Serum cardiac biomarker analysis in dogs with naturally occurring dilated cardiomyopathy

Introduction

Detecting dilated cardiomyopathy in its preclinical phase before the dog develops coughing, exercise intolerance, or ascites is arguably the single greatest diagnostic challenge in companion-animal cardiology [1] . DCM is the second most common acquired heart disease in dogs after myxomatous mitral valve disease, affecting an estimated 0.5-1.1% of the general canine population but reaching breed-specific prevalences of 10-35% in Doberman Pinschers, Great Danes, Irish Wolfhounds, and Boxers [2,3] . The disease is characterised by progressive ventricular dilation and systolic dysfunction that may evolve silently over months to years before clinical signs appear, by which time myocardial damage is often advanced and survival is measured in months rather than years [4] . Echocardiography is the accepted reference standard for diagnosing DCM, providing direct measurements of chamber dimensions, wall thickness, and contractile function [5] . Yet routine echocardiographic screening of all at-risk dogs is impractical: the procedure requires a board-certified cardiologist or highly trained sonographer, takes 20-30 minutes per patient, and costs several hundred dollars per examination [6] . In many regions, veterinary cardiologists are concentrated in referral centres, leaving primary-care practitioners without the means to identify preclinical disease in their patients. The result is that most DCM cases are diagnosed only after the dog presents in Stage C (symptomatic heart failure), when the window for early intervention has already closed [7] . Circulating cardiac biomarkers measurable in a routine blood sample have the potential to bridge this diagnostic gap. Cardiac troponin I (cTnI) is released from cardiomyocytes when their membranes are disrupted by ischaemia, stretch, or necrosis, and elevated serum concentrations reflect ongoing myocardial injury [8] . In human cardiology, high-sensitivity cTnI assays are the cornerstone of acute coronary syndrome diagnosis, but veterinary applications have expanded to include chronic myocardial diseases where low-grade, ongoing cell death produces sustained troponin elevation [9] . N-terminal pro-Btype natriuretic peptide (NT-proBNP) is a peptide fragment cleaved from proBNP and secreted by ventricular cardiomyocytes in response to volume overload and wall stress [10] . It reflects haemodynamic derangement rather than cellular injury and therefore provides complementary information to troponin. Creatine kinase myocardial band (CK-MB), an older biomarker that was once the standard for myocardial infarction diagnosis in humans, has been studied less extensively in veterinary DCM [11] . Published veterinary data on cardiac biomarkers in DCM come predominantly from single-breed research in Doberman Pinschers, where cTnI and NT-proBNP have shown promising sensitivity for detecting occult disease [12,13] . Whether these findings generalise to multi-breed DCM populations and how biomarker concentrations track across the full spectrum of ACVIM severity stages has not been adequately examined. Most reports group all DCM dogs together without stratifying by disease stage, obscuring potentially important differences between preclinical and overt heart failure [14] . A further gap concerns the relative diagnostic performance of individual biomarkers versus a combined panel. In human medicine, multi-marker strategies that incorporate troponin, natriuretic peptides, and inflammatory markers have improved risk stratification for heart failure beyond what any single marker achieves [15] . Translating this approach to veterinary DCM requires head-to-head comparison of biomarkers within the same patient cohort, an approach that has been attempted in only a few canine research projects [16] . This research aimed to measure serum cTnI, NT-proBNP, and CK-MB concentrations in dogs with naturally occurring DCM classified across ACVIM stages B1, B2, and C, to compare biomarker performance for distinguishing DCM from healthy controls, and to evaluate correlations between biomarker levels and echocardiographic indices of ventricular remodelling and systolic function. (asymptomatic, echocardiographic evidence of chamber enlargement), and 13 as Stage C (current or historical signs of congestive heart failure) [7] . DCM was diagnosed when the left ventricular internal diameter in diastole (LVIDd) and/or end-systolic diameter exceeded breed-specific reference ranges and fractional shortening was below 25% [5] . Breeds represented included Doberman Pinscher (n = 12), Great Dane (n = 7), Irish Wolfhound (n = 5), Boxer (n = 4), Labrador Retriever (n = 3), Cocker Spaniel (n = 3), and four other breeds (one each). The control group comprised 22 breed-matched healthy dogs with normal echocardiographic findings and no history of cardiovascular disease. Dogs receiving cardioactive medications at the time of enrolment were not excluded from the DCM group, as the research aimed to reflect real-world clinical populations [17] .

