结蛋白对心肌细胞钙离子的影响及其机制研究

Effect of desmin on calcium in cardiomyocytes and its mechanism

目的探讨结蛋白(desmin)减少导致心律失常发生的机制,为心衰患者恶性心律失常的治疗靶点提供理论依据。方法将27只SD乳鼠分为3组:对照组(NC组),desmin siRNA干扰组(si-desmin组)和desmin siRNA+NAC组(si-desmin+NAC组),每组9只。si-desmin组及si-desmin+NAC组的心肌细胞进行desmin小干扰RNA的细胞转染。采用quantitative Real-time RT-PCR方法检测desmin mRNA的转录量。采用Western blot方法测定desmin蛋白、NF-κB通路相关蛋白(p-IKKa、IKBβ、p-NF-κB65)、SERCA2蛋白表达量。利用激光共聚焦方法对心肌细胞细胞浆及肌质网内钙离子浓度进行测定。结果siRNA转染心肌细胞48 h后,与NC组相比,si-desmin组desmin mRNA及蛋白的表达量明显降低(P<0.01)。当心肌细胞中desmin蛋白表达减少时,NF-κB通路蛋白p-IKKa蛋白明显增多(P<0.01),IKBβ表达量明显减少(P<0.01),p-NF-κB65的蛋白表达明显增多(P<0.01)。心肌细胞中desmin表达减少,SERCA2蛋白明显减少(P<0.01)。心肌细胞中desmin的表达减少,细胞浆内钙浓度明显升高(P<0.01);细胞肌质网钙浓度明显降低(P<0.01)。结论心肌细胞内desmin减少通过IKK/IKB/NF-κB途径影响SERCA2表达从而导致心肌细胞钙离子分布异常。

Objective To investigate the mechanism of desmin reducing on the occurrence of cardiac arrhythmias,and to provide a theoretical basis for the treatment target of malignant arrhythmia in heart failure(HF)patients.Methods Twenty-seven SD mice were divided into three groups:negative control group(NC group),desmin siRNA interference group(si-desmin group),and desmin siRNA+NAC group(si-desmin+NAC gruop),n=9.Establishment of desmin interfering RNA cell model:cardiomyocytes in si-desmin and si-desmin+NAC groups were transfected with desmin small interfering RNA.The transcript amount of desmin mRNA was measured by quantitative Real-time RT-PCR.Desmin protein,NF-κB pathway related protein(p-IKKa,IKBβ,p-NF-κB65)and SERCA2 protein expression were detected by Western blot.Ca2+concentration in the plasma and sarcoplasmic reticulum of cardiomyocytes was determinated by laser-confocal microscopy.Results At 48 hours after siRNA transfection,the expression of desmin mRNA and protein was significantly decreased in the si-desmin group compared with the control group(P<0.01).When the expression of desmin protein was decreased in cardiomyocytes,p-IKKa protein was increased significantly(P<0.01),IKBβexpression was decreased(P<0.01),and the expression of p-NF-κB65 protein was increased significantly(P<0.01).The expression of SERCA2 protein reduced in cardiomyocytes with significant reduction in desmin protein(P<0.01).The expression of desmin was reduced in cardiomyocytes and the cytoplasmic calcium concentration was increased significantly(P<0.01);the cellular cytoplasmic reticulum calcium concentration was decreased significantly(P<0.01).Conclusion Reduced desmin in cardiomyocytes affects SERCA2 expression through the IKK/IKB/NF-κB pathway,thereby resulting in abnormal calcium distribution in cardiomyocytes.