心脏因子CTRP9调控线粒体自噬抗心肌缺血/再灌注损伤的作用及机制研究

Damage to cardiac mitochondria during the myocardial ischemia/reperfusion (MI/R) period is a key contributing mechanism in the progression of cardiomyocytes apoptosis. Mitophagy could remove dysfunctional mitochondria to protect against MI/R injury while its mechanism remains undefined. The applicant demonstrates that cardiokine CTRP9 reveals significant cardioprotective effects via the inhibition of cardiomyocytes apoptosis. Importantly, our preliminary data shows that the level of mitochondria LC3II increased in the CTRP9-KO mice heart during the MI/R injury and CTRP9 combines with mitochondria LC3II in the cardiomyocytes, indicating that CTRP9 might be the key regulator of mitophagy under the MI/R injury. Based on these results, we hypothesize that cardiokine CTRP9 regulates mitophagy to maintain the cardiac mitochondria quality, protecting against MI/R injury. The purpose of this study was to use genetic interventions, including CTRP9-KO mice and protein inhibition with specific siRNA, by study the isolated cardiomyocyte and overall function via methods including co-immuoprecipitation, immunofluorescent staining, transmission electron microscope and, et al. to demonstrate cardiac CTRP9’s regulation on mitophagy, to clarify the underlining mechanisms and to discover new targets against damaged cardiac mitochondria under the MI/R injury. The project will provide a valuable theory basis in the treatment and prevention against MI/R injury.

线粒体受损是导致心肌缺血/再灌注（MI/R）损伤时心肌细胞调亡的重要原因。线粒体自噬可以清除功能障碍的线粒体，减轻MI/R损伤，但其具体机制仍不清楚。申请者发现心脏因子CTRP9抑制心肌细胞凋亡，具有显著的缺血心肌保护作用。预实验研究提示：MI/R损伤时，CTRP9-KO小鼠心肌组织线粒体自噬标签蛋白LC3II表达增加；同时心肌细胞内CTRP9能够结合线粒体LC3II，可能是参与调控线粒体自噬的重要心脏因子。由此提出科学假说：心脏因子CTRP9通过调控心肌细胞线粒体自噬，维持线粒体质量，减轻MI/R损伤。本研究拟通过应用基因敲除、siRNA基因抑制等干预技术，采用在体动物及离体细胞研究相结合及免疫共沉淀、免疫荧光染色、透射电镜等方法，验证CTRP9调控心肌细胞线粒体自噬的作用，阐明具体机制，探寻挽救缺血心肌受损线粒体的分子靶点，为防治MI/R损伤提供理论依据。

线粒体功能障碍是加重心肌缺血/再灌注（MI/R）损伤的重要原因之一。线粒体自噬可以清除受损线粒体，但具体机制不明。申请者前期研究及预实验结果提示：心脏因子CTRP9可能是调控MI/R损伤时线粒体自噬的关键分子。由此提出科学假说：心脏因子CTRP9通过调控心肌细胞线粒体自噬，维持线粒体质量，减轻MI/R损伤。.本研究发现，与WT组相比，CTRP9-KO小鼠心功能损伤加重、心肌梗死面积增加、心肌细胞凋亡增多。MI/R损伤时，CTRP9-KO小鼠心肌线粒体排列紊乱、线粒体膜蛋白降解减少、线粒体自噬标签蛋白LC3II表达升高，LAMP2表达降低。由此证明CTRP9通过调控心肌细胞线粒体自噬，发挥抗MI/R损伤作用。然后通过在体腹腔注射线粒体自噬诱导剂CCCP及自噬溶酶体合成阻断剂Bafilomycin A1发现，CTRP9-KO抑制LC3II转位。应用GFP-RFP-labeled LC3小鼠发现，MI/R损伤后，CTRP9-KO小鼠心肌线粒体自噬溶酶体增加。由此证明MI/R损伤时，CTRP9通过促进自噬溶酶体降解，发挥心肌保护作用。进一步探究其具体分子机制发现，抑制心肌细胞CTRP9表达，线粒体自噬溶酶体关键蛋白Cathepsin B和Cathepsin L活性降低，而不影响Cathepsin D表达。Co-IP结果表明，CTRP9与LC3在心肌线粒体内存在PPI。激光共聚焦显微镜观察到CTRP9与LC3在线粒体上共定位。由此我们证明，CTRP9通过激活Cathepsin B、Cathepsin L，调控心肌线粒体自噬溶酶体降解，促进线粒体自噬流，抑制MI/R损伤。 .本课题首次发现心脏因子CTRP9通过结合LC3，激活Cathepsin B、Cathepsin L，调控线粒体自噬，减轻缺血/再灌注损伤的新机制。进一步完善了CTRP9生物学功能，为MI/R防治提供了新思路和新靶点。