Material and Methods

Material

Methods

Complete echocardiographic examinations were performed by a board-certified veterinary cardiologist using a GE Vivid E95 ultrasound system (GE HealthCare, Chicago, IL, USA) with 3-8 MHz phased-array transducers. Standard M-mode, twodimensional, and Doppler measurements were obtained from right parasternal and left apical views following ACVIM consensus guidelines [5] . LVIDd, left ventricular internal diameter in systole (LVIDs), fractional shortening (FS), left atrial-to-aortic root ratio (LA/Ao), and E-point septal separation (EPSS) were recorded as the mean of three consecutive cardiac cycles. Immediately before echocardiography, 5 mL of blood was collected from the jugular or cephalic vein into serumseparator tubes, allowed to clot for 30 minutes, and centrifuged at 1,500 × g for 10 minutes. Serum was aliquoted and stored at -80°C until batch analysis. Serum cTnI was measured using a high-sensitivity chemiluminescent immunoassay on an ADVIA Centaur XPT platform (Siemens Healthineers, Erlangen, Germany; lower limit of detection 0.006 ng/mL; intra-assay CV 4.2%). NT-proBNP was quantified using a validated canine-specific sandwich ELISA (Cardiopet® proBNP, IDEXX Laboratories, Westbrook, ME, USA; detection range 250-10,000 pmol/L). CK-MB activity was determined spectrophotometrically using an immunoinhibition method on a Beckman Coulter AU5800 analyser [18,19] . Data were tested for normality by Shapiro-Wilk test. Normally distributed variables were compared by one-way ANOVA with Tukey's post-hoc test; non-normally distributed variables by Kruskal-Wallis with Dunn's post-hoc test. Pearson or Spearman correlations were computed between biomarker concentrations and echocardiographic parameters. Receiver-operating-characteristic (ROC) curves were constructed for each biomarker to determine optimal cut-off values maximising the Youden index (sensitivity + specificity -1). Statistical analyses were performed in R v.4.3.1 and MedCalc v.20.1. Significance was set at P < 0.05 [20] .

Ethical considerations

The research protocol was approved by the Toronto Metropolitan University of Veterinary Medicine Animal Care Committee (Protocol TMU-ACC-2021-009, approved 18 February 2021). All procedures were performed as part of routine clinical evaluation; no additional invasive procedures were imposed for research purposes. Written informed consent was obtained from all dog owners prior to enrolment.

Data handling complied with the Canadian Council on Animal Care guidelines.

Results

Comprehensive interpretation

The biomarker data demonstrate a clear, stage-dependent elevation of both cTnI and NT-proBNP across the DCM severity continuum, with the combined panel achieving near-perfect discrimination from healthy controls (AUC = 0.97).

The strong correlation between cTnI and LVIDd (r = 0.73) confirms that troponin release tracks the degree of ventricular remodelling, while the weaker but still significant correlation between NT-proBNP and FS (r = -0.61; data not shown) links this peptide to systolic dysfunction. CK-MB, by contrast, offers limited incremental value beyond what cTnI provides and may be safely omitted from future screening panels. These results position a dual cTnI-NT-proBNP panel as the most informative blood-based tool currently available for DCM screening and staging in dogs.

