltage mapping and

Geurts, A. M., et al. (2009). Knockout rats via embryo microinjection of zinc-finger nucleases.

Falk, E., et al. (1995). Coronary plaque disruption. *Circulation,* Vol. 92, No. 3, pp.657-671 Fischell, T. A., et al. (2006). Real-time detection and alerting for acute ST-segment elevation

the assessment of reperfused acute myocardial infarction. *J Am Coll Cardiol,* Vol. 48,

three-dimensional high-field late gadolinium enhancement MRI. *Am J Physiol Heart* 

infarction in multislice computed tomography: comparison with magnetic resonance imaging and gross morphology in pigs. *Invest Radiol,* Vol. 40, No. 11,

provoked by coronary artery thrombosis induced by ferric chloride in pig. *Thromb* 

myocardial ischemia using an implantable, high-fidelity, intracardiac electrogram monitoring system with long-range telemetry in an ambulatory porcine model. *J* 

myocardial perfusion: implications for the assessment of myocardial ischemia.

Scientific Program (2006BAI08B01-06, 2009ZX09303-003 and 2009ZX09502-017).

(Fig. 6.) (Liu et al., 2007b). Differently from EECG criteria due to relatively lower voltage on the skin, in BS-ECG, ST segment elevated to more than 0.8 mV was regarded as criterion to calculate the degree of myocardial ischemia (total mV of ST segment elevating, Σ-ST) and myocardial ischemic scope (total point number of ST segment elevating, N-ST). At the end of the protocol, the animals sacrificed and heart taken out. Under the coronary occlusion spot, the ventricle was transversely divided into 5 pieces of equal thickness. The pieces were then infiltrated with N-BT staining solution at 25 °C for 15 min. Both the ischemic area (N-BT non-stained area, white to grey) and non-ischemic area (N-BT-stained area, dark brown) were determined by histological methods.

Fig. 6. BS-ECG machine (A), myocardium slices (B) (dark brown: live muscle; white: dead muscle), and BS-ECG (C).

### **3. Conclusion**

The last few decades have seen significant advancement in the therapy of ischemic heart diseases. This is a direct outcome of the increasing knowledge of the molecular mechanisms involved during an ischemic insult of the myocardium. Another important factor is the development of animal model study, both academically and practically. The miniature swine was now widely used as research subject because of its anatomic similarity in coronary circulation to human beings (Hughes et al., 2003). Chest-closed improvement has been one of breakthroughs since birth of interventional technology. The newly introduced intervention technique avoided thoracotomy and disturbance to the environment of thoracic cavity. Moreover, cardiovascular variations could be chronically, continually and systematically observed in these models.

Despite tremendous advances in cardiovascular research and clinical therapy, ischemic heart disease remains the leading cause of serious morbidity and mortality in western society and is growing in developing countries. For researchers involving in basic science of heart diseases, one of the primary impediments to successful drug R&D is the frequent failure of successfully translating positive results obtained in animal models to human disease. To a large degree, this discrepancy is secondary to the substantial biological differences between species. We hope the information contained in this chapter will be helpful for researchers to consider prospectively their research plan regarding with myocardial ischemia/infarction animal study, which will someday bridge the gap between disease and the key molecular processes involved.

### **4. Acknowledgment**

We thank Drs. Zhen Yu and Lei Li for their hard work with animal study. This work was supported by National Natural Science Foundation of China (30830118), National Key Scientific Program (2006BAI08B01-06, 2009ZX09303-003 and 2009ZX09502-017).

### **5. References**

186 Advances in Electrocardiograms – Clinical Applications

(Fig. 6.) (Liu et al., 2007b). Differently from EECG criteria due to relatively lower voltage on the skin, in BS-ECG, ST segment elevated to more than 0.8 mV was regarded as criterion to calculate the degree of myocardial ischemia (total mV of ST segment elevating, Σ-ST) and myocardial ischemic scope (total point number of ST segment elevating, N-ST). At the end of the protocol, the animals sacrificed and heart taken out. Under the coronary occlusion spot, the ventricle was transversely divided into 5 pieces of equal thickness. The pieces were then infiltrated with N-BT staining solution at 25 °C for 15 min. Both the ischemic area (N-BT non-stained area, white to grey) and non-ischemic area (N-BT-stained area, dark brown)

