MicroRNA Expression Profiling in Canine Myxomatous Mitral Valve Disease Highlights Potential Diagnostic Tool and Molecular Pathways

Figures

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  Figure 1. Bioinformatic enrichment analysis of candidate miRNAs. Hierarchical clustering heatmap generated with DIANA-mirPath v3 using the microT-CDS algorithm and Fisher’s exact test with FDR correction. The predefined panel included hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-miR-21-5p, hsa-miR-30b-5p, hsa-miR-133b, hsa-miR-98-5p, and hsa-miR-103a-3p. Color intensity reflects the statistical significance of pathway enrichment (log p-value), with red indicating the strongest associations. The analysis revealed significant convergence (FDR < 0.05) of the selected miRNAs on key pathways implicated in myxomatous mitral valve disease (MMVD) pathogenesis, including ECM–receptor interaction (p = 5.0 × 10−4), TGF-β signaling (p = 3.0 × 10−3), PI3K–Akt signaling (p = 3.0 × 10−3), MAPK signaling (p = 1.27 × 10−2), and focal adhesion/actin cytoskeleton regulation (p = 1.27 × 10−2). Additional modules such as Hippo signaling (p = 3.0 × 10−3) and Wnt signaling (p = 1.02 × 10−2) were also represented, together with glycosaminoglycan/proteoglycan biosynthetic pathways, consistent with the histological hallmark of glycosaminoglycan accumulation in the spongiosa of MMVD valves. Notably, shared gene…

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  Figure 2. Schematic representation illustrating how the selected miRNAs act as inhibitory nodes within the enriched pathways identified by DIANA-miRPath v3.0 analysis. The miRNAs investigated in this study (blue, left) are shown interacting with their predicted target gene groups (orange, center) involved in the main enriched signaling pathways (gray right), including TGF-β, PI3K–Akt, MAPK, ECM–receptor interaction, and focal adhesion. Together, these regulatory networks highlight how the selected miRNAs modulate extracellular matrix remodeling and fibrotic processes in canine MMVD.

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  Figure 3. Relative expression (2−ΔΔCt) of selected miRNAs in low-grade (white bars) and high-grade (red bars) samples. Expression values were normalized to the endogenous control miR-29a and are presented relative to healthy control samples (set to 1). Bars indicate mean ± SD. Values greater than 1 correspond to upregulation compared to controls. Significant differences between low- and high-grade samples are indicated: * p < 0.05; ** p < 0.01.