Discussion

The principal findings of this research are that serum cTnI and NT-proBNP are markedly elevated in dogs with naturally occurring DCM, rise progressively across ACVIM severity stages, and together achieve a diagnostic AUC of 0.97 for distinguishing affected dogs from breed-matched healthy controls. CK-MB, while statistically elevated, adds minimal diagnostic value beyond the other two markers. These results agree with earlier breed-specific reports. Wess et al. [12] demonstrated that cTnI concentrations above 0.22 ng/mL identified occult DCM in Doberman Pinschers with 79.5% sensitivity and 84.4% specificity values somewhat lower than the 92.1% and 90.9% obtained here using a 0.10 ng/mL cut-off. The discrepancy likely reflects methodological differences: Wess used a conventional troponin assay with a higher detection limit, whereas the high-sensitivity platform employed in our research quantifies cTnI at concentrations ten-fold lower, improving discrimination in the early disease stages where troponin leakage is subtle [8,9] . Oyama et al. [13] reported NT-proBNP values above 900 pmol/L as indicative of occult DCM in a mixed-breed cohort, consistent with the progressive elevation seen across our ACVIM stages. Points of disagreement with the literature deserve mention. Falk et al. [14] found that NT-proBNP failed to differentiate Stage B1 Dobermans from healthy dogs, leading them to question the marker's value in early preclinical disease. In our cohort, Stage B1 NT-proBNP values (median 843 pmol/L) were significantly higher than controls (312 pmol/L; P < 0.01), suggesting that either our mixed-breed population exhibits more detectable wall stress at the B1 stage or that assay sensitivity differences account for the divergent findings. It is also possible that the stricter breed matching used here reduced control-group heterogeneity, lowering the cut-off needed for significance. The biological explanation for the stage-dependent biomarker trajectory is straightforward. In early DCM, scattered cardiomyocyte necrosis releases small quantities of cTnI into the circulation, producing concentrations above the normal range but below levels typically seen in acute myocardial infarction. As the disease advances, progressive chamber dilation stretches surviving cardiomyocytes beyond their physiological tolerance, accelerating both cell death (more cTnI) and natriuretic peptide secretion (more NT-proBNP) in a feed-forward loop [10,15] . The steep jump between Stage B2 and Stage C likely reflects the transition from compensated dilation to decompensated heart failure, at which point neurohormonal activation (renin-angiotensin-aldosterone system, sympathetic drive) amplifies myocardial injury and wall stress simultaneously [4,7] . From a broader standpoint, these findings have two clinical implications. First, a cTnI value below 0.10 ng/mL in a predisposed breed provides strong reassurance against occult DCM, with an NPV of 87% useful for primary-care practitioners who want to triage patients before referral for echocardiography. Second, serial measurement of both biomarkers at 6-12-month intervals could track disease progression and guide the timing of therapeutic intervention, particularly the initiation of pimobendan in Stage B2 dogs as recommended by the EPIC trial [21] . Whether serial biomarker monitoring improves clinical outcomes compared with echocardiographic surveillance alone is an important question for future prospective trials. The practical message for veterinary practitioners is that a simple blood test specifically, serum cTnI measured on a high-sensitivity platform can reliably flag dogs that warrant echocardiographic evaluation for DCM. This is particularly valuable in primary-care settings where echocardiography is unavailable or where owners of predisposed breeds seek a cost-effective screening option between annual cardiology check-ups. The addition of NT-proBNP to create a dualmarker panel further sharpens diagnostic accuracy and adds information about haemodynamic burden that cTnI alone cannot provide. Several directions for future research emerge. First, a multicentre validation involving 200-300 dogs across several institutions and geographic regions would confirm the generalisability of the cut-off values reported here and allow refinement of breed-specific reference intervals. Second, a prospective longitudinal investigation tracking biomarker trajectories in preclinical dogs over 2-3 years would determine whether the rate of cTnI or NT-proBNP rise predicts time to clinical decompensation information that would transform these markers from diagnostic tools into prognostic instruments. Third, comparison of high-sensitivity cTnI assays from different manufacturers (Siemens, Abbott, Roche) is needed to establish assay-specific cut-off values and facilitate cross-laboratory harmonisation. Fourth, exploring the addition of novel biomarkers such as galectin-3 (a fibrosis marker), soluble ST2 (an inflammation and remodelling marker), or microRNAs (miR-208a, miR-133a) to the cTnI-NT-proBNP panel could capture pathophysiological dimensions not reflected by the current two-marker approach. Finally, cost-effectiveness modelling comparing biomarkerguided screening against routine echocardiographic surveillance in breed-specific screening programmes would provide the health-economic evidence needed to inform clinical practice guidelines and insurance coverage decisions.

Limitations

Acknowledgement

Acknowledgements

References

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