Fig. 6. BS-ECG machine (A), myocardium slices (B) (dark brown: live muscle; white: dead

The last few decades have seen significant advancement in the therapy of ischemic heart diseases. This is a direct outcome of the increasing knowledge of the molecular mechanisms involved during an ischemic insult of the myocardium. Another important factor is the development of animal model study, both academically and practically. The miniature swine was now widely used as research subject because of its anatomic similarity in coronary circulation to human beings (Hughes et al., 2003). Chest-closed improvement has been one of breakthroughs since birth of interventional technology. The newly introduced intervention technique avoided thoracotomy and disturbance to the environment of thoracic cavity. Moreover, cardiovascular variations could be chronically, continually and

Despite tremendous advances in cardiovascular research and clinical therapy, ischemic heart disease remains the leading cause of serious morbidity and mortality in western society and is growing in developing countries. For researchers involving in basic science of

were determined by histological methods.

muscle), and BS-ECG (C).

systematically observed in these models.

**3. Conclusion** 


Novel Porcine Models of Myocardial Ischemia/Infarction

515

No. 1, pp.52-57

2, pp.247-256

pp.395-414

126

pp.84-93

Vol. 57, No. 3, pp.614-619

*Cardiol,* Vol. 15, No. 9, pp.497-501

*Thromb Res,* Vol. 17, No. 6, pp.841-853

*Surg,* Vol. 123, No. 6, pp.1132-1140

*Cycle,* Vol. 5, No. 11, pp.1175-1177

*Cardiovasc Imaging,* Vol. 2, No. 7, pp.825-831

– Technical Progress, Modified Electrocardiograms Validating, and Future Application 189

Liu, J. X., et al. (2002). Effects of recombinant staphylokinase on coronary thrombosis in

Liu, J. X., et al. (2007b). Cardioprotective effects of diltiazem reevaluated by a novel

Ludemann, L., et al. (2007). Usage of the T1 effect of an iron oxide contrast agent in an

Mahnken, A. H., et al. (2005). Assessment of myocardial viability in reperfused acute

Nash, M. P., et al. (2003). Imaging electrocardiographic dispersion of depolarization and

Naslund, U., et al. (1992). A closed-chest myocardial occlusion-reperfusion model in the pig: techniques, morbidity and mortality. *Eur Heart J,* Vol. 13, No. 9, pp.1282-1289 Odenstedt, J., et al. (2003). Endocardial electromechanical mapping in a porcine acute infarct

Park, W. H., et al. (1985). Morphological changes in the coronary circulation following experimental myocardial ischemia in swine. *Artery,* Vol. 12, No. 5, pp.286-300 Rodriguez, B., et al. (2006). Modeling cardiac ischemia. *Ann N Y Acad Sci,* Vol. 1080, No.

Romson, J. L., et al. (1980). Electrical induction of coronary artery thrombosis in the

Seta, F., et al. (2009). Renal and cardiovascular characterization of COX-2 knockdown mice. *Am J Physiol Regul Integr Comp Physiol,* Vol. 296, No. 6, pp.R1751-1760 Skrzypiec-Spring, M., et al. (2007). Isolated heart perfusion according to Langendorff---still

Takahashi, M., et al. (2005). Effects of angiotensin I-converting enzyme inhibitor and

Tomita, S., et al. (2002). Improved heart function with myogenesis and angiogenesis after

Wright, J., et al. (2009). Quantification of myocardial area at risk with T2-weighted CMR:

Yan, L., et al. (2006). Autophagy: a novel protective mechanism in chronic ischemia. *Cell* 

mapping. *Circulation,* Vol. 107, No. 17, pp.2257-2263

Chinese experimental miniature swine. *Acta Pharmacol Sin,* Vol. 23, No. 6, pp.509-

myocardial ischemic model in Chinese miniature swine. *Acta Pharmacol Sin,* Vol. 28,

animal model to quantify myocardial blood flow by MRI. *Eur J Radiol,* Vol. 62, No.

myocardial infarction using 16-slice computed tomography in comparison to magnetic resonance imaging. *J Am Coll Cardiol,* Vol. 45, No. 12, pp.2042-2047 Moreno, C., et al. (2011). Creation and characterization of a Renin knockout rat. *Hypertension,*

repolarization during ischemia: simultaneous body surface and epicardial

and reperfusion model evaluating the extent of myocardial ischemia. *J Invasive* 

ambulatory canine: a model for in vivo evaluation of anti-thrombotic agents.

