Calpain对心肌再灌注阶段的线粒体自噬的调控机制

The mitochondrial damage is an important step in myocardial ischemia/reperfusion injury (I/R)，and impaired mitochondria were isolated and degraded through autophagy. Mitophagy is an important endogenous protective mechanism. We found that calpain,calcium-dependent cysteine protease, deteriorates the I/R injury in our cellular experiments. Moreover, mitochondrial autophagy is inhibited by upregulation of calpain activity, and the expression of the key proteins, Atg5 and p62, involved in mitophagy pathway is also decreased. Whereas, the exact mechanism remains unknown. Recent studies have revealed that mitophagy is negatively regulated by calpain . We speculate that calpain inhibits mitophagy via degradation of Atg5 and p62, resulting in the accumulation of damaged mitochondria and leading to cell injury. Our reaserch intends to investigate the mitophagy and mitochondria functions in in vitro I/R model by siRNA and adenovirus transfection, RT-PCR, Western blot, fluorescent probes, and laser scanning confocal microscope. Then in vivo I/R model based on cardiac-specific calpain gene knockout mice will be used to investigate how calpain regulates mitophagy in the global level. This study might provide new insights and therapeutic targets for I/R injury treatment.

线粒体损伤是心肌缺血/再灌注损伤(I/R)的重要环节，受损线粒体被以自噬方式隔离和降解，线粒体自噬是重要的内源性保护机制。我们在细胞实验中发现，calpain(钙依赖的半胱氨酸蛋白酶）加重 I/R损伤，与抑制线粒体自噬有关；且calpain活性上调时，参与线粒体自噬通路的关键蛋白Atg5和p62下调，但具体机制未明。新近研究发现线粒体自噬受到calpain的负向调控。推测calpain通过降解Atg5和p62而抑制线粒体自噬，使受损线粒体积累，导致I/R损伤。本项目拟在细胞实验的基础上，以siRNA、腺病毒基因转染干预各关键蛋白的表达，以RT-PCR、Western blot、荧光探针、激光共聚焦显微镜等检测线粒体自噬及相应的线粒体功能的变化；体外I/R模型将应用心肌特异性calpain基因敲除小鼠，从整体层面探讨calpain的调控机制。本项目有望为防治I/R提供新的思路和靶点。

本课题组建立乳鼠原代心肌细胞培养的缺氧/复氧（H/R）模型，实验证明calpain在缺氧和复氧阶段蛋白表达量和活性随时间延长而上调，受到抑制剂干预而下调；同时细胞自噬随缺氧时间延长而增强，抑制calpain使自噬流增强，并改善心肌酶释放、活力下降、凋亡增加等心肌H/R损伤。基因沉默技术证明calpain1是起作用的亚型。Western Blot和RT-PCR提示H/R时自噬相关蛋白Atg5和p62的下调与calpain的激活有关，其他自噬相关蛋白如Atg7、Beclin1则不受calpain影响。进一步通过重组腺病毒转染过表达Atg5和p62，发现Atg5过表达可增强H/R诱导的细胞自噬，但不产生细胞保护作用；过表达p62可增强自噬，同时增加细胞活力，减少凋亡，具有抗H/R损伤作用，p62可能是calpain作用的靶蛋白。P62是线粒体自噬的关键蛋白，而线粒体自噬能清除损伤线粒体，减少ROS产生和内源性凋亡。通过检测线粒体外膜蛋白表达和细胞ROS水平，发现H/R时calpain激活可以抑制线粒体自噬，增加ROS产生，提示了calpain的作用机制。进一步建立小鼠在体心肌缺血/再灌注（I/R）模型，证实抑制calpain可增强心肌细胞自噬反应，改善心肌酶释放、减少危险区的梗死面积、减少TUNEL染色阳性的凋亡细胞；同时抑制自噬可取消calpain抑制剂的保护作用，Western Blot和DHE检测可见calpain引起p62降解和ROS增加，与细胞实验表现一致。总之，本课题组证实心肌I/R引起calpain高表达和激活，降解自噬相关蛋白p62，抑制细胞巨自噬和线粒体自噬，引起ROS产生增加和细胞凋亡、坏死等，是calpain参与I/R损伤的作用机制。