Diagnostic use of cardiac biomarkers and echocardiography in canine dilated cardiomyopathy

Introduction

Veterinary cardiology has advanced greatly since last two decades. According to information disseminated by the American Veterinary Medical Association (AVMA), one in ten dogs suffers from heart disease. The most commonly encountered cardiac abnormalities which cause heart failure in dogs are degenerative mitral valve diseases (MVD) and dilated cardiomyopathy (DCM) whereas, other less common abnormalities include congenital heart disease, infectious endocarditis, cardiac neoplasia, heart worm disease or other conditions leading to pulmonary hypertension (DeFrancesco, 2001) [1] . Cardiomyopathies are a heterogeneous group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that usually exhibit inappropriate ventricular hypertrophy or dilation (O'Grady and Sullivan, 2004) [2] . Dilated cardiomyopathy is one of the most common cardiac diseases in dogs and humans especially in the dog; it primarily affects large and giant breeds (Tidolhm, 2006) [3] . The diagnosis of DCM is mainly based on history, clinical signs, radiography, electrocardiography (ECG) and echocardiography (Tilley et al., 2008) [4] . Echocardiography permits non-invasive and non-ionizing visualization inside the heart including the aorta, the ventricles and atria, the auricular appendages and all of the cardiac valves. M-mode echocardiography helps to measure cardiac chamber size, measurement of wall thickness and detects cardiomegaly and hypertrophy, while, blood flow through the heart can be seen and measured with doppler ultrasound (Boon, 2011) [5] . The cardiac biomarkers help in the suspicion and diagnosis as well as monitoring of cardiovascular diseases and congestive heart failure. Cardiac troponin I (cTnI) has been recognized as the most sensitive and specific marker of myocardial cell necrosis in humans, demonstrating a higher specificity than the cardiac isoenzyme creatine kinase (CKMB) and better sensitivity and specificity than cardiac troponin T (cTnT) (Lavecchio et al., 2009) [6] . N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a cardiac biomarker that increases in the blood of dogs with heart diseases (Oyamaet al., 2009) [7] . Still, there is paucity of information regarding use of advance diagnostic modalities for canine cardiac abnormalities in India. Looking to the need of the era, the study was undertaken and executed.

Materials and Methods

The study was conducted during a period of 2 years (April, 2018 to March, 2020) in the Department of Veterinary Medicine in collaboration with Department of Veterinary Clinical Complex (VCC), College of Veterinary Science and Animal Husbandry, NAU, Navsari, Gujarat, India. The detail examination was performed in 18 confirmed DCM cases and 8 apparently healthy dogs presented to the hospital for health check-up and vaccination as control group. The detail study included clinical examination, blood pressure, radiography, electrocardiography, echocardiography, heamatobiochemical and cardiac biomarkers examination. The clinical examination included physical examination and special cardiovascular assessment as suggested by Tilley et al. (2008) [4] and Ettinger and Feldman (2010) [8] . The heamato-biochemical analysis was performed using autoanalyzer in departmental laboratory, whereas, biomarkers were got analyzed in private human laboratory using commercially available diagnostic kits. For the blood pressure measurement, the average of at least three measurements from each dog was recorded as the final blood pressure as described by Carr (2001) [9] . In thoracic radiography, Vertebral Heart Score (VHS) was calculated as suggested by Buchanan and Bucheler (1995) (Fig. 5). The echocardiography was performed with phased array Cardiac Probe PA320 with frequency ranged between 5.0 to 8.0 MHz. 2-Dimensional (2-D) and Motion-Mode (M-mode) imaging was done as per standard protocols followed by Boon (2011) [5] (Fig. 6).

Following measurements were recorded using 2-D and Mmode imaging From these values the contraction indices like fractional shortening and ejection fraction were calculated by the following formulas:

LVIDd-LVIDs

LVIDd ×100 per cent (Boon, 2011) [5] Ejection Fraction= EDV-ESV EDV × 100 per cent (Boon, 2011) [5] Statistical analysis: Different parameters in DCM affected dogs were compared with healthy dogs using student 'T' test using IBM SPSS statistical software version 20.0.

Results

History and clinical signs:

During the study period, history of DCM affected dogs showed exercise intolerance and dyspnoea (83.33%) as most frequently observed signs followed by weakness (77.78%), coughing (72.22%), inappetence (66.67%), weight loss (61.11%), abdominal enlargement (55.56%), vomiting (27.78%) and syncope (16.67%) (Fig. 1234). The mean rectal temperature of DCM affected dogs was 101.94±0.30 0 F which could not differed from control group (101.33±0.17 0 F). The heart rate (149.17±4.59), pulse rate (145.94±4.27) and respiration rate (36.39±1.17) per minute in DCM affected dogs were significantly higher than control group (p<0.01). The typical characteristics features of DCM observed were tachycardia (77.78%), murmur (77.78%), pulmonary oedema (38.89%) and muffled heart sound (11.11%). The average diastolic blood pressure in DCM group (127.39±2.62) was significantly higher than control group (87.17±2.31) (p<0.05). Vertebral heart score on radiograph was significantly higher in DCM affected dogs (12.27±0.25 V) than control group (8.97±0.11 V) (Fig. 5). Thoracic radiographs of the healthy dogs did not show any enlargement / abnormal regional dilatation or deviation of cardiac silhouette.

