ISO通过CaMKⅡ和PKA激活RyR2诱发心肌细胞凋亡

Isoproterenol induces cardiomyocyte apoptosis via Ca^(2+)/calmodulin-dependent protein kinase Ⅱ and protein kinase A co-mediated ryanodine receptor pathway

目的:探讨异丙肾上腺素(ISO)持续激活Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)和蛋白激酶A(PKA)后对心肌细胞凋亡的影响及其作用机制。方法:将H9C2心肌细胞分为Control组、ISO组、ISO+KN93(CaMKⅡ抑制剂)组、ISO+H-89(PKA抑制剂)组,经不同药物干预后,通过CCK-8法检测心肌细胞活力,Annexin V-FITC/PI细胞凋亡试剂盒和流式细胞术检测心肌细胞凋亡,Western blot法检测CaMKⅡ、PKA、兰尼碱受体2(RyR2)及凋亡相关蛋白Cleaved-Caspase3、Bax、Bcl-2的表达情况,荧光显微镜观察细胞形态变化和钙荧光强度。结果:与Control组比较,ISO组细胞活性降低,CaMKⅡ、PKA和RyR2的磷酸化水平增加,凋亡蛋白Cleaved-Caspase3、Bax/Bcl-2表达增加,胞内钙含量增多,细胞凋亡率增多,差异均有统计学意义(P<0.01);与ISO组比较,ISO+KN93和ISO+H-89组细胞活性增高,CaMKⅡ、PKA和RyR2的磷酸化水平降低,凋亡蛋白Cleaved-Caspase3、Bax/Bcl-2表达减少,胞内钙含量减少,细胞凋亡率减少,差异均有统计学意义(P<0.01)。结论:ISO通过CaMKⅡ和PKA共同介导RyR2途径的磷酸化激活,并诱发心肌细胞凋亡。

Objective:To investigate the effect and mechanism of isoproterenol(ISO)on cardiomyocyte apoptosis after activation of Ca^(2+)/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)and protein kinase A(PKA).Methods:H9C2 cardiomyocytes were divided into the control group,ISO group,ISO+KN93(CaMKⅡ inhibitor)group and ISO+H-89(PKA inhibitor)group.After intervention,cell viability was detected by CCK-8 assay,apoptosis was detected by Annexin V-FITC/PI apoptosis kit and flow cytometry.The expressions of CaMKⅡ,PKA,RyR2,and apoptosis-related proteins Cleaved-Caspase3,Bax and Bcl-2 were detected by Western blot,and cell morphological changes and calcium fluorescence intensity were observed by fluorescence microscopy.Results:Compared with the control group,cell viability decreased,phosphorylation levels of CaMKⅡ,PKA and RyR2 increased,expressions of Cleaved-Caspase3 and Bax/Bcl-2 increased,intracellular calcium content and apoptosis rate increased in the ISO group,there were significant differences(P<0.01).Compared with the ISO group,cell viability increased,phosphorylation levels of CaMKⅡ,PKA and RyR2 decreased,expressions of Cleaved-Caspase3 and Bax/Bcl-2 decreased,intracellular calcium content and apoptosis rate decreased in the ISO+KN93 and ISO+H-89 groups,there were significant differences(P<0.05).Conclusion:ISO mediates the activation of RyR2 phosphorylation through activation of CaMKⅡ and PKA pathways and induces apoptosis in cardiac myocytes.