**3.4. Electrocardiogram**

Serum levels of ALT and GGT were statistically significantly higher in ISACHC II and III groups (*p* < 0.05), while levels of AST, albumin, cholesterol, and total bilirubin were not signif‐ icantly differed among groups. Level of NT‐proBNP was also significantly higher in ISACHC II and III groups (*p* < 0.05), although the level was not significantly differed in ISACHC I group. There were no correlations between levels of AST, albumin, cholesterol, and total bilirubin to echocardiographic indices. The level of NT‐proBNP was correlated with most echocardiographic indices (LA/Ao, LVID/Ao, E‐peak, EDVI, *r* > 0.7) and ALT (*r* = 0.701) and GGT (*r* = 0.782). This study revealed the biochemical evidence of hepatic injures in dogs with

Poor tissue perfusion from CMVI causes pancreatitis in dogs, as indicated by serum pancre‐ atic lipase concentrations. One recent study has evaluated the prevalence of pancreatitis in 62 client‐owned dogs consisting of 40 dogs with different stages of heart failure from CMVI and 22 age‐matched healthy dogs [27]. Serum canine pancreatic lipase immunoreactivity (cPLI) concentrations were determined by quantitative cPLI test in healthy and CMVI groups in this study. Serum cPLI concentrations were 54.0 μg/L (IQR: 38.0–78.8 μg/L) in control, 55.0 μg/L (IQR: 38.3–88.8 μg/L) in ISACHC I, 115.0 μg/L (IQR: 45.0–179.0 μg/L) in ISACHC II, and 223.0 μg/L (IQR: 119.5–817.5 μg/L) in ISACHC III. Also, close correlation of serum cPLI concentration was found in the left atrial to aorta (LA/Ao) ratio (*r* = 0.597; *P* = 0.000) and the severity of heart failure (*r* = 0.530; *P* = 0.000). This study found that the CMVI is associated

Reduction in glomerular filtration rate (GFR) is a common complication in advanced stages of heart failure (HF). The convenient and precise assessment for GFR would be useful for early detection of renal impairment in HF dogs. One recent study has evaluated the reduction in GFR in advanced stages of HF from CMVI, using renal markers including serum cystatin C (Cys‐C) and symmetric dimethylarginine (SDMA) concentrations [28]. Forty‐three client‐ owned dogs consisting of 33 dogs with different stages of HF from CMVI and 10 age‐matched healthy dogs were enrolled in this study. Serum Cys‐C and SDMA concentrations along with other renal (i.e., urea nitrogen and creatinine) and echocardiographic markers were evalu‐ ated in healthy and CMVI dogs. Serum Cys‐C concentrations were 1.4 ± 0.4 mg/l in control, 2.1 ± 0.9 mg/l in ISACHC I, 2.9 ± 0.8 mg/l in ISACHC II, and 3.6 ± 0.6 mg/l in ISACHC III dogs, whereas serum SDMA concentrations were 8 ± 2 μg/dl in control, 14 ± 3 μg/dl in ISACHC I, 18 ± 6 μg/dl in ISACHC II, and 22 ± 7 μg/dl in ISACHC III dogs. There was close correlation of serum Cys‐C and SDMA concentrations with serum creatinine, urea nitrogen, and the sever‐ ity of HF. This study demonstrated that the GFR was decreased in dogs with CMVI having

In recent years, cardiac biomarkers have been developed that are differentiating cardiac and respiratory diseases to evaluate the progress of heart failure in dogs and cats. There are many cardiac biomarkers. The ideal biomarkers should reflect the therapeutic response, the patho‐ physiology of heart diseases, assist in the early diagnosis of CHF, and be applicable through‐ out the various phases of the syndrome from before the onset of its clinical manifestations

with pancreatic injury in congestive heart failure due to the CMVI [27].

advanced stage of CMVI [26].

98 Canine Medicine - Recent Topics and Advanced Research

earlier stages of HF [28].