Echocardiography

In 2-D echocardiography, the mean Left atrium (LA) diameter in DCM group was significantly higher (3.98±0.13 cm) and Aortic (Ao) diameter (1.98±0.07 cm) was significantly lower than the corresponding diameter of control group (Table-1) (Fig. 8). The mean of LA/Ao ratio in the DCM group was significantly higher (2.05±0.09) than control group (p<0.01) and value recommended (LA/Ao ratio< 1.60). In M-mode echocardiography, highly significant increased LVDd (5.30±0.21 cm) and LVDs (4.57±0.19 cm) was observed in DCM affected dogs than control group. The significantly higher values of RVDd and RVDs were also observed in DCM affected dogs than control group (p<0.05). The mean of IVSd was significantly lower (p<0.01) whereas, mean of IVSs was comparatively lower in DCM group than control group which are an indicative of reduced thickness of inter ventricular septum during dilatation of ventricles (Fig. 7). The significantly decreased values of LVPWd (p<0.05) and LVPWs (p<0.01) in DCM group as compared to the control group was noticed. The means of EF (26.94±2.64%) and FS (13.68±1.49%) in DCM group were significantly lower than the control group (p<0.01) (Fig. 9). The EPSS in DCM group (1.71±0.08 cm) was significantly higher than control group (0.40±0.02) (p<0.01) (Table -

1).

Haemato-biochemical findings: Significant decreased means of haemoglobin, packed cell volume and RBC was observed in DCM group than control group (p<0.01). Comparative increased means of monocytes and neutrophils were also observed in DCM group than control group. In DCM group, the mean of total protein was comparatively and albumin was significantly lower than control group (p<0.01), whereas, AST was significantly (p<0.01) and ALT was comparatively higher than control group (Table-2).

Cardiac biomarkers:

The mean of cTn-I and CPK-MB were found comparatively elevated in DCM affected dogs than control group. The mean NT-proBNP in DCM group was significantly higher (319.29±38.20 pg/ml or 1171.04±140.23 pmol/l) than control groups (p<0.05) (Table-3).

Discussion

History and clinical signs: History and various clinical signs observed in present study were in accordance with the findings of Petric et al. (2002) [10] who observed coughing in 79% cases followed by anorexia (71%), exercise intolerance (43%), dyspnoea (36%), vomiting (14%), syncope (14%), tachycardia (14%) and abdominal distension (7%) as the chief clinical complaints. Similarly, Tidholm (2006) [3] reported dyspnoea in all cases followed by cough (73%), exercise intolerance (60%), inappetence (42%), polydipsia (15%), weight loss (13%), syncope (11%) and abdominal enlargement (6%). Later on Martin et al. (2009) [11] recorded most presenting signs of DCM with their frequency. The signs were breathlessness (67%), cough (64%), exercise intolerance (48.8%), weakness (39.2%), reduced appetite (35.7%), weight loss (30.2%), collapse (26%), dullness and lethargy (20%). Two reports (Jeyaraja et al., 2015; Venkateshkumar et al., 2018) [12,13] had also reported similar findings in their studies on dilated cardiomyopathy in dogs. Rectal temperatures in DCM and control group did not differ. Similarly, DeFransesco (2001) [1] and Deepti et al. (2016) [14] did not findany difference in rectal temperature of DCM affected and healthy dogs. Whereas on contrary, McEwan et al. (2003) [15] reported elevation of body temperature in some cases and stated that clinical presentation could vary from case to case and according to breed. The heart rate, pulse rate and respiration rate of DCM affected dogs were significantly higher than control group. Increased heart rate, pulse rate and respiration rate might be due to congestion or pulmonary edema with resultant hypoxemia and hypercapnia (Tilley et al., 2008) [4] . Tachycardia as a chief clinical complaint of DCMwas also reported by Petric et al. (2002) [10] . Further, variation in heart rate and rhythm due DCM were also observed by various authors. Further, Tidholm (2006) [3] found soft systolic murmur (grades III/VI) in 56% cases of DCM. Martin et al. (2009) [16] also noticed murmur and gallop sound in 33 and 10% DCM affected dogs. In an Indian study, Jeyaraja et al. (2015) [12] also recorded gallop rhythm and systolic murmur in 87 and 73% DCM cases as major clinical features. In our study, an average diastolic blood pressure in DCM group was significantly higher than control group. But, reports of Velhankar (2013) [17] and Thirunavukkarasu (2019) [18] showed non-significant differences in systolic and diastolic blood pressures of DCM affected and healthy dogs. The vertebral heart score on radiography of DCM affected dogs was significantly higher than control group. It might be suggestive of the enlargement of cardiac silhouette due to presence of structural heart disease and due to dilatation of cardiac chambers in DCM. Similarly, increased VHS in DCM group was also recorded previously by Venkatesakumar et al. (2018) [13] and Bhargavi et al. (2019) [19] .

