oa Imbalance in Extracellular Matrix Synthesis and Degradation as a Mechanism of Leaflet Weakening in Sporadic Mitral Valve Prolapse in Humans

Sporadic mitral valve prolapse (MVP) is a common valvular disorder affecting 2-3% of the humans worldwide. Unlike MVP in Marfan’s and Ehlers-Danlos syndromes, no specific genetic mutations that impact tissue homeostasis are currently identified. Yet, significant degenerative leaflet changes are observed, manifesting as leaflet weakening and billowing. In this study, we hypothesized that assessing the matrix health by investigating the activity of matrix synthetic and degradative markers would provide insights into the leaflet degeneration processes. Mitral leaflets were obtained from eight humans (N =8) undergoing surgical repair for MVP at our institution. Fresh leaflets were rinsed, stored in sterile PBS and divided into annulus, base or edge regions and used for immunohistochemistry(IHC) and western blotting(WB). Matrix synthetic activity was assessed using assays for: prolyl-4-hydroxylase(P4H–collagen synthesis enzyme), heat shock protein-47(HSP47–chaperone for collagen folding), and lysyl oxidase(LOX–collagen cross-linking). Matrix degradation was assessed using assays for matrix metalloproteases: MMP-1(collagen I degradation), MMP-3(collagen III degradation), and MMP-9(collagen V degradation). Tissue sections were imaged and quantitative analysis was performed using an image thresholding technique. Cell viability was confirmed using DAPI, and overall tissue structure assessed using H and E staining. Tissue section closest to the mitral annulus served as the control, against which the belly and edge expressions were compared. In the synthetic pathway, P4H activity decreased from 4±1.5 at the annulus to 2±1.3 at the belly (2X) and increased to 17±12 at the edge (4X). HSP47 activity was 3.2±2 in the annular section that increased to 5.1±2 in the belly (1.6X) and 4±1.3 in the edge (1.2X). LOX expression slightly increased from the annulus to the belly (1.2X), but was significantly higher in the edge (10X). In the degradative pathway, MMP-1 expression was 8.4±6.3 at the annulus, which decreased to 5.6±2.8 in the belly (1.5X), and 6±2 in the edge (1.4X). MMP-3 expression significantly increased from the annulus to the edge, with 2.3±2 at the annulus, 7.7±5 at the belly (3.3X) and 24.5±10.6 at the edge (10.4X). MMP-9 expression decreased from the annulus (1.3±0.4) to the belly (0.7±0.2; 1.7X) and increased to 3±1 in the edge(2.4X). Collagen biosynthesis is adequate in the edge as evident from increased P4H activity, however nominal HSP47 activity indicates poor fibril maturation and folding, resulting in a weak matrix in spite of good cross linking from higher LOX expression. Increased MMP-3 and 9 activities in the edge signify degradation of collagen III and V, which may further weaken the matrix. Studies to investigate the regional differences in genetic, cellular and molecular pathways in MVP and the potential role of the elevated stress in leaflet degradation are necessary.