**Abstract**

Development of right ventricular (RV) failure in patients after ST-segment elevation myocardial infarction (STEMI) is common. However, a systematic analysis of chamber-specific changes in the expression of genes linked to cardiac function, apoptosis, fibrosis, receptor responsiveness, and inflammation is lacking. Postischemic remodeling was analyzed in rats that received STEMI in the closed chest mode. Rats were sacrificed at day 1, 3, 7, and 120 after surgery. The mRNA expression of genes was quantified by a real-time RT-PCR. Echocardiography was performed after 120 days. Organ weights and systemic blood pressure were determined in addition. Rats developed left and RV dysfunction within 7 days after ischemia/reperfusion and this lasted until the end of the experiments. However, adaptation to ischemia/reperfusion differed significantly between both ventricles. In the LV, a high expression of MMP12, a neutrophile-specific elastase, indicated a significant inflammatory responsiveness that did not occur in RV. A number of differentially regulated genes in the RV exceeded that of the LV at day 3. Postinfarction RV failure is common in rats with ischemia/reperfusion of the left arterial descending aorta. It is associated with severe RV remodeling that occurred delayed to that of the LV. Changes in RV are independent of the initial inflammation.

**Keywords:** myocardial infarction, cardiac remodeling, right heart failure, inflammation, reperfusion injury

## **1. Introduction**

Right ventricular (RV) failure is common in patients with acute ST-segment elevated myocardial infarction (STEMI) and animal models of remodeling postmyocardial infarction [1–3]. The underlying reason for biventricular failure due to myocardial infarction and/or transient ischemic events is not clear but may be a consequence of hemodynamic changes during infarction and ischemic events in the RV as well. Nevertheless, RV failure is a severe complication during the subsequent postinfarct period and a limitation of further prognosis [4]. Therefore, it is important to understand the molecular adaptation of the RV in response to LV infarction.

Molecular and cellular mechanisms involved in cardiac remodeling after myocardial infarction are triggered by transcriptional regulation of genes linked to apoptosis, fibrosis, inflammation, calcium handling, and receptor responsiveness. Some of these adaptive mechanisms occur early after reperfusion and activation of the transcription factor AP-1 have been identified as main factors [5]. However, steady-state mRNA levels depend also from RNA degradation leading to a decrease in the expression of some genes. Thus, steady-state mRNA expression of genes

**6**

*Visions of Cardiomyocyte - Fundamental Concepts of Heart Life and Disease*

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**References**

involved in cardiac remodeling can be increased or decreased. Real-time RT-PCR allows quantification of such molecular and cellular adaptation in tissues and can be used to characterize such changes in a time- and organ-specific manner. This study was aimed to identify differentially regulated cardiac genes in the LV and RV that are involved in cardiac remodeling during postmyocardial infarction.

The left anterior descending coronary artery was occluded in rats for 45 min and subsequently reopened. Success of occlusion and reperfusion was monitored by ECG recordings. Rats were sacrificed after 1, 3, 7, and 120 days and the left and right ventricles were removed and analyzed thereafter. The hemodynamic consequences were recorded by echocardiography.

### **2. Material and methods**

#### **2.1 Animal models and animal handling**

The investigation conforms to the directive 2010/63/EU of the European Parliament. Use of animals was registered at the Justus-Liebig-University (registration-no.: 417-M). The experimental protocols were approved by the ethics committee for animal experimentation of the local authorities in Giessen, Germany and Szeged, Hungary.

Myocardial infarction and reperfusion was performed in the closed-chest model. To achieve this, rats were anesthetized by inhalation of isoflurane (induction: 5%, maintenance: 2–3%), intubated, and placed on a respirator during surgery to maintain ventilation. Before surgery, 0.03 mg/kg nalbuphine (Nalbuphin Orpha, AOP Orpha Pharmaceuticals, Vienna, Austria) was injected (i.p.). The adequacy of anesthesia was monitored by electrocardiography and pulse rate. A suture was placed around the left anterior descending coronary artery (LAD) and remained subcutaneously [6]. Two hours after the wound closing, 0.03 mg/kg nalbuphine was repeated to alleviate postoperative pain. Seven days later, rats were anesthetized as before and the suture was mobilized and the LAD was occluded for 30 min. The occlusion was monitored by electrocardiography (ST elevation; see **Figure 1**). Thereafter, the occluder was opened again and the suture was cut and the skin was closed in one layer. Sham rats received the same protocol but the occluder was not mobilized after 7 days. Please note that this study is a second end-point analysis of tissue material also used before to characterize the role of arginase in ischemia/ reperfusion injury [7].

#### **2.2** *Ex vivo* **analysis of cardiac function**

In order to analyze the cardiac function *ex vivo*, rats were anesthetized again by isoflurane and killed by cervical dislocation. Thereafter, hearts were rapidly excised and the aorta was cannulated for retrograde perfusion with a 16-gauge needle connected to a Langendorff perfusion system. Left ventricular function was determined by insertion of a water-filled balloon into the left ventricle as described before [8]. Hearts were paced during measurements.