viable in the new millennium. *J Pharmacol Toxicol Methods,* Vol. 55, No. 2, pp.113-

angiotensin II type 1 receptor blocker on the right ventricular sarcoglycans and dystrophin after left coronary artery ligation. *Eur J Pharmacol,* Vol. 522, No. 1-3,

autologous porcine bone marrow stromal cell transplantation. *J Thorac Cardiovasc* 

comparison with contrast-enhanced CMR and coronary angiography. *JACC* 


Guth, B. D., et al. (1990). Myocardial lactate release during ischemia in swine. Relation to

Halvorsen, P. S., et al. (2009). Detection of myocardial ischaemia by epicardial

Hennan, J. K., et al. (2001). Effects of selective cyclooxygenase-2 inhibition on vascular

Higuchi, T., et al. (2007). Characterization of normal and infarcted rat myocardium using a

Huang, Y., et al. (2004). Remodeling of the chronic severely failing ischemic sheep heart after

responses. *Am J Physiol Heart Circ Physiol,* Vol. 286, No. 6, pp.H2141-2150 Hughes, G. C., et al. (2003). Translational physiology: porcine models of human coronary

Hutchison, S. J., et al. (2005). Dehydroepiandrosterone sulfate induces acute vasodilation of

Inou, T., et al. (1980). A newly developed X-ray transparent ameroid constrictor for study on

Koning, M. M., et al. (1993). Intracoronary trimetazidine does not improve recovery of

Kraitchman, D. L., et al. (2000). A minimally invasive method for creating coronary stenosis

Krombach, G. A., et al. (2005). Minimally invasive close-chest method for creating

Laham, R. J., et al. (2000). Intrapericardial delivery of fibroblast growth factor-2 induces

Leadley, R. J., Jr., et al. (2000). Contribution of in vivo models of thrombosis to the discovery

Li, L. (1978). The improvement of epicardial electrogram methods. *New Med Phar J,* Vol. 1,

Li, X. Z., et al. (2009). Establishment of coronary heart disease model of coronary

Lichtig, C., et al. (1975). Basic fuchsin picric acid method to detect acute myocardial ischemia. An experimental study in swine. *Arch Pathol,* Vol. 99, No. 3, pp.158-161 Liu, J. X., et al. (2007a). Effects of Corocalm (shuguan capsule) on acute myocardial ischemia

in anesthetized dogs. *Chin J Integr Med,* Vol. 13, No. 3, pp.206-210

responses and thrombosis in canine coronary arteries. *Circulation,* Vol. 104, No. 7,

combination of small-animal PET and clinical MRI. *J Nucl Med,* Vol. 48, No. 2,

coronary microembolization: functional, energetic, structural, and cellular

artery disease: implications for preclinical trials of therapeutic angiogenesis. *J Appl* 

porcine coronary arteries in vitro and in vivo. *J Cardiovasc Pharmacol,* Vol. 46, No. 3,

progression of gradual coronary stenosis. *Basic Res Cardiol,* Vol. 75, No. 4, pp.537-

regional function in a porcine model of repeated ischemia. *Cardiovasc Drugs Ther,*

in a swine model for MRI and SPECT imaging. *Invest Radiol,* Vol. 35, No. 7, pp.445-

reperfused or occlusive myocardial infarction in swine. *Invest Radiol,* Vol. 40, No. 1,

neovascularization in a porcine model of chronic myocardial ischemia. *J Pharmacol* 

and development of novel antithrombotic agents. *J Pharmacol Toxicol Methods,* Vol.

atherosclerosis in mini-swines. *Zhongguo Zhong Xi Yi Jie He Za Zhi,* Vol. 29, No. 3,

regional blood flow. *Circulation,* Vol. 81, No. 6, pp.1948-1958

pp.820-825

pp.288-294

pp.325-332

Vol. 7, No. 5, pp.801-807

43, No. 2, pp.101-116

No. 11, pp.52-53

pp.228-232

543

451

pp.14-18

*Physiol,* Vol. 94, No. 5, pp.1689-1701

*Exp Ther,* Vol. 292, No. 2, pp.795-802

accelerometers in the pig. *Br J Anaesth,* Vol. 102, No. 1, pp.29-37


**Part 3** 

**Autonomic Dysregulation** 

