CASQ2和RyR2在二乙酰吗啡致心肌细胞节律异常中的作用

Effects of CASQ2 and RYR2 on diacetylmorphine-induced Arrhythmia in rat cardiomyocytes

目的探讨二乙酰吗啡致心肌细胞节律改变与心肌钙离子通道异常之间的关系,探究肌钙集蛋白2(CASQ2)与兰碱尼受体2(RYR2)因子与钙通道之间的联系,明确其是否参与心肌细胞钙离子通道异常及心律失常的过程。方法取出生3日龄SD大鼠乳鼠的心肌细胞进行体外培养,分为正常对照组(N组)、二乙酰吗啡干预组(H组)、二乙酰吗啡加维拉帕米干预组(H+V组),二乙酰吗啡浓度为10^(-4) mol/L,药物作用时间24 h。在荧光倒置显微镜下,观察心肌细胞形态和收缩频率节律改变;采用逆转录-聚合酶链反应(RT-PCR)和蛋白印迹法(Western blot)检测CASQ2和RyR2因子的mRNA及蛋白表达,观察H组、N组及H+V组上述2个因子表达含量的变化情况。结果 (1)细胞培养:原代心肌细胞形态多样,呈三角形、长梭形和多边形,细胞核圆形或类圆形,大小正常核膜光滑,细胞间通过伪足相互连接,连接成片的细胞搏动节律一致。(2)心肌细胞形态及节律改变:与N组相比,H组和H+V组心肌细胞形态发生一定程度的改变,收缩频率降低,差异有统计学意义(P<0.05)。(3)与N组相比,H组和H+V组中CASQ2和RYR2蛋白和mRNA表达量减少,差异有统计学意义(P<0.05)。与H组相比,H+V组中CASQ2和RYR2蛋白和mRNA表达量减少,差异有统计学意义(P<0.05)。结论二乙酰吗啡可致心肌细胞自发性搏动频率和节律异常,CASQ2和RyR2因子参与二乙酰吗啡致心肌细胞自发性搏动频率和节律异常的过程。关键字:

ObjeciveTo investigate the relationship between diacetylmorphine-induced myocardial cell rhythm changes and abnormal cardiac calcium channels,and to explore the association between troponin 2(CASQ2),lankine receptor 2(RYR2)factors and calcium channels,and to determine whether it is involved in the process of abnormal cardiac calcium channels and arrhythmias.MethodsMyocardial cells from neonatal SD rats(3rd Day)were cultured in vitro,and divided into control group,diacetylmorphine intervention group and diacetylmorphine plus verapamil intervention group.The concentration of diacetylmorphine was 10-4 mol/L.The duration time of drug was 24 h.The myocardial cell morphology change and rhythm was observed by fluorescence inversion microscope.The mRNA and protein expression of CASQ2 and RyR2 were detected by RT-PCR and Western blot.Results(1)The shape of myocardial cells of the primary generation were various kinds,including triangular,long spindle and polygonal,etc.Its nucleus was round or nearly round,its size was normal,and its nuclear membrane was smooth.The cell communication was connected to each other by pseudopod.And the pulsatile rhythm of the connected cells were consistent.(2)Myocardial cell morphology and rhythm changes:Compared to group N,the morphology of myocardial cell were changed at a certain extent,and the contraction frequency of myocardial cell was decreased significantly(P<0.05),in group H and group H+V.(3)Compared to group N,the protein and mRNA expressions of CASQ2 and RYR2 were decreased in group H and group H+V,with statistically significant results(P<0.05).Compared with group H,CASQ2 and RYR2 protein and mRNA expressions was decreased significantly(P<0.05)in group H+V.ConclusionDiacetylmorphine could cause spontaneous beat frequency and abnormal rhythm of cardiac myocyte,and CASQ2 and RyR2 factors were involved in the process of spontaneous beat frequency and abnormal rhythm of cardiac myocyte induced by diacetylmorphine.