线粒体calpain介导ATP合成障碍激活NLRP3炎症小体参与病毒性心肌炎发病的机制研究

病毒感染介导心肌细胞损伤是导致病毒性心肌炎（VMC）的核心机制之一。我们前期研究发现病毒感染可通过calpain介导致线粒体结构功能异常，详尽机制不清。近期我们在VMC模型预试验中发现线粒体calpain异常活化可切割ATP合酶-α(ATP5A1)介导线粒体功能障碍；同时也观察到病毒感染引起线粒体功能障碍可激活NOD样受体家族蛋白3（NLRP3）炎症小体加重心肌损伤。综合上述工作我们提出假说：病毒感染激活线粒体calpain切割APT5A1致线粒体功能障碍并激活NLRP3炎症小体进一步加重心肌细胞损伤。本项目拟利用心肌线粒体靶向过表达calpain抑制剂calpastatin转基因小鼠及心肌特异性calpain亚基Capn-4敲除小鼠从表达与功能两个层面，采用Western blot、ELISA、免疫组织病理、透射电镜、线粒体炫观察等技术手段验证上述假说、阐明具体机制，以明确其科学价值。

Viral infection-mediated cardiomyocytes damage is one of the core mechanisms leading to viral myocarditis (VMC). Our previous researches revealed that viral infection caused cardiomyocytes damage through calpain activation. Recently, we found that virus infection could lead to abnormalities of both mitochondrial structure and function with calpain translocation to mitochondria, suggesting that calpain may participate in the pathogenesis of VMC by regulating mitochondrial function. It has been reported that calpain abnormal activation could result in mitochondrial dysfunction by cleaving ATP synthase-α (ATP5A1) in the septic cardiomyopathy model. Meanwhile, we found preliminarily that mitochondrial dysfunction mediated by virus infection could activate NLRP3 inflammasome and aggravate myocardial injury. Therefore, we propose the hypothesis that pathologic calpain activation caused by virus infection contributes to mitochondrial dysfunction by cleaving ATP5A1 and activating NLRP3 inflammasome which further aggravate myocardial injury. In this project, we intend to verify the above hypotheses and clarify the mechanisms from both expression and function perspectives by using myocardial-specific mitochondrial overexpressing calpastatin, the endogenous inhibitor of calpain, transgenic mice, and myocardial-specific calpain knockout mice and utilizing western bolt, ELISA, immunohistopathology, transmission electron microscopy and mitochondrial flash obseration to clarify the roles of calpain in VMC pathogenesis.