Abstract
Background: Aortic stenosis due to deleterious remodeling of the aortic valve is a health concern since it can be corrected only by valve replacement due to the poor knowledge of cellular mechanisms involved. Fibroblastic valvular interstitial cells (VICs) play a central role in valve leaflet stiffness by trans-differentiation into osteoblast-like cells leading to calcification. The TRPM4 (transient receptor potential melastatin 4) cation channel was shown to participate in cardiac fibroblast remodeling. It is also involved in radiation-induced aortic valve remodeling in vivo in mice. We hypothesized that TRPM4 might participate in human VIC transition to osteoblastic phenotype. Methods: Human aortic valves were collected from patients undergoing surgical valve replacement. Isolated VICs were maintained 14 days in culture in standard or pro-calcifying media and submitted to the TRPM4 inhibitor 9-phenanthrol, or small hairpin RNA-TRPM4. Osteogenic differentiation was evaluated by measuring hydroxyapatite crystals by Alizarin red staining and protein expression of osteogenic markers. Results: Western blot on VICs revealed TRPM4 protein expression and channel functionality was confirmed by patch-clamp recordings exhibiting a cationic current sensitive to voltage and internal Ca2+. VICs maintained in pro-calcifying media exhibited a higher mineralization than in standard media, with an increase in osteogenic markers. Mineralization and osteogenic markers (bone morphogenetic protein 2, runt-related transcription factor 2) were decreased when pro-calcifying media contained 9-phenanthrol or small hairpin RNA-TRPM4. Similarly, SMAD1/5 and nuclear factor of activated T-cell pathways were stimulated in pro-calcifying media conditions compared with standard media but reduced by 9-phenanthrol or small hairpin RNA-TRPM4. Conclusions: TRPM4 participates in osteogenic differentiation of human VICs and thus appears as a target to prevent aortic valve remodeling.