**3.1. Physical examination**

Heart murmur is a major finding in physical examination. Depending on the severity, cli‐ nicians can hear various grade of heart murmur before the onset of clinical signs. In early stages of the CMVI, heart murmur can be localized and weak as 1~2/6 scale at left apex. In late stages of the CMVI, heart murmur is gradually radiated and louder and is typically cre‐ scendo‐decrescendo type systolic regurgitant murmur (**Figure 2A**). Recent study found that the grade of heart murmur was closely related to the severity of CMVI [24].

## **3.2. Laboratory tests**

Common laboratory findings in dogs with CMVI are normal or slightly raise in kidney and/ or liver chemistry profiles, probably due to the congestion and poor body perfusion [25]. One recent study evaluated hepatic panel in dogs at different stages of heart failure from CMVI [26].

**Figure 2.** (A) Phonocardiogram in dogs with CMVI. Heart murmur is gradually radiated and louder and is typically crescendo‐decrescendo type systolic regurgitant murmur. (B) ECG in dogs with CMVI. P‐mitrale (wide P‐wave) and wide and tall QRS complexes indicating LA and LV dilation.

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 advanced stage of CMVI [26].

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

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 earlier stages of HF [28].

## **3.3. Cardiac biomarkers**

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 through its end‐stage. Cardiac biomarkers have used as diagnostic tools [29], prognostic indi‐ cator [30], and monitoring system [31] for CHF.

Troponins are marker of myocardial necrosis and ischemia and found to be closely associ‐ ated with the severity of heart failure in dogs [32] and cats [33], although it often elevated in many noncardiac disease [34–36]. Natriuretic peptides (NPs) are markers releasing from hemodynamic stress on the heart [37], responded against volume expansion/pressure over‐ load [37]. The plasma concentration of N‐terminal prohormone of brain natriuretic peptide (NT‐proBNP) is well correlated with severity of heart failure in dogs [38], although the level of NT‐proBNP can be affected by noncardiac factors such as body weight and renal func‐ tion [39]. Cardiopet® proBNP is a commercially available diagnostic test. According to the manufacturer (Idexx, USA), dogs with <900 pmol/L of serum NT‐proBNP may not have heart failure, while dogs with 900–1800 pmol/L may have heart failure, but is required further dis‐ criminative tests. Dogs with >1800 pmol/L may have higher possibility of heart failure. C‐ reactive protein (CRP) is an acute‐phase reactant protein [40, 41] that is increased in several diseases in dogs [42–47]. Although the level of CRP is increased in dogs with CMVI, the CRP concentration was not related to the presence of CHF or murmur grade [48].
