阿片受体调节CaMKⅡ磷酸化在二乙酰吗啡致心肌细胞节律异常中的作用

Opioid receptor regulates the role of CaMKⅡphosphorylation in diacetylmorphine⁃induced cardiomyocyte rhythm abnormalities

目的探讨κ、δ和μ3种阿片受体亚型及CaMKⅡ在二乙酰吗啡致心肌节律异常过程中的作用机制。方法取3 d龄SPF级SD大鼠乳鼠(雌雄不限)心脏进行原代心肌细胞体外培养。将培养好的原代心肌细胞分为细胞对照组、二乙酰吗啡干预组、No组(二乙酰吗啡+κ阿片受体拮抗剂组)、Nal组(二乙酰吗啡+δ阿片受体拮抗剂组)和CTOP组(二乙酰吗啡+μ阿片受体拮抗剂组),检测各组GOT、LDH活性及PLC、PI3K含量,JC⁃1检测各组线粒体膜电位变化,Fluo⁃4 AM检测各组细胞内Ca2+水平,Western blot检测p⁃CaMKⅡ、Calm蛋白表达情况。结果与细胞对照组相比,二乙酰吗啡干预组GOT和LDH活性明显升高,PLC、PI3K含量明显增加,差异均具有统计学意义(F=15.165,F=38.642,F=13.482,F=18.047,P<0.05);与二乙酰吗啡干预组相比,Nal组GOT、LDH活性及PLC、PI3K含量明显降低,差异均具有统计学意义(P<0.05),No组和CTOP组LDH活性及PLC、PI3K含量无明显变化;二乙酰吗啡干预组较细胞对照组线粒体膜电位降低(F=15.843,P<0.05),Nal组较二乙酰吗啡干预组线粒体膜电位升高(P<0.05),与二乙酰吗啡干预组相比,No组和CTOP组线粒体膜电位无明显变化;Fluo⁃4 AM结果显示,与细胞对照组相比,二乙酰吗啡干预组细胞内[Ca2+]i明显升高,差异具有统计学意义(F=38.326,P<0.05),Nal组较二乙酰吗啡干预组细胞内[Ca2+]i明显下降,差异具有统计学意义(P<0.05),No组和CTOP组较二乙酰吗啡干预组细胞内[Ca2+]i无明显变化;Western blot结果显示,与细胞对照组相比,二乙酰吗啡干预组Calm蛋白表达水平及CaMKⅡ磷酸化水平明显升高(F=5.847,P<0.05),与二乙酰吗啡干预组相比,Nal组Calm蛋白表达水平及CaMKⅡ磷酸化水平明显降低,差异均具有统计学意义(P<0.05)。结论二乙酰吗啡可损伤心肌,造成心肌细胞节律异常,其机制可能与δ阿片受体激活CaMKⅡ有关。

Objective To explore the mechanism of the three opioid receptors,κ,δ,μand CaMKⅡin diacetylmorphine⁃induced cardiomyocyte rhythm abnormalities.Methods Hearts of 3⁃day⁃old male and female unrestricted SPF⁃grade SD rats were harvested for in vitro culture of primary cardiomyocytes.The cultured primary cardiomyocytes were divided into cell control group,diacetylmorphine intervention group,No group(diacetylmorphine+κopioid receptor antagonist group),Nal group(diacetylmorphine+δopioid receptor antagonist group),CTOP group(diacetylmorphine+μopioid receptor antagonist group).GOT,LDH activity and PLC and PI3K were detected,membrane potential changes by JC⁃1,intracellular[Ca2+]levels by Fluo⁃4 AM,and p⁃CaMKⅡand Calm protein expression by Western blot.Results In contrast to the cell controls,The GOT and LDH activities were significantly increased in the diacetylmorphine intervention group,The PLC and PI3K content increased significantly,The differences were all statistically significant(F=15.165,F=38.642,F=13.482,F=18.047,P<0.05);Mitochondrial membrane potential was reduced in the diacetylmorphine intervention group compared with the cell control group(F=15.843,P<0.05).The result of Fluo⁃4 AM showed that,compared with the cell control group,the intracellular[Ca2+]i in the diacetylmorphine intervention group was significantly increased(F=38.326,P<0.05),the Nal group decreased significantly compared with the diacetylmorphine intervention group intracellular[Ca2+]i,the difference was statistically significant(P<0.05),and there was no significant change in the No group and CTOP group compared with the intracellular[Ca2+]i in the diacetylmorphine intervention group.The result of Western blot showed that compared with the cell control group,the expression level of Calm protein and the phosphorylation level of CaMKII in the diacetylmorphine intervention group were significantly increased(F=5.847,F=5.234,P<0.05),and compared with the diacetylmorphine intervention group,the expression level of Calm protein and the phosphorylation level of CaMKII in the Nal group were significantly reduced(P<0.05).Conclusion Diacetylmorphine can damage the myocardium and cause abnormal cardiomyocyte rhythm,and the mechanism may be related to CaMKⅡactivation by opioid receptors.