MARCH5 ameliorates aortic valve calcification via RACGAP1-DRP1 associated mitochondrial quality control

Background: Mitochondrial E3 ubiquitin ligase (MARCH5) as an important regulator in maintaining mitochondrial function. Our aims were to investigate the role and mechanism of MARCH5 in aortic valve calcification. Methods: Human aortic valves, both calcified and non-calcified, were analyzed for MARCH5 expression using western blotting. Mitochondrial fragmentation was evaluated using transmission electron microscope. Osteogenic differentiation of human aortic valvular interstitial cells (HVICs) was induced with osteoblastic medium (OM), confirmed by western blotting and Alizarin red staining. Mitochondrial morphology and oxidative phosphorylation were assessed using MitoTracker and Seahorse, respectively. MARCH5-knockdown and ApoE-knockout mice fed high-fat diet were used to study aortic valve calcification. Results: The mitochondrial quality control was impaired in calcified valves, and the level of MARCH5 protein was also decreased in calcified valves. Inhibition of MARCH5 impaired mitochondrial quality control, increased mitochondrial stress and accelerates osteogenic transformation in OM treated HVICs. While, overexpression MARCH5 has the opposite effects. Co-immunoprecipitation, mass spectrometry and molecular docking found MARCH5 interacted Rac GTPase-activating protein 1 (RACGAP1) and promoted its ubiquitination, leading to impaired mitochondrial quality control. Inhibiting RACGAP1 reversed osteogenic transformation induced by MARCH5 silencing in OM treated HVICs. Silencing dynamin-related protein 1 (DRP1) under RACGAP1 inhibition had no additional benefit. In vivo, deficiency of MARCH5 promoted aortic valve calcification, while inhibition RACGAP1 reversed aortic valve calcification in MARCH5 deficiency mice. Conclusion: Downregulation of MARCH5 promotes RACGAP1 ubiquitination, activating DRP1 and impairing mitochondrial quality control, which contributes to aortic valve calcification. This identifies a potential therapeutic target for aortic valve calcification.