COX5A/5B亚基表达转变介导冠状动脉微栓塞后线粒体能量代谢紊乱及心肌结构损伤

Cardiac injury induced by coronary microembolization severely influences the clinical prognosis of coronary artery disease after coronary intervention. It has been demonstrated that mitochondrial dysfunction could be one of most critical initial factors in cardiac injury. Previous studies proved that cytochrome c oxidase (COX) played an important role in mitochondrial energetic metabolism. However,it was unclear whether the structural or activity changes of COX involved in cardiac injury after coronary microembolization. Our preliminary experiments found that tumor necrosis factor-α (TNF-α) was increased after coronary microembolization. Importantly, subunits COX5A and 5B expression were alteration. Meanwihle, cardiomyocytes stimulated by TNF-α, not only disrupted mitochondria membrane potential, but also changed the arrangement of cardiac actin. Therefore, we supposed that the structural and activity changes of COX participate in cardiac injury after coronary microembolization. In this study, mice coronary microembolization model and cardiomyoctyes with differential expression of COX5A/5B were developed. The activity of COX was detected by spectrophotometry. Then, we try to evaluate the mitochondrial function from mitochondria membrane potential, ATP synthesis and status of mitochondrial permeability transition pore. Myocardial structural proteins, such as actin, tropomyosin and connexins, will be detected by confocal imaging and western blot in order to clarify the mechanism between COX activity and cardiac injury. Above all, these fingdings will provide novel evidence for clinical treatment of coronary microembolization.

冠状动脉微栓塞所致的心肌损伤严重影响冠心病介入治疗的预后。线粒体功能紊乱是心肌损伤的重要始动因素，既往研究发现，细胞色素c氧化酶（COX）是线粒体能量代谢的关键。然而，COX结构或功能改变是否介导微栓塞后的心肌损伤尚不明确。我们前期发现，小鼠冠脉微栓塞后肿瘤坏死因子α（TNF-α）表达升高，心肌COX-5A和COX-5B亚基比例失调（蛋白组学iTRAQ技术）；应用TNF-α刺激心肌细胞也发现线粒体膜电位下降和肌动蛋白排列改变；故推测COX结构及功能改变可能参与冠脉微栓塞后的心肌损伤。本研究拟构建COX5A/5B差异性表达的微栓塞小鼠和心肌细胞，分光光度法检测COX活性，从线粒体膜电位、ATP合成和渗透性转换孔道开发状态评价线粒体功能；共聚焦显像和蛋白印记法检测心肌结构蛋白变化（肌动蛋白、原肌球蛋白、连接蛋白），明确COX活性对心肌结构的影响及机制，进而为冠状动脉微栓塞的防治提供新的思路。

冠状动脉微栓塞所致的心肌损伤严重影响冠心病介入治疗的预后。线粒体功能紊乱是心肌 损伤的重要始动因素，既往研究发现，细胞色素c氧化酶（COX）是线粒体能量代谢的关键。然而，COX结构或功能改变是否介导微栓塞后的心肌损伤尚不明确。我们前期发现，小鼠冠脉微栓塞后肿瘤坏死因子α（TNF-α）表达升高，心肌COX-5A和COX-5B亚基比例失调（蛋白组学iTRAQ技术）；应用TNF-α刺激心肌细胞也发现线粒体膜电位下降和肌动蛋白排列改变；故推测COX结构及功能改变可能参与冠脉微栓塞后的心肌损伤。本次研究围绕构建COX5A差异性表达的小鼠和心肌细胞，应用阿霉素DOX干预后，探索COX5A的保护作用；研究发现DOX可以显著下调COX5A蛋白表达。过表达COX5A在体内外水平有效地保护了DOX诱导的氧化应激、心肌细胞凋亡、线粒体损伤和心功能不全。在机制上，我们发现上调COX5A通过激活PI3K/Akt信号，从而减轻DOX诱导的氧化应激、心肌细胞凋亡和线粒体功能障碍。同时本次项目研究共发表论文11篇，其中SCI论文10篇（均标注课题基金号），培养研究生5名（均已毕业），课题组参加国际会议交流3人次。