内质网应激-miR-105-RyR2信号通路参与房颤发生的机制研究

Mechanism of endoplasmic reticulum stress-miR-105-RyR2 signal pathway involving in the occurrence of atrial fibrillation

目的探讨内质网应激(endoplasmic reticulum stress,ERS)-miR-105-RyR2通路参与大鼠房颤(atrial fibrillation,AF)发生的机制。方法8周龄雄性SD大鼠随机分为房颤组和对照组,每组10只。经食道高频电刺激快速心房起搏诱导建立大鼠房颤模型,对照组不予处理。分离房颤大鼠心房肌细胞设立房颤组,大鼠心房肌细胞经衣霉素刺激建立ERS模型设立衣霉素组。乳大鼠心肌细胞经衣霉素刺激建立ERS模型并转染miR-105 mimic或mimic NC作为衣霉素+miR-105 mimic组和衣霉素+mimic NC组(阴性对照),同时乳大鼠心肌细胞分别转染miR-105 mimic及mimic NC作为对照+miR-105 mimic组、对照+mimic NC组。Western blot检测葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、雷尼丁受体2(ryanodine receptor2,RyR2)及其磷酸化表达,qRT-PCR检测非编码小分子RNA-105(microRNAs-105,miR-105)、RyR2 mRNA表达。结果与对照组相比,房颤组大鼠心房肌组织中GRP78、RyR2mRNA、RyR2蛋白及磷酸化表达上调(均P<0.05),miR-105表达下调(P<0.05)。与对照组比较,衣霉素组和房颤组心房肌细胞GRP78、RyR2mRNA、RyR2蛋白及磷酸化表达均上调(P<0.05),miR-105表达均下调(P<0.05)。与对照+mimic NC组比较,衣霉素+mimic NC组GRP78表达上调(P<0.01),miR-105表达下调(P<0.01);与衣霉素+mimic NC组比较,衣霉素+miR-105 mimic组GRP78表达无变化,RyR2 mRNA、RyR2蛋白及磷酸化表达下调(均P<0.05)。结论内质网应激通过miR-105调控RyR2蛋白及磷酸化水平参与大鼠房颤的发生。

Objective To explore the mechanism of endoplasmic reticulum stress-miR-105-RyR2 signal pathway involving in the occurrence of atrial fibrillation(AF).Methods The male SD rats aged 8 weeks were randomly divided into AF group and control group,with 10 rats in each group.The rats were given transoesophageal electrophysiological stimulation to establish AF model in atrial fibrillation group,while the rats were given no treatment in control group.Rat atrial myocytes were isolated from atrial fibrillation rats as AF group.Rats atrial myocytes were stimulated with tunicamycin to establish the ERS model as tunicamycin group.Neonatal rat cardiomyocytes were stimulated with tunicamycin to establish the ERS model,and then transfected with miR-105(tunicamycin+miR-105 mimic group)and negative control(tunicamycin+mimic NC group).Neonatal rat cardiomyocytes were transfected with miR-105 mimic(control+miR-105 mimic group)and mimic NC miR-105(control+mimic NC group).Western blot was used to detect the expression of glucose-regulated protein 78(GRP78),RyR2 and its phosphorylated level.Real-time quantitative PCR was employed to detect the expression of microRNAs-105(miR-105)and RyR2 mRNA.Results Compared with control group,the expression of GRP78,RyR2 mRNA,RyR2 protein and their phosphorylation in atrial tissues in AF group were up-regulated(all P<0.05),while the expression of miR-105 was down-regulated(P<0.05).Compared with control group,the expression levels of GRP78,RyR2 mRNA,RyR2 protein and their phosphorylation were up-regulated in atrial myocytes in tunicamycin group and AF group(all P<0.05),while the expression of miR-105 was down-regulated(P<0.05).Compared with control+mimic NC group,the expression of GRP78 was up-regulated in myocardial cells in tunicamycin+mimic NC group(P<0.01),while the expression of miR-105 was down-regulated(P<0.01).Compared with tunicamycin+mimic NC group,the expression of GRP78 expression showed no obvious change in tunicamycin+miR-105 group,while the expression levels of RyR2 mRNA,RyR2 protein and phosphorylation expression were down-regulated(all P<0.05).Conclusion Endoplasmic reticulum stress may participate in the atrial fibrillation by regulating the expression of RyR2 protein and its phosphorylation via miR-105.