**3.3. Cardiac biomarkers**

Major findings on the electrocardiography (ECG) in dogs with CMVI are P mitrale (wide P‐wave) and wide and tall QRS complexes indicating LA and LV dilation (**Figure 2B**) [49]. Tachycardia may be occurred either persistently or intermittently as the CMVI progresses [50, 51]. Although atrial fibrillation is often observed in large breed dogs with CMVI, it is rarely found in the small breed dogs. However, if dogs have early stage of CMVI, there will be no abnormal finding on the ECG [23]. The ECG signs indicating myocardial hypoxic damage (i.e., the ST‐slurring) can be seen in dogs with advanced stage of heart failure [52].

## **3.5. Thoracic radiography**

Thoracic radiography is the diagnostic test of choice in dogs with CMVI [23]. Enlargement of the LA/LV and pulmonary venous vasculatures is common findings on thoracic radiography (**Figure 3**) [23, 53, 54]. Other radiographic signs indicating left‐sided heart failure including the dorsal displacement of trachea, the compression and/or elevation of left main stem bron‐ chus, and the dividing view of left and right stem bronchus can be noticed as the disease progresses (**Figure 3**) [55]. In advanced stage, radiographic signs related to pulmonary edema (i.e., pulmonary venous engorgement, peribronchial pattern, air bronchograms) can be obvi‐ ous in most cases [23]. Also, when complications with pulmonary hypertension (PHT) are combined, radiographic signs indicating right‐sided heart failure (i.e., hepatomegaly, ascites) can be observed [23].

## **3.6. Echocardiography**

The transthoracic echocardiographic examination is noninvasive diagnostic method and can help to identify mitral valvular lesions and to determine the severity of MR. Echocardiography

**Figure 3.** Thoracic radiography in dogs with CMVI. Enlargement of the LA/LV and pulmonary venous vasculatures is common findings on thoracic radiography. Other radiographic signs indicating left‐sided heart failure including the dorsal displacement of trachea, the compression and/or elevation of left main stem bronchus, and the dividing view of left and right stem bronchus can be noticed as the disease progresses. (A) Ventrodorsal projection. (B) Right lateral projection.

can also assess its impact on cardiac remodeling, myocardial function, left ventricular filling pressures, and pulmonary arterial pressure [56–61].

Mitral valve lesion can be identified using two‐dimensional and M‐mode echocardiography. The mitral valve lesions associated with CMVI are small and smooth, creating a club‐shaped appearance to the leaflet tips during early stages of the disease, but may become large and irregular during disease progression (**Figure 4B**) [56, 62, 63]. Mitral valve prolapse, which is characterized by one or both leaflets bent back into the left atrial chamber during systole, occurs commonly in dogs with CMVI (**Figure 4A**) [7, 64]. In one recent study, the severity of mitral valve prolapse was significantly correlated with MR severity [64]. Anterior leaflet of mitral valve is more commonly affected than posterior leaflet in dogs [64]. Abnormal excur‐ sion [i.e., decreased ejection fraction (EF) slope] and thickening of anterior mitral leaflet can also be detected in M‐mode echocardiography (**Figure 5A**).

Ruptured chordae tendineae is also common echocardiographic finding in dogs with CMVI [64]. The mitral valve leaflet is seen pointing back into the left atrium (LA) during systole and bent back on itself with in the left ventricular outflow tract during diastole [65–67]. Chordae tendineae of anterior mitral valve leaflet is more commonly ruptured in dogs [68].

