心肌细胞线粒体基质金属蛋白酶谷胱甘肽化修饰介导心室扩张和心力衰竭

Matrix metalloproteinases (MMPs) are released as latent enzymes which are proteolytically activated in the extracellular space. They are capable of degrading extracellular matrix components. Increased activity of extracellular MMPs is associated with myocardial remodeling and heart failure. Peroxynitrite induces S-glutathiolation of recombinant human MMP-2, resulting in activation of MMP-2. The activation of intracellular MMP-2 can degrade contractile proteins in cardiac myocytes. Our previous studies have shown that reactive oxygen species mediate oxidative modifications of proteins (for instance, Ca2+ -ATPase), resulting in alterations of their activities. However, it is unknown whether reactive oxygen species mediate S-glutathiolation of mitochondrial MMPs in cardiac myocytes that leads to their activation. It is unclear about intracellular targets of activation of mitochondrial MMPs in cardiac myocytes in heart failure. In this proposal, we will use cardiac myocytes in culture and the animal model of rapid left ventricular pacing-induced heart failure to test the novel hypothesis that oxidative stress induces S-glutathiolation of mitochondrial MMPs in cardiac myocytes that results in activation of MMPs, which mediates down-regulation of mitochondrial Cx43 or Bcl-2 protein and up-regulation of Beclin-1 and Bax proteins, leading to cardiac myocyte autophagy and apoptosis, thereby contributes to ventricular dilation and heart failure. The proposed study will elucidate the novel mechanisms of intracellular activation of MMPs which mediates myocardial remodeling at the molecular, cellular and animal levels and provide the novel targets for the prevention and treatment of myocardial remodeling and heart failure.

基质金属蛋白酶（MMPs）以潜酶形式分泌到细胞外经蛋白酶水解激活，降解细胞外基质成分。MMPs活性增加与心肌重构有关。过氧亚硝基阴离子介导重组人MMP2谷胱甘肽化修饰使其激活。MMP2细胞内激活能降解心肌细胞(CM)收缩蛋白。我们前期研究表明活性氧介导蛋白氧化修饰（例如钙离子ATP酶）致其活性改变。然而，是否活性氧介导CM线粒体MMPs谷胱甘肽化修饰使其激活目前一无所知。在心衰时CM线粒体MMPs激活的细胞内靶点不清楚。本研究将采用CM培养和快速左室起搏诱导心衰动物模型，验证这个新的科学假说：氧化应激诱导CM线粒体MMPs谷胱甘肽化修饰使其激活，介导线粒体Cx43或Bcl-2蛋白下调，致使Beclin-1和Bax上调，诱导CM自噬和凋亡，导致心室扩张和心衰。本课题拟从分子，细胞和整体水平研究，将阐明MMPs细胞内激活的新机制及其介导心肌重构机理，为心衰心肌重构的防治提供新的靶点。

在大鼠心肌细胞培养模型上，我们首先探讨了在生理条件下内源性活性氧在心肌细胞自噬调节中的作用。结果提示在生理条件下，内源性活性氧，至少部分，通过抑制Akt活性而促进心肌细胞自噬流。外源性低浓度过氧化氢（H2O2，48h 孵育）诱导心肌细胞降低与心肌细胞肥大相关。而高浓度H2O2（30 min 孵育）诱导心肌细胞自噬增加与心肌细胞凋亡相关。. 基质金属蛋白酶 （MMPs）在心衰心肌重构过程中起重要作用。然而， 诱导MMPs激活的分子机理及其作用尚未完全阐明。我们探究是否氧化应激介导MMPs谷胱甘肽化修饰致其激活。结果表明，超氧阴离子（O2- ），OONO-或过氧化氢（H2O2）诱导MMPs活性增加。OONO—或H2O2诱导心肌细胞线粒体MMP9谷胱甘肽化修饰及其活性增加。谷氧还蛋白（GRX） 过表达抑制OONO-诱导的MMP9谷胱甘肽修饰。我们进一步研究活性氧介导MMPs谷胱甘肽化修饰致其激活的细胞内靶点和信号传导通路。结果表明，O2- 或H2O2诱导LC3II表达增加，MMPs抑制剂ONO-4817减轻O2-或H2O2诱导LC3II表达增加，提示活性氧诱导MMPs激活，介导心肌细胞自噬。MMP2基因沉默减轻H2O2诱导心肌细胞自噬增加。线粒体自噬抑制剂Mdivi-1抑制H2O2诱导的心肌细胞自噬和凋亡。过氧化氢酶过表达减轻H2O2诱导的心肌细胞MMP谷胱甘肽化修饰和自噬增加。. 在心衰兔模型上，结果表明心肌线粒体MMP9谷胱甘肽化修饰，心肌线粒体MMP活性和心肌细胞自噬活性均增加。心肌线粒体Cx43蛋白降低，Bcl-2蛋白降低，Beclin-1 蛋白表达增加，心肌细胞凋亡增加 。应用抗氧化剂（具有超氧歧化酶和过氧化氢酶双功能的化合物EUK8）抑制线粒体MMPs激活， 降低心肌细胞自噬和凋亡的增加，减轻心肌重构，改善心功能。本研究提供了MMPs细胞内激活的新机制，心肌细胞内MMPs激活的新靶点，抑制氧化应激，抑制MMPs谷胱甘肽化修饰或调节心肌细胞自噬的变化将为心力衰竭心肌重构的预防和治疗提供新的措施。