Vinculin调控肌动蛋白细胞骨架及线粒体功能在压力超负荷性心力衰竭中的保护作用及机制研究

Myocardial damage under pressure overload is an important pathogenic factors of heart failure. Perception and transmission of the mechanical signal by membrane proteins is a critical starting link of this process. Previous research suggests that vinculin, a membrane protein, may play a key role in protecting myocardium from injury from pressure overload through the regulation of actin cytoskeleton and mitochondrial function. However, the specific function and molecular mechanism is unclear. In this project, the protective effects of vinculin against myocardial injury from pressure overload would be observed from animal and cell level respectively, using the ultrasonic doppler technology, visualization of single cell edge detection technology, and detecting the heart failure and myocardial damage index. The regulation of actin cytoskeleton assembly was observed by immunofluorescence staining, transmission electron microscopy and molecular biology techniques and mitochondrial function was detected from biosynthesis and oxidative stress. Then immune coprecipitation technology was used to explore actin regulatory proteins interacting with vinculin and specific blocker or neutralizing antibody was used to further clarify the molecular mechanisms of vinculin in regulating the actin cytoskeleton assembly and mitochondrial function. This project aims to explore a new effective strategy for preventing pressure overload induced heart failure.

压力超负荷下的心肌损伤是心力衰竭的重要致病因素，胞膜蛋白对力学信号的感知和传递是这一病理生理过程的始动环节。前期研究提示：胞膜蛋白vinculin可能通过调控肌动蛋白细胞骨架和线粒体功能在心肌对抗压力超负荷损伤中发挥关键作用。然而其具体作用和分子机制尚不明确。本项目我们将分别从动物和细胞水平上，利用超声多普勒技术、可视化单细胞动缘检测技术、心力衰竭和心肌细胞损伤指标物测定等观察vinculin高表达对压力超负荷下心肌的保护作用，利用免疫荧光染色、透射电镜及分子生物学技术观察对肌动蛋白细胞骨架组装的调控，从生物合成和氧化应激两方面观察对线粒体功能的影响，并利用免疫共沉淀技术探寻与vinculin相互作用的肌动蛋白调节蛋白，采用特定阻滞剂或中和抗体阻断其相互作用后，进一步阐明vinculin调控肌动蛋白细胞骨架和线粒体功能的分子机制。为压力超负荷性心力衰竭的防治提供新的有效的干预策略。

压力超负荷下的心肌损伤是心力衰竭的重要致病因素，胞膜蛋白对力学信号的感知和传递是这一病理生理过程的始动环节。本项目我们分别从动物和细胞水平上，利用超声多普勒技术、组织病理学观察、心力衰竭和心肌细胞损伤指标物测定等观察vinculin高表达对压力超负荷下心肌的保护作用，利用免疫荧光染色、透射电镜及分子生物学技术观察对肌动蛋白细胞骨架组装的调控，从生物合成和氧化应激两方面观察对线粒体功能的影响，并利用免疫共沉淀技术探寻与vinculin相互作用的肌动蛋白调节蛋白。证实了胞膜蛋白vinculin通过调控肌动蛋白细胞骨架和线粒体功能在心肌对抗压力超负荷损伤中发挥关键作用，并深入研究了相关的分子机制。体内实验发现vinculin高表达可显著减轻压力超负荷诱导的心肌重构并改善心力衰竭，细胞实验显示vinculin高表达可减轻TGF-a刺激下心肌细胞的损伤和actin细胞骨架的紊乱，进一步研究证实Vinculin通过Rho家族蛋白RhoA和Rac1,调节心肌细胞内actin细胞骨架的聚集，通过Integrin 4和FAK与细胞外基质联系，调节心肌纤维化过程。此外，vinculin通过SIRT1影响心肌细胞线粒体功能，并且这一作用依赖于肌动蛋白细胞骨架。本研究揭示了vinculin在心肌对抗压力超负荷中的作用及分子机制，为压力超负荷下的心力衰竭提供潜在有效的基因治疗策略。