线粒体Omi/HtrA2及其下游通路在加重衰老心肌缺血/再灌注损伤中的机制研究

It is well recognized that age is a major independent risk factor for morbidity and mortality in coronary heart disease. The increased rate of myocardial cell death in the aging heart exacerbates myocardial ischemia/reperfusion (MI/R) injury. Our original research found that the increased expression of Omi/HtrA2,a potent mitochondria pro-apoptotic protein, enhanced MI/R injury in aging hearts; however, the mechanisms responsible for this age-related pathologic change remain unclear. Therefore, we will first make the cardiac-specific overexpression of mitochondrial Omi in transgenic mice, and then obeserve its effect on cardiac apoptosis and/or the aging process. A mitochondrial permeability transition inhibitor was used to determine not only the leakage of Omi/HtrA2 from the mitochondria to the cytosol but also if the increased mitochondrial Omi could enhance MI/R injury through degrading mitochondrial anti-apoptotic protein and promoting cell death in the aging heart. Moreover, either mitochondrial Omi or mitochondrial Omi with destroyed serine protease activity plasmid was transfected into the caridiomyocyte to determine whether the serine protease activity were involved in age-related cardiomyocyte death. Finally, we will utilize the cardiomyocyte co-transfected with mitochondria Omi and HAX-1 to identify the mechanisms and contributions of the Omi-HAX-1 pathway in exacerbated MI/R injury. Based on the findings from our studies, we hope to identify the possible role and significance of increased Omi in the aging heart.

衰老是冠心病的主要独立危险因子，随年龄增加而发生的心肌细胞进行性死亡具有重要作用。我们的原创性研究发现，衰老大鼠心肌组织中表达增加的Omi可加重缺血/再灌注损伤，但这种与年龄相关的病理改变机制还不清楚。本项目在此基础上通过构建心肌特异性过表达线粒体Omi的转基因小鼠，动态观察其心肌细胞凋亡和/或老化的特点，明确线粒体中增多的Omi除了通过线粒体转位机制，还可直接抑制线粒体抗凋亡蛋白，加重心肌缺血/再灌注损伤；通过构建过表达线粒体Omi或线粒体无丝氨酸蛋白酶活性Omi的心肌细胞，探讨线粒体Omi的丝氨酸蛋白酶活性在心肌细胞凋亡中的新机制；通过共转染线粒体Omi或HAX-1、RNA干扰技术，研究Omi-HAX-1通路在衰老心肌缺血/再灌注损伤敏感性增加中的贡献。本课题拟从分子、细胞和整体水平证实我们的假说：衰老心肌表达增加的Omi通过线粒体转位和"非转位"机制加重衰老心脏的缺血/再灌注损伤。

衰老是冠心病的重要独立危险因子，随年龄增加而发生的心肌细胞进行性死亡在冠心病的发生发展中扮演着重要角色。我们已有研究发现，衰老大鼠心肌组织中表达增加的Omi/HtrA2 可加重缺血/ 再灌注损伤，但这种与年龄相关的病理改变机制还未完全阐明清楚。在本研究中，我们首先明确了衰老心肌中Omi/HtrA2表达增高，且缺血/再灌注后Omi/HtrA2由线粒体转位入胞浆增加，增加的Omi/HtrA2通过转位机制使衰老心肌中抗凋亡连锁蛋白（XIAP）表达下降，从而发挥促凋亡作用，最终使衰老心肌对于缺血/再灌注损伤的敏感性增加。本研究进一步对Omi/HtrA2的促凋亡作用进行研究发现，衰老心肌组织中的Omi/HtrA2不仅可以通过转位机制降解XIAP，还可以通过非转位机制引起线粒体细胞色素C的释放，从而激活caspase依赖的凋亡途径，促进心肌细胞的凋亡。在探讨Omi/HtrA2促进细胞色素C释放的机制研究中，我们发现表达增加的Omi/HtrA2可以通过降解HAX-1改变线粒体的膜电位，促进细胞色素C的释放，提示线粒体Omi-HAX-1通路在促衰老心肌凋亡中的有着重要贡献。 为了进一步验证心肌中过多的Omi/HtrA2促凋亡及增加缺血/再灌注损伤敏感性的作用，我们构建了心脏特异性过表达Omi/HtrA2的转基因小鼠和过表达线粒体和胞浆Omi/HtrA2 的H9C2心肌细胞系，结果表明心肌特异性过表达Omi/HtrA2的转基因小鼠的心脏结构及功能出现明显的损伤，过表达Omi/HtrA2的心肌细胞对缺氧复氧损伤的敏感性增高。为明确Omi/HtrA2在衰老心肌组织中表达升高的的机制，我们对Omi/HtrA2的启动子进行分析，发现热休克因子（HSF1）及p53可作为Omi/HtrA2启动子的转录因子调控Omi/HtrA2的表达。以上这些研究从衰老心肌Omi/HtrA2增加的角度入手，揭示了衰老过程中心肌细胞凋亡在衰老心肌缺血/再灌注损伤中的作用、机制及可能的意义，为把 Omi/HtrA2作为潜在的治疗心肌缺血/再灌注损伤的靶点提供更坚实的实验依据。

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