It is clinically important to evaluate severity of MR in dogs with CMVI. Color‐flow Doppler imaging (CDI) is widely used for detection and assessment of MR in dogs with CMVI (**Figures 4C**, **D** and **6B**). Maximal area of the regurgitant jet signals to the left atrium area (ARJ/LAA) ratio, which is the maximal ratio of the regurgitant jet area signal to left atrial area, is used in semi‐quantification of MR [56, 69, 70]. The ARJ/LAA ratio lesser than 20–30%

**Figure 4.** 2D and color Doppler echocardiography in dogs with CMVI. (A) Mitral valve prolapse, which is characterized by one or both leaflets bent back into the left atrial chamber during systole, occurs commonly in dogs with CMVI. The severity of mitral valve prolapse was significantly correlated with MR severity. (B) The mitral valve lesions associated with CMVI are large and irregular in advanced stage of CMVI. Anterior leaflet of mitral valve is more commonly affected than posterior leaflet in dogs. (C) Color‐flow Doppler imaging in 2D echocardiography revealed severe regurgitant jets from left ventricle to left atrium during systole and is widely used for detection and assessment of MR in dogs with CMVI. (D) The MR can be also detected in color M‐mode echocardiography on the LV short‐axis view. LV, left ventricle; CT, chordae tendineae; AMV, anterior mitral valve; PMV, posterior mitral valve; RV, right ventricle; RA, right atrium.

can also assess its impact on cardiac remodeling, myocardial function, left ventricular filling

**Figure 3.** Thoracic radiography in dogs with CMVI. Enlargement of the LA/LV and pulmonary venous vasculatures is common findings on thoracic radiography. Other radiographic signs indicating left‐sided heart failure including the dorsal displacement of trachea, the compression and/or elevation of left main stem bronchus, and the dividing view of left and right stem bronchus can be noticed as the disease progresses. (A) Ventrodorsal projection. (B) Right lateral

Mitral valve lesion can be identified using two‐dimensional and M‐mode echocardiography. The mitral valve lesions associated with CMVI are small and smooth, creating a club‐shaped appearance to the leaflet tips during early stages of the disease, but may become large and irregular during disease progression (**Figure 4B**) [56, 62, 63]. Mitral valve prolapse, which is characterized by one or both leaflets bent back into the left atrial chamber during systole, occurs commonly in dogs with CMVI (**Figure 4A**) [7, 64]. In one recent study, the severity of mitral valve prolapse was significantly correlated with MR severity [64]. Anterior leaflet of mitral valve is more commonly affected than posterior leaflet in dogs [64]. Abnormal excur‐ sion [i.e., decreased ejection fraction (EF) slope] and thickening of anterior mitral leaflet can

Ruptured chordae tendineae is also common echocardiographic finding in dogs with CMVI [64]. The mitral valve leaflet is seen pointing back into the left atrium (LA) during systole and bent back on itself with in the left ventricular outflow tract during diastole [65–67]. Chordae tendineae of anterior mitral valve leaflet is more commonly ruptured in

It is clinically important to evaluate severity of MR in dogs with CMVI. Color‐flow Doppler imaging (CDI) is widely used for detection and assessment of MR in dogs with CMVI (**Figures 4C**, **D** and **6B**). Maximal area of the regurgitant jet signals to the left atrium area (ARJ/LAA) ratio, which is the maximal ratio of the regurgitant jet area signal to left atrial area, is used in semi‐quantification of MR [56, 69, 70]. The ARJ/LAA ratio lesser than 20–30%

pressures, and pulmonary arterial pressure [56–61].

100 Canine Medicine - Recent Topics and Advanced Research

also be detected in M‐mode echocardiography (**Figure 5A**).

dogs [68].

projection.

**Figure 5.** 2D and M‐mode echocardiography in dogs with CMVI. (A) Abnormal excursion (decreased EF slope) and thickening of anterior mitral leaflet can be detected in M‐mode echocardiography. (B) The eccentric hypertrophy, which is characterized by an increase in end‐diastolic left ventricular dimensions (EDV), occurs in dogs with CMVI. (C)–(D) Hemodynamically significant chronic MR can induce volume overload, which subsequently can increase LV and LA volume and can result in LA and LV dilation. The degree of left atrial enlargement that is assessed by the left atrium to aorta (LA/Ao) ratio in 2D and M‐mode echocardiography and is closely correlated with the severity of heart failure.

is indicative of mild MR. The ratio involves between 20 and 30, 70% is indicative of moderate MR, and >70% is indicative of severe MR [56, 69–71].