#### **2.3 In vivo analysis of cardiac function**

Transthoracic echocardiography was performed as described previously [9] under isoflurane anesthesia (1.5%) at 120 days after ischemia/reperfusion. Briefly, two-dimensional and M-mode echocardiographic examinations were performed in accordance with the criteria of the American Society of Echocardiography with

**9**

**2.4 qRT-PCR**

**Figure 1.**

*Right Heart Adaptation to Left Ventricular STEMI in Rats*

a Vivid 7 dimension ultrasound system (General Electric Medical Systems) using a phased array 5.5–12 MHz transducer (10S probe). Data of three consecutive heart cycles were analyzed (EchoPac Dimension software; General Electric Medical Systems) by an experienced investigator in a blinded manner. The mean values of three measurements were calculated and used for statistical evaluation. Functional parameters including left ventricular ejection fraction (EF) and fractional shorten-

*ECG recording during ischemia (A) and after reperfusion (B) indicating ST elevation in these rats* 

After removing the hearts from the Langendorff apparatus, the ventricular tissue was carefully isolated and quickly frozen into fluid nitrogen. Tissue samples were prepared to analyze the steady-state mRNA levels of proteins of interest according to the previously described method [8]. Briefly, total RNA was isolated from the ventricles using peqGoldTriFast (peqlab, Biotechnology GmbH, Germany) according to the manufacturer's protocol. To remove genomic DNA contamination, isolated RNA samples were treated with 1 U DNase per mg RNA (Invitrogen, Karlsruhe, Germany) for 15 min at 37°C. One microgram of total RNA was used in 10 μl reaction to synthesize cDNA using superscript RNaseH

ing (FS) were calculated on four-chamber view images.

*(red arrow). (C) TTC staining visualizing the location of the infarct.*

*DOI: http://dx.doi.org/10.5772/intechopen.84868*

*Right Heart Adaptation to Left Ventricular STEMI in Rats DOI: http://dx.doi.org/10.5772/intechopen.84868*

#### **Figure 1.**

*Visions of Cardiomyocyte - Fundamental Concepts of Heart Life and Disease*

are involved in cardiac remodeling during postmyocardial infarction.

were recorded by echocardiography.

**2.1 Animal models and animal handling**

**2. Material and methods**

Szeged, Hungary.

reperfusion injury [7].

**2.2** *Ex vivo* **analysis of cardiac function**

**2.3 In vivo analysis of cardiac function**

before [8]. Hearts were paced during measurements.

involved in cardiac remodeling can be increased or decreased. Real-time RT-PCR allows quantification of such molecular and cellular adaptation in tissues and can be used to characterize such changes in a time- and organ-specific manner. This study was aimed to identify differentially regulated cardiac genes in the LV and RV that

The investigation conforms to the directive 2010/63/EU of the European Parliament. Use of animals was registered at the Justus-Liebig-University (registration-no.: 417-M). The experimental protocols were approved by the ethics committee for animal experimentation of the local authorities in Giessen, Germany and

To achieve this, rats were anesthetized by inhalation of isoflurane (induction: 5%, maintenance: 2–3%), intubated, and placed on a respirator during surgery to maintain ventilation. Before surgery, 0.03 mg/kg nalbuphine (Nalbuphin Orpha, AOP Orpha Pharmaceuticals, Vienna, Austria) was injected (i.p.). The adequacy of anesthesia was monitored by electrocardiography and pulse rate. A suture was placed around the left anterior descending coronary artery (LAD) and remained subcutaneously [6]. Two hours after the wound closing, 0.03 mg/kg nalbuphine was repeated to alleviate postoperative pain. Seven days later, rats were anesthetized as before and the suture was mobilized and the LAD was occluded for 30 min. The occlusion was monitored by electrocardiography (ST elevation; see **Figure 1**). Thereafter, the occluder was opened again and the suture was cut and the skin was closed in one layer. Sham rats received the same protocol but the occluder was not mobilized after 7 days. Please note that this study is a second end-point analysis of tissue material also used before to characterize the role of arginase in ischemia/

In order to analyze the cardiac function *ex vivo*, rats were anesthetized again by isoflurane and killed by cervical dislocation. Thereafter, hearts were rapidly excised and the aorta was cannulated for retrograde perfusion with a 16-gauge needle connected to a Langendorff perfusion system. Left ventricular function was determined by insertion of a water-filled balloon into the left ventricle as described

Transthoracic echocardiography was performed as described previously [9] under isoflurane anesthesia (1.5%) at 120 days after ischemia/reperfusion. Briefly, two-dimensional and M-mode echocardiographic examinations were performed in accordance with the criteria of the American Society of Echocardiography with

Myocardial infarction and reperfusion was performed in the closed-chest model.

