TRPC6调控CaMKKβ-AMPK-mTOR信号通路在心肌缺血/再灌注损伤自噬中的作用研究

Autophagy and apoptosis are the two most important pathophysiological processes of myocardial ischemia/reperfusion injury(MIRI) and high level of autophagy plays an key role to maintain cardiomyocytes homeostasis. TRP channels are nonselective monovalent cation channels, most of which also allow passage of Ca2+. Ca2+ in cells can both promote autophagy and apoptosis. Intracellular Ca2+ can induce the activation of AMPK by activating CaMKKβ, thus inhibiting the activity of mTOR and inducing autophagy. Previous studies show TRPC6-/- could inhibit the apoptosis of MIRI, There exists crosstalk between autophagy and apoptosis, the activation of autophagy could inhibit apoptosis. Thus, could TRPC6-/- inhibit the apoptosis of cardiomyocytes through the activation of autophagy? To confirm this propose, the autophagy process of cardiomyocytes under the H/R was observed by confocal through the transfection of GFP-LC3 under the level of cell, and the change of the Ca2+ Channel and Ca2+ concentration of cardiomyocytes induced by TRPC6-/- were detected by Ca2+-mark-fluorescent probes. To investigate the role of CaMKKβ-AMPK-mTOR passway in cardiomyocytes autophagy and apoptosis, the expression level of CaMKKβ, AMPK, Beclin-1, CathepsinD, LC3 and P62 in TRPC6-/- induced cardiomyocytes were detected by gene transfection and silence. The study of this project will allow us to gain insights into the effect and mechanism of CaMKKβ-AMPK-mTOR pathway in the TRPC6 induced cardiomyocytes autophagy and provide new theoretical basis for finding drugs to promote autophagy and cardiomyocytes protection of MIRI.

自噬和凋亡是MIRI最重要的两个病理生理过程。高水平的自噬对维持心肌细胞稳态有重要意义。TRPCs是位于胞膜上重要的非选择性阳离子通道，能通透Ca2+。胞内Ca2+通过CaMKKβ-AMPK-mTOR信号通路诱导细胞自噬。前期实验证实TRPC6-/-可减轻MIRI中细胞凋亡，而凋亡信号和自噬信号存在串话作用。TRPC6-/-对凋亡的抑制是否通过活化自噬实现呢？本课题用TRPC6-/-原代心肌细胞制备H/R模型，通过confocal观察GFP-LC3转染心肌细胞在H/R下自噬的变化；通过游离钙荧光探针、膜片钳技术观察TRPC6-/-对心肌细胞Ca2+浓度的影响；通过基因转染、基因沉默观察TRPC6-/-对CaMKKβ，AMPK和自噬蛋白的影响；探讨TRPC6调控CaMKKβ-AMPK-mTOR信号通路在心肌MIRI自噬中的作用，为防治MIRI，寻找激活自噬的药物提供理论。

自噬和凋亡是MI/RI重要的两个病理生理过程。自噬对维持心肌细胞稳态有重要意义。TRPCs是位于胞膜上重要的非选择性阳离子通道，能通透Ca2+。前期实验证实TRPC6-/-可减轻MI/RI中细胞凋亡，而凋亡信号和自噬信号存在串话作用。TRPC6-/-对凋亡的抑制是否通过活化自噬实现呢？本课题用H9c2细胞及TRPC6-/-小鼠制备缺氧/复氧（H/R）和缺血/再灌注（I/R）模型；主要结果如下：.（一）细胞实验结果：.1）H9c2细胞H/R后自噬变化.H9c2细胞H/R后活力下降；H/R组自噬小体、LC3荧光斑点较Control组明显上升，说明H/R后H9c2细胞自噬增加。.2）自噬指标检测.confocal：mCherry-eGFP-LC3转染H9c2细胞H/R后，H/R组红色荧光斑点与Control组比增加，细胞自噬增加。电镜、Western blot及IF示：H9c2细胞H/R处理后自噬增强。 .3）凋亡指标检测 .H9c2细胞H/R后，凋亡增加；SAR7334可减轻凋亡。.流式结果：H/R后细胞凋亡率上升；H/R后给予SAR7334干扰，凋亡率下降；H/R+3MA组凋亡率较H/R组升高。.（二）动物实验结果：.1）I/R模型.C57与TRPC6-/-小鼠分别予以Sham或I/R处理。心脏超声：I/R处理组较Sham处理组小鼠心功能下降，TRPC6-/-小鼠心功能下降较Sham低。电镜结果：I/R处理后见胞内细胞器减少，肌纤维断裂，明暗带结构不清晰，线粒体部分空泡或皱缩改变，TRPC6-/-小鼠心肌损伤较Sham轻。.2）TRPC6对I/R处理小鼠自噬及CaMKKβ和AMPK蛋白的影响.电镜结果示：I/R处理后自噬增强，I/R处理后TRPC6-/-组较Sham组自噬减弱。Western blot示I/R组CathpsinD、Beclin-1和LC3蛋白较Sham组上升；而I/R后TRPC6-/-组CathpsinD、Beclin-1与LC3较Sham组下降。I/R处理组CaMKKβ和AMPK通道蛋白较Sham组上升，表明I/R后CaMKKβ和AMPK相关通路被激活；而I/R后CaMKKβ和AMPK通道蛋白TRPC6-/-组较Sham组下降。.本研究发现TRPC6可通过CaMKKβ-AMPK通路调节MI/RI自噬，此研究为防治MI/RI提供了一定的理论依据。

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