The vena contracta is the measurement of smallest mitral regurgitant jet width through the valve and is correlated with MR severity [71]. Measuring the vena contracta uses parasternal long‐axis views that identify the vena contracta perpendicular to the sound plane. Very few data are available regarding vena contracta associated with dogs with CMVI [71]. In human study, a correlation was found between vena contracta and MR severity [72–74]. The proxi‐ mal isovelocity surface area (PISA) method can be used in echocardiography to estimate the area of flow acceleration and convergence proximal to the mitral valve as the regurgitant jet approaches the orifice [75]. Left apical four‐chamber views confirming the mitral regurgitant jet are generally recommended for measurement. Regurgitant orifice area, regurgitant frac‐ tion, and volume can be measured by this method. In study performed on dogs with severe MR, mitral regurgitant fraction calculated using PISA method was significantly correlated with MR severity [76].

Hemodynamically significant chronic MR can induce volume overload, which subsequently can increase LV and LA volume and can result in LA and LV dilation [63]. The degree of left atrial enlargement that is assessed by the left atrium to aorta (LA/Ao) ratio is closely corre‐ lated with the severity of heart failure (**Figure 5C** and **D**). Both two‐dimensional mode and M‐ mode echocardiography should be used in order to determine left atrial enlargement. Other indirect signs of high LA pressures in dogs with CMVI are left atrial rupture or acquired atrial

**Figure 6.** Pulse and continuous Doppler and tissue Doppler echocardiography in dogs with CMVI. (A) The transmitral flow profile consists of E and A and is affected by the pressure gradient between the LA and LV. Elevated E represents increased LA pressure and a worsening of heart failure. (B) Continuous Doppler echocardiography is useful to detect MR in dogs with CMVI. However, the degree of MR is not correlated with the severity of CMVI. (C) Pulse Doppler echocardiography in pulmonary venous flow is also useful to assess the progression of CMVI. The presence of pulmonary venous flow at atrial systole (PVa) indicates high LA pressure noticed in advanced stage of CMVI. (D) The early mitral inflow velocity to early mitral annular tissue velocity (E:Ea) ratio can be used to assess LV diastolic function. The E:E' ratio is significantly correlated with left ventricular filling pressures.

septal defect secondary to atrial septal rupture [77–79]. Due to volume retention, remodel‐ ing, and elevations in LA and pulmonary venous pressures, left ventricular volume overload can occur concomitantly with MR worsening [80]. The eccentric hypertrophy, which is char‐ acterized by an increase in end‐diastolic left ventricular dimensions (EDV), occurs in dogs with CMVI (**Figure 5B**) [81]. The diastolic left ventricular volume and diameters should be assessed by both M‐mode and two‐dimensional echocardiography.

is indicative of mild MR. The ratio involves between 20 and 30, 70% is indicative of moderate

The vena contracta is the measurement of smallest mitral regurgitant jet width through the valve and is correlated with MR severity [71]. Measuring the vena contracta uses parasternal long‐axis views that identify the vena contracta perpendicular to the sound plane. Very few data are available regarding vena contracta associated with dogs with CMVI [71]. In human study, a correlation was found between vena contracta and MR severity [72–74]. The proxi‐ mal isovelocity surface area (PISA) method can be used in echocardiography to estimate the area of flow acceleration and convergence proximal to the mitral valve as the regurgitant jet approaches the orifice [75]. Left apical four‐chamber views confirming the mitral regurgitant jet are generally recommended for measurement. Regurgitant orifice area, regurgitant frac‐ tion, and volume can be measured by this method. In study performed on dogs with severe MR, mitral regurgitant fraction calculated using PISA method was significantly correlated

