过表达氯离子通道蛋白3对异丙肾上腺素诱导的小鼠心肌肥厚的预防作用与机制

目的 探讨氯离子通道蛋白3(ClC-3)基因过表达对异丙肾上腺素(ISO)诱导的小鼠心肌肥厚的预防作用及机制.方法 运用腺相关病毒9(AAV9)感染方法构建ClC-3过表达小鼠模型和原代心肌细胞模型,采用蛋白质印迹法与qRT-PCR检测小鼠心脏组织和原代心肌细胞中ClC-3的表达以确定模型是否建立成功.32只雄性C57BL/6小鼠随机分为4组,每组8只.对照组连续7 d腹腔注射生理盐水,ISO组连续7 d腹腔注射ISO 7.5 mg?kg-1?d-1,AAV9-bv+ISO组用空白载体AAV9病毒颗粒感染小鼠4周后连续7 d腹腔注射ISO 7.5 mg?kg-1?d-1,AAV9-ClC-3+ISO组用携带ClC-3基因的AAV9病毒颗粒感染小鼠4周后连续7 d腹腔注射ISO 7.5 mg?kg-1?d-1.采用心脏超声检查小鼠心功能变化,计算心脏体重指数,采用qRT-PCR检测心肌组织中心房钠尿肽(ANP)、脑钠肽(BNP)的mRNA表达水平,H-E染色观察左心室形态学变化,苦味酸-天狼星红(PSR)染色观察心脏组织胶原纤维变化.取乳鼠摘出心脏,体外培养原代心肌细胞并分为4组,对照组未予任何干预,ISO组用0.1μmol/L ISO干预48 h,AAV9-ClC-3组用携带ClC-3基因的AAV9病毒颗粒感染细胞48 h,AAV9-ClC-3+ISO组用携带ClC-3基因的AAV9病毒颗粒和0.1μmol/L ISO共同干预48 h.采用芯片膜片钳技术检测小鼠原代心肌细胞的容积激活性氯电流(ICl,vol).结果 蛋白质印迹法和qRT-PCR检测结果均表明ClC-3过表达小鼠和原代心肌细胞模型成功建立.AAV9介导的ClC-3过表达能缓解ISO诱导的小鼠心脏体重指数、收缩末期室间隔厚度、收缩期左室后壁厚度和舒张期左室后壁厚度的增加,改善心脏组织形态学异常和心脏纤维化,减少心脏组织中ANP、BNP mRNA表达的增加,并能体外抑制ISO导致的心肌细胞ICl,vol降低.结论 ClC-3过表达可预防ISO诱导的小鼠心肌肥厚,其机制可能与心肌细胞ICl,vol激活有关.

Preventive effect of chloride channel protein 3 overexpression on isoprenaline-induced myocardial hypertrophy in mice and its mechanisms

.Objective To explore the preventive effect and mechanism of ClC-3 gene overexpression on isoprenaline induced myocardial hypertrophy in mice. Methods The ClC-3 overexpression mouse model was constructed by adenovirus transfection and divided into a control group, a model group, a negative virus group and a ClC-3 overexpression group,with 8 mice in each group. Western Blot was used to detect the expression of ClC-3 protein in mouse heart, Real-time quantitative PCR detection of ANP and BNP mRNA expression levels in myocardial tissue, echocardiography was used to detect the change of cardiac function in mice, and hematoxylin eosin staining (H-E staining) was used to observe Morphological changes of left ventricle, Sirius red picric acid staining (PSR staining) to observe the changes of collagen fibers in heart tissue,Whole cell patch clamp technique to detect cell membrane current. Results The results of PCR and Western blot showed that a mouse model of cardiac ClC-3 overexpression was successfully established.AAV9-mediated ClC-3 overexpression could reduce isoproterenol-induced increase in mouse cardiac body index (HW/BW,P < 0.05), interventricular septum thickness (IVS), and left ventricular posterior wall thickness (LVPW), inhibit the decrease of cell volume activating chloride current caused by isoproterenol(P < 0.05),reduce myocardial fibrosis and decrease the expression of cardiac hypertrophy marker factor. Conclusion ClC-3 overexpression can prevent isoprenaline-induced myocardial hypertrophy and improve heart function in mice.