PUMA介导缺血-再灌注心肌线粒体自噬异常的分子机制研究

Ischemia/reperfusion (I/R) -induced myocardial injury severely impairs human's health and life. Currently, a key focus for the researchers is to understand how I/R induces myocardial injury. It has been demonstrated that abnormal mitophagy is a key event in myocardial injury after I/R. However, the molecular mechanism by which I/R leads to abnormal mitophagy remains unknown. p53 upregulated modulator of apoptosis (PUMA) is a powerful inducer of cell death. It is expressed at very low levels under physiological condition. In response to damage stimuli, PUMA is up-regulated rapidly to initiate cell death. We previously found that the induction of abnormal mitophagy by I/R is accompanied by the upregulation of PUMA expression in cardiomyocytes. In contrast, knockdown of PUMA inhibits mitophagy. The results indicate that PUMA might be required for I/R-induced abnormal mitophagy. Therefore, in this project, we focus on investigating the effect and mechanism of PUMA on the abnormality of mitophagy in myocardial I/R. The research aims include: (1) To clarify the role of PUMA in abnormal mitophagy induced by I/R. (2) To elucidate whether PUMA-mediated mitochondrial fission is prerequisite for abnormal mitophagy in cardiomyocytes exposed to I/R. (3) To delineate how PUMA is involved in the progress of I/R-initiated abnormal mitophagy. The results of this project may establish the basis and provide the rationale for future study to explore and understand the mechanisms of myocardial I/R injury.

缺血再灌注性心肌损伤严重威胁人类健康和生命，其机制研究是当前研究热点。诱导线粒体自噬异常是缺血再灌注性心肌损伤关键，但是其分子机制尚不清楚。PUMA是一强大的促细胞损伤因子，正常状态下呈低水平表达，在损伤刺激作用下，其可被快速诱导，促进细胞死亡。我们前期实验发现：缺血再灌注在诱导心肌线粒体自噬异常的同时上调PUMA表达，而抑制内源性PUMA表达明显降低线粒体自噬，提示缺血再灌注致心肌线粒体自噬异常可能需要PUMA的参与。因此本项目立题是：PUMA介导缺血再灌注心肌线粒体自噬异常的分子机制研究。研究内容包括：(1) 明确PUMA在缺血再灌注致心肌线粒体自噬异常中作用。(2) 探究缺血再灌注条件下PUMA触发的线粒体分裂异常是否是线粒体自噬异常的前提。（3）阐明PUMA在缺血再灌注条件下如何参与心肌细胞线粒体自噬过程。本研究将为进一步探索和认识缺血再灌注心肌损伤机制提供重要理论基础和学术思想。

缺血/再灌注性心肌损伤严重威胁人类健康和生命，其机制研究是当前研究热点。诱导线粒体自噬异常是缺血/再灌注性心肌损伤关键，但是其分子机制尚不清楚。PUMA是一强大的促细胞损伤因子，正常状态下呈低水平表达，在损伤刺激作用下，其可被快速诱导，促进细胞死亡。我们前期实验发现：缺血/再灌注在诱导心肌线粒体自噬异常的同时上调PUMA表达，而抑制内源性PUMA表达明显降低线粒体自噬，提示缺血/再灌注致心肌线粒体自噬异常可能需要PUMA的参与。因此本项目立题是：PUMA介导缺血/再灌注心肌线粒体自噬异常的分子机制研究。研究内容包括：(1) 明确PUMA在缺血/再灌注致心肌线粒体自噬异常中作用。(2) 探究缺血/再灌注条件下PUMA触发的线粒体分裂异常是否是线粒体自噬异常的前提。(3) 阐明PUMA在缺血/再灌注条件下如何参与心肌细胞线粒体自噬过程。研究发现：(1) 缺血/再灌注条件下，PUMA蛋白表达与线粒体自噬水平相关，在心肌细胞中过表达/敲低内源性PUMA的表达，可以增加/减少线粒体自噬，刺激/抑制心肌细胞损伤。研究结果证实PUMA在缺血/再灌注诱导心肌细胞线粒体自噬异常中发挥重要作用。(2) PUMA通过调控Drp1的线粒体转位，影响Drp1和hFis1的结合，从而参与缺血/再灌注诱导的心肌细胞线粒体分裂，影响线粒体自噬。(3) 过表达GABARAP诱导心肌细胞线粒体自噬异常，PUMA可以与GABARAP相互作用，缺血/再灌注促进PUMA和GABARAP的结合，刺激自噬溶酶体和自噬流的形成。本研究结果不仅为缺血/再灌注诱导心肌损伤的机制和防治研究提供重要的理论基础和实验依据，而且也对心脏分子生物学和细胞生物学具有重要的理论意义。