Hemodynamically significant chronic MR can induce volume overload, which subsequently can increase LV and LA volume and can result in LA and LV dilation [63]. The degree of left atrial enlargement that is assessed by the left atrium to aorta (LA/Ao) ratio is closely corre‐ lated with the severity of heart failure (**Figure 5C** and **D**). Both two‐dimensional mode and M‐ mode echocardiography should be used in order to determine left atrial enlargement. Other indirect signs of high LA pressures in dogs with CMVI are left atrial rupture or acquired atrial

**Figure 6.** Pulse and continuous Doppler and tissue Doppler echocardiography in dogs with CMVI. (A) The transmitral flow profile consists of E and A and is affected by the pressure gradient between the LA and LV. Elevated E represents increased LA pressure and a worsening of heart failure. (B) Continuous Doppler echocardiography is useful to detect MR in dogs with CMVI. However, the degree of MR is not correlated with the severity of CMVI. (C) Pulse Doppler echocardiography in pulmonary venous flow is also useful to assess the progression of CMVI. The presence of pulmonary venous flow at atrial systole (PVa) indicates high LA pressure noticed in advanced stage of CMVI. (D) The early mitral inflow velocity to early mitral annular tissue velocity (E:Ea) ratio can be used to assess LV diastolic function.

The E:E' ratio is significantly correlated with left ventricular filling pressures.

MR, and >70% is indicative of severe MR [56, 69–71].

102 Canine Medicine - Recent Topics and Advanced Research

with MR severity [76].

The common indices of left ventricular systolic myocardial function are ejection fraction (EF) and fractional shortening (FS). The EF is defined by the percent of the end‐diastolic volume ejected from left ventricle (LV) with each heart beat. The FS is a measure of the percent change in the dimension from end diastole to end systole [82]. FS is dependent on preload, after‐ load, and myocardial contractility. Left ventricular systolic myocardial dysfunction is tradi‐ tionally characterized by reduced EF and FS. However, FS should be increased in dogs with CMVI because of elevated preload and reduced afterload, and hyperdynamic ventricular contraction.

Systolic myocardial dysfunction may be identified by end‐systolic left ventricular dimen‐ sions, such as end‐systolic diameter, end‐systolic volume, and end‐systolic volume indexed to body surface area (ESVI) [60, 61, 83]. Increased end‐systolic left ventricular dimensions are consistently referred to impaired left ventricular systolic function. There are three ultrasound methods, including the Teichholz method, the monoplane Simpson's derived method of discs, and the length‐area method, to measure ESVI. Because the Teichholz method tends to over‐ estimate ESVI, other methods except Teichholz method may be recommended in dogs with CMVI [61].

Spectral Doppler methods, including pulsed wave Doppler (PWD) and continuous wave Doppler (CWD), can identify regurgitant jet, transmitral flow in dogs with CMVI (**Figure 6A** and **B**). PWD may be used to record transmitral flow profile. The sample gate should be located in the tip of the leaflet. The transmitral flow profile looks like "M‐shaped" and consists of E (peak early transmitral flow velocity) and A (peak late transmitral flow velocity) related to early filling and atrial contraction, respectively [84]. Diastolic function and left ventricular filling pressures can be assessed using PWD method [84]. CWD is generally used for assessing severity of MR and thus provides information on LA pressure, preload, and systemic arterial pressure. CWD can be used to identify elevated LA pressure, left ventricular systolic and dia‐ stolic dysfunction in dogs with CMVI [60]. The presence of pulmonary venous flow at atrial systole (PVa) indicates high LA pressure and is commonly noticed in advanced stage of CMVI (**Figure 6C**). Continuous Doppler echocardiography is useful to detect MR in dogs with CMVI (**Figure 6B**). However, the degree of MR is not correlated with the severity of CMVI.

