线粒体PINK1/parkin-VDAC1自噬信号通路对心肌缺血再灌注损伤的调控研究

Mitochondrial permeability transition pore (mPTP) opening was found not only involved in the event of mitochondria-mediated apoptosis, but also related to mitophagy. Autophagy often interacts with apoptosis, leads to blocking or synergistic effects on cell death in different situations and time. And there are controversies on the positive or negative role of autophagy in myocardial ischemia/reperfusion injury (IRI). As we previously demonstrated, Voltage dependent anion channel 1 (VDAC1), a channel that located in the outer membrane of mitochondria, was up-regulated in cardiomyocytes subjected to anoxia/reoxygenation, which subsequently promoted mPTP opening and induced apoptosis. Moreover, VDAC1 is required to recruit Parkin from cytosol to defective mitochondria in PINK1/Parkin autophagy signaling pathway. In light of our previous study, we further presume that VDAC1 is the phosphorylation target of PINK1 and plays key role in the activation of PINK1/Parkin mitophagy pathway. In this project, we plan to use RNA interference and adenovirus transfection in cardiomyocytes and animal experiments. A variety of classical autophagy evaluation indexes are adopted to make a full illustration about the role of PINK1/Parkin-VDAC1 mitophagy pathway in myocardial ischemia period and reperfusion period; and explore the relationship with the VDAC1-mediated mitochondrial apoptosis pathway. The research work provides theoretical basis for mitophagy to be taken as a new therapeutic target of cardiovascular disease.

mPTP开放不但参与线粒体介导的凋亡，而且与线粒体自噬密切相关。自噬与凋亡彼此交互调控，不同条件下，自噬可以抑制凋亡，也可与凋亡共同作用诱发细胞死亡。自噬对心肌缺血再灌注损伤有益还是有害尚有争议。前期研究证明，心肌细胞缺氧/复氧损伤可上调位于线粒体外膜上的VDAC1表达，从而促进mPTP开放及细胞凋亡。VDAC1亦是线粒体PINK1/Parkin自噬通路中募集Parkin从胞浆移位至受损线粒体所必须的蛋白。结合前期研究，我们推测VDAC1可能是PINK1的磷酸化靶点，是保证该自噬通路激活的关键蛋白。本课题拟在细胞及整体水平，采用RNAi和腺病毒转染等技术，利用多项经典的自噬活性检测指标，探讨PINK1/Parkin-VDAC1线粒体自噬通路在心肌缺血期和再灌注期的作用，以及与VDAC1介导的线粒体凋亡通路之间的相互联系，为线粒体自噬成为新的心血管疾病治疗靶点提供理论依据。

本课题建立了基因克隆、RNA干扰、腺病毒构建等研究的技术平台。本研究发现，H9c2心肌细胞经缺氧/复氧（A/R）处理后，VDAC1表达明显增高，激活了PINK1/Parkin线粒体自噬通路，自噬增强，引起线粒体功能紊乱，导致细胞凋亡。动物实验与细胞水平结果一致。大鼠心脏经缺血/再灌注损伤后，自噬水平增高；下调VDAC1或Parkin后，自噬水平下调，心功能有明显改善。本课题为线粒体自噬成为新的心血管疾病治疗靶点提供了理论依据。本课题全面完成研究计划，发表学术论文4篇，培养硕士研究生4名。