1型多聚ADP核糖合成酶在缺血再灌注损伤中调控心肌细胞分泌exosome的作用及机制研究

Ischemia/reperfusion (I/R) injury induces myocardium damage after myocardial infarction. As highly developed cells, cardiomyocytes could no longer regenerate in a short time. It is important to keep more cells alive in I/R injury so as to improve clinical outcomes of myocardial infarction. Recently, a 30-100nm cup-shaped vesicle called exosome was reported to be secreted by cardiomyocytes after I/R injury. It could carry proteins, such as HSP60 and TNF-α, and promote cardiomyocyte apoptosis. Poly(ADP-ribose) polymerase 1 (PARP1) is a ubiquitous nuclear DNA base repair enzyme present in eukaryotes, which could be activated by I/R injury. Excessive activation of PARP1 was known to promote cardiomyocyte apoptosis. But the underlying molecular mechanism remains elusive. In our study, we discovered that inhibition of PARP1 by enzymatic inhibitors or specific siRNA could reduce exosome secretion by cardiomyocytes after “hypoxia/reoxygenation” stimulation. This suggests an important role of PARP1 in mediating exosome secretion. In this project, we aim to investigate the role of PARP1 in cardiomyocyte apoptosis and its exosome secretion, so as to uncover the detailed mechanism of cardiomyocyte apoptosis induced by I/R injury. This project may provide more experimental evidence in treatment of myocardial infarction.

缺血再灌注损伤是心肌梗死后引起心肌细胞凋亡坏死的常见原因。由于心肌细胞短期内不可再生，降低缺血再灌注所致心肌损伤将能够改善心肌梗死治疗的预后。晚近的研究表明，缺血再灌注损伤通过促使心肌细胞分泌携带HSP60、TNF-α等蛋白的外泌体（exosome）来加速心肌细胞凋亡的发生。1型多聚ADP核糖合成酶（PARP1）是广泛存在于真核细胞内的核蛋白酶，能在缺血再灌注后被激活。过度激活的PARP1能够促进凋亡发生，但具体机制不清。申请人发现，在体外培养的人心肌细胞系中，抑制PARP1表达或酶活性，可以减少“缺氧/复氧”刺激下心肌细胞exosome的分泌，提示PARP1在由exsome介导的细胞凋亡的发生中可能起着关键作用。本项目拟研究PARP1在心脏缺血再灌注过程中对细胞凋亡和分泌exosome的影响及具体调控机制，进一步阐明缺血再灌注致心脏损伤的分子机制，为心肌梗死的治疗提供理论和实验依据。

心肌细胞作为终末分化细胞，几乎没有分裂增值能力。心肌缺血及再灌注损伤导致大量心肌细胞发生凋亡及坏死，损失的心肌不可再生，由不具有收缩舒张功能的间质组织替代。这直接导致了心室重塑，最终可出现心律失常，心力衰竭，甚至心脏破裂等严重并发症，危及病人生命。因此，如何降低心肌梗死后心肌细胞的死亡成为研究热点问题。晚近的研究表明，外泌体（exosome）作为一种新型信号传递载体，通过携带HSP60、TNF-α等蛋白在心肌细胞之间传递，加速邻近心肌细胞凋亡的发生。这可能成为阻断心肌细胞发生级联凋亡反应的有效靶点。但是我们对这种信号传递方式的调控知之甚少。我们课题组长期致力于1型多聚ADP核糖合成酶（PARP1）在心血管疾病发生、发展方面调控作用的研究。PARP1是广泛存在于真核细胞内的核蛋白酶，能在缺血再灌注后被激活。过度激活的PARP1能够促进凋亡发生，但具体机制不清。本课题的研究发现，PARP1参与调控缺氧/复氧刺激下心肌细胞分泌外泌体，并改变外泌体中miRNA的组成成分。这种调控作用是依赖于与RNA结合蛋白hnrnpA2B1发生结合，并介导其多聚ADP核糖化反应实现的。抑制PARP1能够有效减少心肌细胞分泌的外泌体中促进凋亡的miRNA含量，从而减少心肌细胞的凋亡，减少心功能的损伤，降低梗死面积。因此，我们的研究结果提示，抑制PARP1活性可能成为减轻心肌梗死后心肌细胞死亡的有效治疗靶点，具有重要临床转化潜力。