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

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

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。

Objective To explore the preventive effect of chloride channel protein 3(ClC-3)gene overexpression on isoprenaline(ISO)-induced myocardial hypertrophy in mice and its mechanisms.Methods ClC-3 overexpression mouse model and primary cardiomyocyte model were constructed using adeno-associated virus 9(AAV9)infection method.Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)were used to detect the expression of ClC-3 in mouse heart tissues and primary cardiomyocytes to determine whether the model was successfully established.Thirty-two male C57BL/6 mice were randomly divided into 4 groups:control group(normal saline intraperitoneally for 7 d),ISO group(ISO 7.5 mg•kg-1•d-1 intraperitoneally for 7 d),AAV9-bv+ISO group(ISO 7.5 mg•kg-1•d-1 intraperitoneally for 7 d after infected AAV9 with carrying blank vector for 4 weeks)and AAV9-CLC-3+ISO group(ISO 7.5 mg•kg-1•d-1 intraperitoneally for 7 d after infected AAV9 with carrying ClC-3 gene for 4 weeks),with 8 mice in each group.The cardiac function of the mice was detected by echocardiography,and the heart-body weight index was calculated.The mRNA expression of atrial natriuretic peptide(ANP)and brain natriuretic peptide(BNP)in myocardial tissues was detected by qRT-PCR.The morphological changes of left ventricle were observed by hematoxylineosin staining,and the changes of cardiac collagen fibers were observed by picric-sirius red(PSR)staining.Suckling mice were selected and the primary cardiomyocytes were isolated in vitro and divided into 4 groups:control group(without any intervention),ISO group(0.1μmol/L ISO intervention for 48 h),AAV9-ClC-3 group(AAV9 carrying ClC-3 gene infection for 48 h)and AAV9-ClC-3+ISO group(AAV9 carrying ClC-3 gene infection and 0.1μmol/L ISO intervention for 48 h).The volume activating chloride current(ICl,vol)of primary mouse cardiomyocytes was measured by patch clamp technique.Results Western blotting and qRT-PCR results showed that the mouse model and primary cardiomyocyte model with ClC-3 overexpression were successfully established.AAV9-mediated ClC-3 overexpression could reduce the ISO-induced increases in heart-body weight index,end-systolic interventricular septum(IVSs),systolic left ventricular posterior wall(LVPWs),diastolic left ventricular posterior wall(LVPWd),and mRNA expression of ANP and BNP in cardiac tissues,alleviate the abnormal cardiac histomorphology and cardiac fibrosis,as well as inhibit the ISO-induced decrease of ICl,vol in vitro.Conclusion ClC-3 overexpression can prevent ISO-induced myocardial hypertrophy in mice,which may be related to the activation of ICl,vol.