Advanced echocardiographic techniques, including tissue Doppler image (TDI), strain and strain rate imaging, and speckle tracking echocardiography (STE), are recently developed to assess myocardial abnormalities. TDI measures the myocardial velocities to quantify myocar‐ dial abnormalities. Systolic and diastolic myocardial abnormalities can be detected by TDI. TDI can be used in PWD and CDI. Myocardial velocity profile is characterized by a S wave, an E wave, and an A wave related to systolic myocardial velocity, early diastolic myocardial relaxation velocity, and late diastolic myocardial relaxation velocity, respectively. Strain and strain rate imaging are TDI‐based measurement and represent regional myocardial deforma‐ tion and deformation rate, respectively [85, 86]. The two‐dimensional STE is recently available and can be used to assess regional myocardial function. The STE is created by irregularities in reflected ultrasound from neighboring structures [87]. Very few data are available regard‐ ing advanced echocardiographic techniques data associated with canine heart diseases. TDI provides myocardial and annular velocity. Unlike Doppler patterns of mitral inflow, TDI assessment of diastolic function is relatively load‐independent. The early mitral inflow veloc‐ ity to early mitral annular tissue velocity (E:Ea) can be used to assess LV diastolic function (**Figure 6D**). The E:Ea ratio is significantly correlated with left ventricular filling pressures [88, 89].

Because most dogs affected by CMVI are older, progression of CMVI can lead to diastolic dysfunction, with time. Diastolic dysfunction is characterized by increased resistance to fill‐ ing and increased left ventricular filling pressure secondary to decreased compliance and impaired relaxation [90, 91]. The assessment of left ventricular diastolic function is difficult to undertake in the dogs with CMVI. Diastolic function can be assessed using several param‐ eters, including isovolumetric relaxation time (IVRT), transmitral flow velocities, and myo‐ cardial velocities.

Elevated LA pressure caused by MR and volume overload was found in dogs with moder‐ ate‐to‐severe CMVI [92, 93]. For the noninvasive assessment of LA pressure, the IVRT can be used in volume overload model [93]. IVRT is the time that elapses from aortic valve closure to mitral valve opening. In recent studies, the duration of IVRT and the ratio of E to IVRT were used in the diagnosis of elevated LA pressure [94, 95]. Decrease in IVRT is indicative for increase in LA pressure.

The left ventricular diastolic function can be assessed by Doppler patterns of mitral inflow. The transmitral flow profile consists of E and A and is affected by the pressure gradient between the LA and LV. Elevated E represents increased LA pressure and a worsening of heart failure (**Figure 6A**) [5]. If diastolic function is normal, E is greater than A. In early diastolic dysfunc‐ tion, a reversal of E and A can be occurred, as left ventricular compliance decreases. Further worsening of diastolic function leads to pseudonormalization associated with increased LA pressure. Because mitral inflow velocities are load‐dependent, the use of transmitral flow profile to assess diastolic function remains limited.

One recent study has evaluated the diagnostic value of left atrial volume index (LAVi) and the ratio of early filling to early diastolic mitral annular velocity (E/Ea) on the progression of heart failure in 51 dogs with CMVI and body weight matched 18 healthy control dogs, along with other known echocardiographic markers [96]. The LAVi and E/Ea were well correlated with the severity of heart failure in this study group. Based on the receiver‐operating characteristic analysis on echocardiographic variables, the echocardiographic indications for advanced heart failure in this study were left atrium to aorta ratio (LA:Ao) >2.0, left ventricular diastolic dimen‐ sion to aorta ratio (LVIDd:Ao) >2.4, end‐diastolic volume index (EDVI) >100 ml/m<sup>2</sup> , transmi‐ tral E‐peak >1.2 m/s, E/Ea >9.0 and LAVi 49 ml/m<sup>2</sup> , while indications for healthy or dogs with no signs of cardiac enlargement were LA:Ao <1.3, LVIDd:Ao <1.7, EDVI <45 ml/m<sup>2</sup> , E‐peak <0.65 m/s, E/Ea <6.0 and LAVi <15 ml/m<sup>2</sup> in dogs with CMVI.
