有氧运动通过抑制心磷脂重塑酶ALCAT1表达改善心梗心脏重塑的机制研究

Myocardial infarction (MI) is the primary of five leading causes of death which lead to the loss-of-life in global population. The sifting of prevention means, therapeutic methods and targets will be of great significance. Oxidative stress is the core link of MI occurrence and development. ROS results in the pathological remodeling of cardiolipin (CL). Acyl-CoA:lysocardiolipin acyltransferase-1(ALCAT1), which regulates the pathological remodeling of CL, is a new target of cardiovascular disease occurrence and development. Basic and clinical research found that aerobic exercise can improve heart function significantly, but there is no report about the mechanisms and the relationship between ALCAT1 characterization and heart dysfunction in MI heart. This subject is intended to establish the MI model in ALCAT1 gene knockout mouse and administrate the aerobic exercise intervention; using the ALCAT1 overexpression and interference technology on H9C2 cell culture in hypoxia conditions, which is intervened by the SIRT1 agonist and inhibitor in order to investigate the ALCAT1 characterization and whether aerobic exercise improves the heart function through ALCAT1 in MI heart in vivo and in vitro, as well as the related signaling mechanism by multi- techniques such as electron microscope, confocal, immunofluorescence, WB, RT-qPCR, gene overexpression and knockout in the levels of tissue, cell, protein and molecular. Providing the possible targets and ideas on the sifting of prevention means and therapeutic methods in MI caused heart failure has the important theoretical and clinical guiding significance.

心梗是造成全球人口“寿命损失”前五大死因之首，其防治手段方法与治疗靶点筛选意义重大。氧化应激是心梗发生发展的核心环节，活性氧导致心磷脂(CL)发生病态重塑，心磷脂重塑酶ALCAT1可调节CL病态重塑，是心血管病发生发展的新靶点。基础与临床研究发现运动改善心功能的机制涉及心肌和血管再生、交感副交感调控、自噬与凋亡等途径，但从心磷脂重塑途径探讨运动改善心梗心脏重塑及其机制研究尚缺。本课题采用ALCAT1-/-小鼠建立心梗模型，进行有氧运动干预；采用ALCAT1过表达和敲除的H9C2细胞低氧培养，以组蛋白去乙酰化酶SIRT1激动剂、抑制剂干预，在整体和细胞、蛋白及分子层面，利用电镜、共聚焦显微镜、荧光免疫组化、WB、RT-qPCR、基因敲入和敲除等技术，探讨有氧运动通过抑制心磷脂重塑酶ALCAT1表达改善心梗心脏功能的机制。为防治心梗手段和方法筛选提供可能靶标和思路，具有重要理论和临床指导意义。

心梗严重损害心功能并累及诸多远隔器官。本项目采用野生型SD大鼠/C57小鼠、ALCAT1敲除小鼠心梗模型和有氧运动干预，ALCAT1过表达慢病毒转染细胞培养干预，检测心梗面积和心功能、心肌、骨骼肌、肝、肾组织和循环ALCAT1、Irisin、FGF21水平，HIF-1α、SIRT1和PGC-1α、COXⅠ、Akt等通路相关蛋白、细胞钙信号蛋白、细胞和线粒体自噬与凋亡相关蛋白。结果发现：（1）有氧运动显著抑制心梗大鼠心肌ALCAT1表达和氧化应激与过度自噬，改善受损线粒体融合和线粒体功能，显著缩小心梗面积，改善心功能。（2）有氧运动显著降低心梗小鼠心肌ALCAT1表达和氧化应激，上调FGF21和FGFR1表达，激活PI3K/ Akt/SERCA2a通路、抑制ERs和IRE1α/JNK凋亡通路，改善心肌线粒体自噬受损并抑制细胞凋亡，改善心功能；小鼠敲除alcat1或H9C2细胞外源性AICAR或rhFGF21干预，均显著降低心梗小鼠氧化应激损伤，抑制H9C2细胞ALCAT1表达、ERs和IRE1α/JNK凋亡通路，抑制H2O2诱导的H9C2凋亡，改善线粒体自噬受损。（3）有氧运动显著降低心梗小鼠肝ALCAT1表达并升高FGF21表达，改善线粒体自噬受损，降低肝细胞ERs和细胞凋亡，改善肝功能。（4）有氧运动促进心梗小鼠肾脏FNDC5/Irisin表达，抑制ALCAT1表达和氧化应激与细胞凋亡，改善肾功能；敲除alcat1发挥有氧运动对心梗小鼠肾损伤的保护作用；rhIRISIN干预显著激活FNDC5/Irisin-AMPK-Sirt1-PGC-1α/ AKT通路，降低H2O2和ALCAT1诱导的NRK肾细胞氧化应激和凋亡。（5）不同强度运动均显著抑制正常和心梗小鼠骨骼肌ALCAT1表达，升高Irisin表达，激活Irisin-PI3K-Akt通路，改善骼肌细胞自噬受损和凋亡，Irisin-PI3K-Akt-mTOR在抑制心梗诱导骨骼肌细胞凋亡，促进细胞增殖和蛋白合成，改善骨骼肌减少中发挥作用。该研究将为心梗心脏运动康复手段与作用靶标筛选提供实验依据。本项目发表SCI论文6篇，CSSCI/CSCD论文11篇，引用35篇次；举办国际会议1次，参加学术会议5次，邀请做特邀报告和专题主报告2个；培养毕业博士生1名，在读4名，毕业硕士生7名；获留学基金委联合培养博士生计划项目2项。