EETs调控TFAM乙酰化在有氧康复运动改善心肌梗死后心力衰竭中的作用和机制探讨

Exercise training has been reported to improve left ventricular function after post-infarction heart failure in patients. However, the mechanism remains unknown. Mitochondrial DNA (mtDNA) content as well as the expression of mtDNA-encoded genes transcription were markedly decreased in the failing heart. Mitochondrial transcription factor A (TFAM) is essential for mtDNA contents and transcription. Peroxisome proliferator-activated receptor-coactivator-1α (PGC-1α) was recently reported to be the target of epoxyeicosatrienoic acids (EETs). One of the seven mammalian sirtuins, sirtuin 3 (SIRT3), a mitochondrial deacetylase, activates Sirt3 gene transcription through bound the proximal Sirt3 gene promoter region. We found that aerobic training increase the plasma concentration of EETs in mice with post-infarction heart failure. We also demonstrated that aerobic exercise increased mtDNA contents and mitochondrially encoded gene expression, as well as decreased the acetylation levels of TFAM. Therefore we speculate that aerobic exercise increased mtDNA contents and transcription by regulating the acetylation levels of TFAM through Sirt3 expression, which was activated by PGC-1α , thereby improving the contractility of the failing myocardial cells. In order to prove this point, firstly, it will be demonstrated the effect of aerobic exercise on inducing mtDNA contents and transcription after post-infarction heart failure in the present study. To identify possible targets and to further define the mechanism by which deacetylation of mitochondrial proteins results in increased mtDNA contents and transcription, purified myocardial mitochondria were isolated from mice, and analyzed by nanoflow liquid chromatography tandem mass spectrometry (LC MS/MS). TFAM acetylation was identified and determined it contained how much acetylation sites. Lysine-to- arginine mutations were made and expression vectors for a single or multi-site mutant TFAM were generated, transfected in cells and assayed for acetylation. To determine whether the acetylation state of TFAM modified its role in mtDNA contents and transcription after aerobic exercise. And to test the effect and mechanism of EETs to deacetylate TFAM after aerobic exercise. This study will improve the understanding the role of cardiac rehabilitation exercise on improving heart failure and provide new theoretical basis.

有氧康复运动有效改善心梗后心衰，但机制不明。心肌线粒体基因组（mtDNA）含量和转录与心衰密切相关，其受线粒体转录因子A（TFAM）调控。已知环氧二十碳三烯酸（EETs）增加线粒体去乙酰化酶Sirt3表达，而Sirt3表达依赖于细胞核转录辅助激活因子PGC-1α结合至其启动序列。我们发现有氧运动增加心梗后心衰小鼠血浆EETs浓度、心肌mtDNA含量和转录、伴线粒体TFAM乙酰化降低。因此推测有氧运动通过上调EETs，促使PGC-1α启动Sirt3表达以降低心肌TFAM乙酰化，增加mtDNA含量和转录而改善心梗后心衰。为此本项目首先明确有氧运动增加mtDNA含量和转录，接着采用液相质谱探明TFAM乙酰化修饰位点，点突变验证其乙酰化修饰状态并证实其参与有氧运动调控mtDNA，再进一步证实EETs介导有氧运动调控TFAM乙酰化并探讨相关机制。本研究将为有氧运动改善心梗后心衰机制的认识提供新思路。

心力衰竭（简称“心衰”）是多种心血管疾病发展的最后阶段，严重危害人类健康。心肌梗死是导致心衰最常见的原因。因此，改善心肌梗死后心功能具有重要临床意义。有氧康复运动有效改善心梗后心衰，但机制不明。转录因子KLF14可调节脂质代谢和胰岛素抵抗等多种与动脉粥样硬化形成和发展相关的病理过程。KLF14基因rs4731702位点单核苷酸多态性与冠心病的发病风险有较高的相关性，提示KLF14在心肌梗死中扮演重要作用。前期研究证实小鼠心肌梗死区KLF14 mRNA及蛋白表达明显降低，其中线粒体蛋白为甚；我们进一步研究者证实过表达KLF14后小鼠心肌梗死面积显著缩小，心肌纤维化程度明显降低，小鼠心功能得到改善。心梗时电镜观察到心肌线粒体损伤明显增多，包括线粒体断裂、延长、异质性增加，而过表达KLF14可逆转这种病理损伤，此外，H2O2可升高心肌细胞Bax表达，降低Bcl-2的表达，降低Bcl-2/Bax比值，促进线粒体细胞色素c的释放，从而增加氧化应激诱导的凋亡，而过表达KLF14可逆转这种改变。同时KLF14通过调控参与细胞凋亡的基因表达来减少凋亡。因此，KLF14可通过改善线粒体功能，从而改善心梗后心功能。同时观察到，过表达KLF14可明显上调心肌细胞过氧化物酶体增殖物激活受体γ共激活因子1α（PGC-1α）的表达，提示①KLF14可能通过上调PGC-1α调节基因转录，从而调控线粒体生物合成，减少线粒体损伤，并减少细胞凋亡；或者②KLF14可通过上调PGC-1α，促进Bcl-2转录的同时抑制Bax转录，从而减少凋亡，改善心肌梗死后心功能。进一步研究发现，有氧运动改善心梗后心衰，同时小鼠血浆EETs明显升高，心肌KLF14蛋白表达上调，予以可溶性环氧化物水解酶抑制剂干预后KLF14表达上调更为明显。因此，推测有氧运动通过EETs升高KLF14表达而提高线粒体代谢，从而逆转心梗后心衰。