Echocardiography:

The mean of LA/Ao ratio in the DCM group was significantly higher than control group and value recommended (LA/Ao ratio< 1.60) by Tilley et al. (2008) [4] . It is suggestive of left atrial enlargement due to increased diameter of left atrium (LA) and decreased diameter of aorta (Ao). Such findings are in agreement with Deepti et al. (2016) [14] who reported increased diameter of left atrium (5.3 cm) during end systole and decreased aortic diameter (2.15 cm) during end diastole in DCM affected dogs. It might be due the elevated ventricular end systolic pressure which creates low pressure gradient from the atrium into the ventricle resulting in less atrial emptying leading to high left atrial pressures and ultimately atrial dilatation in DCM affected dogs (Calvert and Brown, 1986) [20] . In M-mode echocardiography, increased LVDd and LVDs in DCM group are in agreement with Meurs et al. (2001) [21] and Calvert & Wall (2001) [22] who observed increased left ventricular internal dimension in DCM affected Doberman Pinchers. In above context, McEwan et al. (2003) [15] stated that increased left ventricular lumen (particularly during systole) can be an important M-mode echocardiography criteria for the diagnosis of DCM. Further, Boon (2011) [5] stated that reduced thickness of inter ventricular septum and wall results into decreased function. The finding of decreased thickness of posterior wall in DCM is also in agreement with reports of McEwan et al. (2003) [15] and Boon (2011) [5] . Dilated cardiomyopathy alters the thickness of left ventricular posterior wall owing to eccentric hypertrophy and its severity is dependent on breed, its genetic predisposition and stage of disease at the time of examination (Tilley et al., 2008) [4] . The above changes resulted into reduction of EF and FS values which is clearly suggestive of left ventricular systolic dysfunction with significant reduction in cardiac output (EF) and left ventricular myocardial contractility (FS) in DCM affected dogs. The finding of significant reduction of EF is in agreement with previous reports in which they stated that low fractional shortening (< 25%) is the best Mmode echocardiography criteria for the diagnosis of DCM (Boon, 2011; Meurs et al., 2001; McEwan et al., 2003) [5,21,15] . Whereas, for reduced FS, O'Grady and Sullivan (2004) [2] stated that fractional shortening (FS) is the major indicator of systolic function in Veterinary echocardiography and values of FS less than 15% are suggestive of strong evidence of DCM.The finding of increased EPSS in DCM affected dogs is in accordance with reports of Jeyaraja et al. (2015) [12] and Wess et al. (2017) [23] who reported EPSS >6.5 mm (0.65 cm) in DCM cases. Previously, McEwan et al. (2003) [15] also opined that the increased mitral valve EPSS can be considered as one of the important criteria for the diagnosis of DCM. Boon (2011) [5] also stated that E-point to septal separation (EPSS) is one of the consistent and popular mitral valve measurements for diagnosis of DCM.

Haematology:

The haematological observations of present study are in accordance with previous reports in which significant increased means of white blood cell count and absolute neutrophil count in DCM affected dogs was noticed (Sesh et al., 2013; Singh et al., 2014; Jeyraja et al., 2015) [24,25,12] . Increased level of leucogram and neutrophils in the DCM group could be due to other primary or concurrent diseases.

Biochemical studies:

The mild elevation in liver enzyme could be due to liver damage and decreases in total protein and albumin have been noticed due to impaired liver function in DCM affected group. Previously, Ettinger and Feldman (2010) [8] observed non-significant increase in ALT and AP levels due to degree of hepatic damage associated with its engorgement. Similarly, previous reports mentioned also elevation of ALT along with low total protein and albumin level in DCM affected dogs (McEwan et al., 2003; Martin et al., 2009; Haritha et al., 2019) [15,16,26] .

Cardiac biomarkers:

The finding of elevated level of cTn-I in DCM group is in agreement with observation of Spratt et al. (2005) [27] who reported significant elevated cTn-I level in dogs with acquired mitral valve disease, dilated cardiomyopathy and pericardial effusion. Later, Lavecchio et al. (2009) [6] explained that cardiac troponin-I (cTn-I) is a polypeptide found specifically in cardiac muscle tissue that has been used as a diagnostic and prognostic indicator of cardiomyopathy in dogs and cats. Along with elevated cTn-I, UshaShree (2013) [28] also found elevated CK-MB (35.04±4.93 U/L) in DCM dogs as compared to control dogs (13.79±2.83 U/L). In context to significant elevated NT-proBNP, Kimmenade and Januzzi, (2009) [29] stated that BNP is rapidly produced by cardiomyocytes after stimuli like myocardial stretch or hypoxia and revealed that a concentration of less than 900 pmol/l of NT-proBNP is not compatible with myocardial increased stretch and stress. Whereas, on contrary Oyama et al. (2009) [7] stated that serum NT-proBNP cut-off concentration >1,158 pmol/ L discriminated between dogs with congestive heart failure and primary respiratory tract diseases.

Acknowledgement

Acknowledgements:The authors are thankful to Principal, College of Veterinary Science & A. H., KU, Navsari and Head of Department (s), Departments of Veterinary Clinical Complex for providing necessary facilities to conduct this study.

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