DIAGNOSTIC APPROACH TO MYXOMATOUS MITRAL VALVE DISEASE IN CAVALIER KING CHARLES SPANIELS: IDENTIFICATION OF EARLY DIAGNOSTIC FACTORS (MORPHOMETRIC PARAMETERS AND MIRNAS EXPRESSION)

Myxomatous mitral valve disease (MMVD) is the most prevalent acquired cardiac disorder in dogs and a significant contributor to both morbidity and mortality, especially among small breeds. Notably, the Cavalier King Charles Spaniel (CKCS) demonstrates an early onset and more rapid progression of the disease, indicating a strong genetic predisposition along with breed-specific risk factors. Despite considerable advancements in veterinary cardiology, the early diagnosis of MMVD remains a challenge, as conventional diagnostic tools—such as auscultation—typically detect structural and functional cardiac changes only after the disease has progressed. Given the lengthy preclinical phase of MMVD, the identification of reliable early biomarkers is crucial for enhancing patient management, refining breeding strategies, and alleviating the overall disease burden. The objective of this PhD project was to identify early diagnostic factors MMVD in CKCSs by integrating morphometric parameters with circulating microRNA (miRNA) expression. To accomplish this, three complementary studies were conducted, combining molecular, echocardiographic, and morphometric approaches to enhance the understanding of disease onset and progression. The first study was a prospective pilot investigation aimed at assessing the predictive role of plasma miR-30b-5p levels in relation to echocardiographic changes in CKCSs at various stages of MMVD as classified by the American College of Veterinary Internal Medicine (ACVIM). The findings revealed that elevated levels of plasma miR-30b-5p were associated with milder forms of MMVD over time. These results underscore the potential of miR-30b-5p as a minimally invasive biomarker for the early detection of MMVD, thereby supporting the clinical significance of circulating miRNA profiling in veterinary cardiology. The second study focused on salivary miRNA expression as a novel, non-invasive approach for MMVD screening. By profiling salivary miRNAs associated with MMVD severity in CKCSs, we identified significant differential expression patterns across ACVIM stages, with specific miRNAs associated with disease presence and severity. Saliva-based profiling showed promising diagnostic performance, indicating that liquid biopsy techniques could supplement plasma analyses and enable disease monitoring in a less invasive way. The third study examined the relationship between morphometric measurements and the progression of MMVD in preclinical CKCSs through a retrospective longitudinal analysis. A thorough evaluation of craniofacial and thoracic morphometry indicated that specific physical traits—such as thoracic length, circumference, and index—are significantly associated with the progression of MMVD in this breed. These findings suggest that thoracic morphology may play a role in disease progression among CKCSs. Therefore, it is important to consider these morphological features alongside other known risk factors when assessing the risk of MMVD progression in CKCSs. The findings of this PhD thesis offer new insights into the pathophysiology and early diagnosis of mitral valve disease (MMVD) in Cavalier King Charles Spaniels (CKCSs). By combining morphometric profiling with the analysis of circulating microRNA (miRNA) expression, this research presents a multifactorial diagnostic approach that can identify predisposed individuals before significant cardiac remodeling occurs. These strategies could enhance clinical decision-making, improve selection in breeding programs, and help reduce the prevalence of severe MMVD in this breed, which is highly susceptible to the condition. Future research should focus on validating these results in larger populations and across different breeds to confirm the diagnostic value of miRNAs and morphometric parameters for early MMVD detection. Additionally, integrating these tools into routine cardiological evaluations for CKCSs could facilitate the implementation of proactive strategies, such as targeted monitoring.