M3受体调控TRPC6通道抑制心肌缺血再灌注后细胞自噬的机制研究

Myocardial ischemia reperfusion (IR) injury induces excessive autophagy and promotes cardiomyocytes death. Our previous studies demonstrated that M3 muscarinic receptor conferred protective effect against myocardial IR injury. However, whether M3 muscarinic receptor protects myocardial IR injured heart by inhibiting autophagy remains unclear. Our preliminary studies found that activation of M3 muscarinic receptor could repress myocardial IR-induced upregulation of autophagy marker protein LC3Ⅱ. Furthermore, we also found that activation of M3 muscarinic receptor could induce protein kinase C-δ (PKC-δ) translocation to phosphorylate transient receptor potential canonical 6 (TRPC6) channel and caused channel inhibition in myocardial IR. Therefore, we hypothesize that M3 muscarinic receptor promotes the translocation of PKC-δ and inhibits TRPC6 channel in myocardial IR. It further decreases calcium influx, inhibits downstream beclin-1 expression, which inhibits the excessive autophagy of the cardiomyocytes and protects the injured myocardium. Molecular biology, patch clamp, laser confocal microscopy and immunohistochemistry as well as the cardiac specific over-expression of M3 muscarinic receptor transgenic mice and cell models are employed. Our study is designed to reveal the molecular mechanisms of inhibitory effect of M3 muscarinic receptor on myocardial IR-induced autophagy by regulating TRPC6 channel both in vivo and in vitro. Our project is aimed to elucidate the novel mechanism of M3 muscarinic receptor against myocardial IR injury by modulating autophagy and provide novel targets for prevention and cure of myocardial IR injury.

心肌缺血再灌注（IR）损伤时过度自噬可导致心肌细胞死亡。我们既往研究证实M3受体具有抗心肌IR损伤作用，然而M3受体能否通过抑制细胞自噬发挥保护作用尚不明确。前期研究发现激动M3受体可抑制心肌IR时细胞自噬标志性蛋白LC3Ⅱ的表达。并且心肌IR时M3受体上调引起PKC-δ转位并磷酸化膜上瞬时受体电位通道6（TRPC6），抑制其通道功能。因此我们假设：心肌IR损伤时，M3受体通过PKC-δ转位抑制TRPC6通道，减少钙离子内流，抑制下游beclin-1表达，进而抑制细胞过度自噬，保护受损心肌。本研究拟在整体、细胞及分子水平，采用分子生物学、膜片钳、激光共聚焦、免疫组化等技术，并利用心脏特异性过表达M3受体转基因动物及细胞模型揭示M3受体调控TRPC6通道抑制心肌IR后细胞自噬的分子机制，旨在从心肌细胞自噬这一新视角阐明M3受体保护心肌IR损伤的新机制，为防治心肌IR损伤提供新靶点。

细胞自噬在心脏疾病发生发展中发挥重要作用，是本领域最新研究热点之一。本项目围绕毒蕈碱M3受体介导信号通路对心肌缺血再灌注（IR）后细胞自噬的调控作用及机制展开研究。首先建立大鼠IR动物模型，采用透射电镜检测发现IR心肌细胞自噬水平增加，自噬相关标志性蛋白LC3Ⅱ，p62，beclin-1表达上调；M3受体激动剂胆碱处理后心肌细胞自噬水平减轻，LC3Ⅱ，p62，beclin-1蛋白表达降低。苏木精-伊红和TUNEL染色结果表明胆碱抑制IR后心肌细胞损伤及凋亡。胆碱抑制IR心肌细胞凋亡及自噬的保护作用可被自噬诱导剂雷帕霉素逆转。胆碱抑制IR后心肌细胞自噬形成过程抗IR损伤的分子机制是抑制心肌细胞TRPC3通道以及激动Akt/mTOR通路。体外水平鉴定了过表达M3受体H9c2心肌细胞系。首次揭示胆碱激动M3受体后下调蛋白激酶C-δ介导的TRPC6通道蛋白的表达，保护心肌细胞的分子机制。另外，还研究了M3受体下游信号分子BDNF调控miR-195抗缺血性心肌细胞损伤的作用及机制。揭示了BDNF激动Akt通路抗化疗药阿霉素诱导心肌细胞凋亡，降低其心脏毒性的作用及机制。综上，本项目深入探讨了M3受体及其下游信号分子BDNF对心肌细胞自噬及凋亡的调控作用及其分子机制，为防治心肌损伤提供新靶点。