Sex–Specific Remodeling Phenotypes of the Tricuspid Valve Leaflets in an Ovine Model of Functional Tricuspid Regurgitation

Figures

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  Figure 1.. Pulmonary artery banding (PAB) enlarges tricuspid valve leaflet area with distinct patterns across leaflets and sex. (a) Representative tricuspid valve imaged on a calibrated grid (1-cm scale). Septal, anterior, and posterior leaflet boundaries are traced (black outlines), and commissures are labeled as posterior-septal (PS), antero-septal (AS), and antero-posterior (AP). Leaflet areas were measured between PS and AS for the septal leaflet, AS and AP for the anterior leaflet, and AP and PS for the posterior leaflet. (b–d) Leaflet area for the septal, anterior, and posterior leaflets, respectively, in control (CTL, blue) and PAB (red) sheep stratified by sex (Female, castrated male (C-Male), and non-castrated male (NC-Male)). Data are shown for individual animals with group means ± standard deviation. Statistical differences between groups were assessed using linear mixed-effects models with post hoc pairwise comparisons of estimated marginal means. Significant contrasts between groups (CTL vs. PAB) are denoted by (*) for p < 0.05 and (***) for p < 0.001. Significant contrasts between sexes are denoted by (#) for p <0.05 (C-Male vs. Female), (§) for p < 0.05 (NC-Male vs. Female),…

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  Figure 2.. Pulmonary artery banding (PAB) induced leaflet thickening differs by leaflet and sex. (a) Representative profilometric thickness maps for a PAB septal, anterior, and posterior leaflet. Total leaflet thickness comparison between control (CTL, blue) and pulmonary artery banding (PAB, red) sheep stratified by sex (Female, castrated male (C-Male), and non-castrated male (NC-Male)). Total thickness of the (b) septal, (c) anterior, and (d) posterior leaflets was quantified from the profilometric thickness maps. Data are shown for individual animals with group means ± standard deviation. Statistical differences between groups were assessed using linear mixed-effects models with post hoc pairwise comparisons of estimated marginal means. Significant contrasts between treatment groups (CTL vs. PAB) are denoted by (*) for p < 0.05 and (***) for p < 0.001, while significant contrasts between sex groups are denoted by (¥) for p < 0.05 (C-Male vs. NC-Male).

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  Figure 3.. Pulmonary artery banding (PAB) alters the biaxial mechanical behavior of the anterior leaflet. Circumferential membrane tension (Mem. Tens.) and stretch curves for control (CTL, blue) and PAB (red) sheep are shown for (a) Female, (b) castrated male (C-Male), and (c) non-castrated male (NC-Male) sheep, with shaded regions denoting mean (solid line) and standard deviation (shaded). Corresponding circumferential mechanical metrics are presented as (d) toe stiffness, (e) calf stiffness, and (f) transition stretch (λ) for each sex group. Radial membrane tension–stretch curves for CTL and PAB sheep are shown for (g) Female, (h) C-Male, and (i) NC-Male sheep, with shaded regions denoting ± standard deviation, and the associated radial (j) toe stiffness, (k) calf stiffness, and (l) transition stretch (λ). Data are shown for individual animals with group means ± standard deviation. Statistical differences between groups were assessed using linear mixed-effects models with post hoc pairwise comparisons of estimated marginal means. Significant contrasts between groups (CTL vs. PAB) denoted by (*) for p < 0.05 and (**) for p < 0.01.

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  Figure 4.. Total nuclei count increases in leaflets after pulmonary artery banding (PAB) in a sex-dependent manner. Representative H&E-stained cross-sectional histology images for (a) septal, (c) anterior, (e) posterior leaflets from a diseased (PAB) non-castrated male (NC-Male) sheep. Total cell nuclei comparison for (b) septal, (d) anterior, and (f) posterior leaflets from Female, castrated male (C-Male), and non-castrated male (NC-Male) sheep. Data are shown for individual animals with group means ± standard deviation. Statistical significance between groups (CTL vs. PAB) is denoted by (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001. Statistical significance between sexes is denoted by (§) for p < 0.05 (NC-Male vs. Female) and (¥) for p < 0.05 (C-Male vs. NC-Male).

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  Figure 5.. Sex-stratified differential gene expression in tricuspid valve leaflets after pulmonary artery banding (PAB). Volcano plots depict differential gene expression (PAB vs. control [CTL]) from bulk RNA-seq of tricuspid valve leaflets from (a) Female, (b) castrated male (C-Male), and (c) non-castrated male (NC-Male) sheep. Each point represents a gene, plotted by log2-fold change (FC) (PAB vs. CTL) on the x-axis and −log10(adjusted p-value) on the y-axis. Significantly upregulated and downregulated genes (adjusted p ≤ 0.05 and |log2(FC)| ≥ 0.5) are shown in red and blue, respectively; non-significant genes are shown in grey. Dashed lines indicate significance thresholds. The total number of differentially expressed genes is reported for each group. Selected extracellular matrix and remodeling-associated genes in each group are annotated.

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  Figure 6.. Sex-specific extracellular matrix remodeling signature and enriched biological pathways in the pressure-overloaded tricuspid valve. (a) Heatmap of log2 counts per million (CPM) for a curated panel of immune, extracellular matrix (ECM)/matricellular, matrix-modifying (Matrix Mod.), valve interstitial cell activation–associated (VIC Act.), and apoptosis genes in tricuspid valve tissue. Expression values are averaged within sex and group combinations (Female, castrated male (C-Male), and non-castrated male (NC-Male); PAB vs. CTL) and are z-scored per gene to highlight relative differences expression across the six groups. Genes were selected a priori based on established associations with ECM structure, matrix remodeling, inflammation, and cellular activation. (b) Curated Gene Ontology (GO) biological process enrichment analysis of upregulated genes within each sex-stratified comparison. The panel focuses on ECM-, proteolysis-, apoptosis-, and immune-associated pathways. Each point represents a sex-specific enrichment result for a given pathway; point size reflects gene count and the x-axis denotes −log10(adjusted p-value). Pathways with FDR < 0.1 are considered significantly…