MARCH5通过RACGAP1-DRP1相关的线粒体质量控制改善主动脉瓣钙化。
MARCH5 ameliorates aortic valve calcification via RACGAP1-DRP1 associated mitochondrial quality control.
作者信息
Zhang Jialiang, Zhang Yaoyu, Lei Wenhua, Zhou Jing, Xu Yanjiani, Hao Zhou, Liao Yanbiao, Huang Fangyang, Chen Mao
机构信息
Laboratory of Cardiac Structure and Function, Institute of Cardiovascular Diseases, West China Hospital, Sichuan University, Chengdu 610041, PR China; Department of Cardiology, West China Hospital, Sichuan University, Chengdu, Sichuan, China; Cardiac Structure and Function Research Key Laboratory of Sichuan Province, West China Hospital, Sichuan University, Chengdu 610041, PR China.
Laboratory of Cardiac Structure and Function, Institute of Cardiovascular Diseases, West China Hospital, Sichuan University, Chengdu 610041, PR China; Department of Cardiology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
出版信息
Biochim Biophys Acta Mol Cell Res. 2025 Mar;1872(3):119911. doi: 10.1016/j.bbamcr.2025.119911. Epub 2025 Jan 27.
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.
背景
线粒体E3泛素连接酶（MARCH5）是维持线粒体功能的重要调节因子。我们的目的是研究MARCH5在主动脉瓣钙化中的作用及机制。
方法
采用蛋白质印迹法分析钙化和未钙化的人主动脉瓣中MARCH5的表达。使用透射电子显微镜评估线粒体碎片化情况。用人成骨培养基（OM）诱导人主动脉瓣间质细胞（HVICs）的成骨分化，通过蛋白质印迹法和茜素红染色进行确认。分别使用线粒体追踪染料和海马分析仪评估线粒体形态和氧化磷酸化。使用MARCH5基因敲低和载脂蛋白E基因敲除的高脂饮食喂养小鼠来研究主动脉瓣钙化。
结果
钙化瓣膜中线粒体质量控制受损，钙化瓣膜中MARCH5蛋白水平也降低。抑制MARCH5会损害线粒体质量控制，增加线粒体应激并加速经OM处理的HVICs的成骨转化。而MARCH5过表达则有相反的作用。免疫共沉淀、质谱分析和分子对接发现MARCH5与Rac GTP酶激活蛋白1（RACGAP1）相互作用并促进其泛素化，导致线粒体质量控制受损。抑制RACGAP1可逆转经OM处理的HVICs中MARCH5沉默诱导的成骨转化。在RACGAP1抑制下沉默动力相关蛋白1（DRP1）没有额外益处。在体内，MARCH5缺乏会促进主动脉瓣钙化，而抑制RACGAP1可逆转MARCH5缺乏小鼠的主动脉瓣钙化。
结论
MARCH5下调促进RACGAP1泛素化，激活DRP1并损害线粒体质量控制，这导致主动脉瓣钙化。这确定了主动脉瓣钙化的一个潜在治疗靶点。
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