The left anterior descending coronary artery was occluded in rats for 45 min and subsequently reopened. Success of occlusion and reperfusion was monitored by ECG recordings. Rats were sacrificed after 1, 3, 7, and 120 days and the left and right ventricles were removed and analyzed thereafter. The hemodynamic consequences

**8**

*ECG recording during ischemia (A) and after reperfusion (B) indicating ST elevation in these rats (red arrow). (C) TTC staining visualizing the location of the infarct.*

a Vivid 7 dimension ultrasound system (General Electric Medical Systems) using a phased array 5.5–12 MHz transducer (10S probe). Data of three consecutive heart cycles were analyzed (EchoPac Dimension software; General Electric Medical Systems) by an experienced investigator in a blinded manner. The mean values of three measurements were calculated and used for statistical evaluation. Functional parameters including left ventricular ejection fraction (EF) and fractional shortening (FS) were calculated on four-chamber view images.

#### **2.4 qRT-PCR**

After removing the hearts from the Langendorff apparatus, the ventricular tissue was carefully isolated and quickly frozen into fluid nitrogen. Tissue samples were prepared to analyze the steady-state mRNA levels of proteins of interest according to the previously described method [8]. Briefly, total RNA was isolated from the ventricles using peqGoldTriFast (peqlab, Biotechnology GmbH, Germany) according to the manufacturer's protocol. To remove genomic DNA contamination, isolated RNA samples were treated with 1 U DNase per mg RNA (Invitrogen, Karlsruhe, Germany) for 15 min at 37°C. One microgram of total RNA was used in 10 μl reaction to synthesize cDNA using superscript RNaseH


**11**

*Right Heart Adaptation to Left Ventricular STEMI in Rats*

reverse transcriptase (200 U/μg; Invitrogen) and oligo dTs (Roche, Mannheim, Germany) as primers. Reverse transcriptase reactions were performed for 60 min at 37°C. Real-time PCR was performed using the Icycler IQ detection system (Bio-Rad, Munich, Germany) in combination with IQ SYBR Green real-time supermix (Bio-Rad). A complete list of all primers used in this study is given in **Table 1**. Data are normalized to hypoxanthine phosphoribosyltransferase (HPRT) expression that was used as a house-keeping gene in this study. Preliminary experiments with β2 microglobulin, which was alternatively considered as house-keeping gene, revealed similar results but higher variability. The relative change in expression was quanti-

The results are expressed as means ± S.E.M or median with 25 and 75% quartiles as indicated in the legend to the figures. Statistical comparisons were performed by two-side T-Test or Mann-Whitney Test. Levene test was used to check the normal distribution of the samples. A p value of 0.05 was considered as statistical

**3.1 Weight of organs, left ventricular pressures, and biventricular functions** 

Body weight, LV weight, RV weight, lung wet weight, and kidney weight increased during the 4-months observation period in sham rats and those undergoing ischemia/reperfusion (**Table 2**). Mean increase in body weight at day 3 after the ischemic event was smaller in the surgery group (+2 g) versus the sham group (+13 g) indicating a small impact of the surgery on general behavior. Differences between both groups in ventricular weight occurred only for the RV at day 1 (**Table 1**). RV weights of rats in the experimental group normalized thereafter. No differences were obtained for lung and kidney weights (**Table 2**). Necrotic tissue was nearly

LV function was determined in vitro. Immediately after ischemia/reperfusion, a significant decline in cardiac function was observed but this was normalized thereafter (**Table 3**). Biventricular function was analyzed after 120 days via echocardiography. RV fractional area change (four-chamber view) and LV ejection fraction (longitudinal four-chamber view) were significantly lower in rats of the

In total, biventricular expression of 36 genes was analyzed by a real-time RT-PCR. These genes cover the following area of interest: cardiac hypertrophy (ANP, BNP, MHC-α), fibrosis (TGF-β1, biglycan, decorin, collagen-1, collagen-3, elastin, fibronectin, laminin, MMP9), intracellular calcium handling (SERCA2a,

phospholamban, NCX), apoptosis (bax, bcl-2), arginine metabolism (ODC, arginase-2, eNOS), receptor coupling (intermedin, RAMP-1, -2, -3), inflammation (iNOS, MMP12), cardiac metabolism (Nrf-1, PGC-1α), stem cell mobilization (SDF-1α, CXCR4, VEGF), cardiac transcription factors (GATA-4, Mef-2c, Nkx.2a), and endothelial markers (von Willebrand factor (vWF), VE-cadherin). **Figure 2** shows how many of these 36 genes were either

*DOI: http://dx.doi.org/10.5772/intechopen.84868*

fied by the ΔΔC method [10].

**2.5 Statistics**

significant.

**3. Results**

**analysis over time**

exclusively seen in LV (**Figure 1**).

experimental group compared to shams (**Table 4**).

**3.2 Biventricular gene regulation over time**
