腺病毒介导超极化激活环核苷酸门控阳离子通道基因亚型4过度表达对乳鼠心肌细胞电生理特性的影响  

Electrophysiological effects of hyperpolarization activated cyclic nucleotide gated channel 4 overexpression in neonatal cardiomyocytes mediated by recombinant adenovirus

作　　者：李继文[1] 郭继鸿[1] 张萍[1] 李春[1] 刘元伟[1] 周春燕[2] 

机构地区：[1]北京大学人民医院心电生理室,100044 [2]北京大学基础医学院生物化学与分子生物学系

出　　处：《中华医学杂志》2006年第19期1332-1336,共5页National Medical Journal of China

基　　金：国家"863"计划基金资助项目(2001AA2160313);北京市科技计划重大项目基金资助项目(H020220010490);首都医学发展科研基金资助项目(2003-3002)

摘　　要：目的观察过度表达超极化激活环核苷酸门控阳离子通道基因亚型(HCN)4对乳鼠心肌细胞电生理特性的影响。方法酶解法分离20只乳鼠的原代心肌细胞并进行培养,用含HCN4基因的重组腺病毒AdHCN4感染心肌细胞,以未感染细胞作为对照组,通过RT-PCR及细胞免疫荧光方法检测心肌细胞中HCN4基因在mRNA和蛋白质水平的表达,用全细胞膜片钳方法测定转染HCN4基因的心肌细胞的动作电位和起搏电流If。结果AdHCN4感染组心肌细胞中HCN4mRNA和蛋白质的表达水平明显高于对照组细胞;感染组心肌细胞自发搏动的频率为(92·5±7·4)次/分,明显高于对照组心肌细胞的(68·9±6·2)次/分(P<0·05);动作电位出现明显的4相自动除极化,其最大舒张电位(-52·8±4·2)mV明显高于对照组细胞(-62·1±2·6)mV(P<0·05);动作电位复极50%和90%的时间两者比较差异无统计学意义;在感染组细胞中,起搏电流If的电流密度为(115·7±7·8)pA/pF,也明显高于对照组细胞的(7·2±0·6)pA/pF(P<0·05)。结论过度表达起搏通道基因HCN4能够明显增强乳鼠心肌细胞的自律性,从而增加心肌细胞自发搏动的频率,这提示HCN4基因有可能成为治疗缓慢性心律失常疾病的目的基因。Objective To explore the eletrophysiological effects of hyperpolarization activated cyclic nucleotide gated channel 4 （HCN4） overexpression in rat neonatal cardiomyocytes mediated by recombinant adenovirus, Methods Ventricular cardiomyocytes were obtained from 20 neonatal rats. Recombinant adenovirus carrying HCN4 gene, AdHCN4, a dominant isoform of hyperpolarization activated cyclic nucleotide gated cation channel gene in cardiac transduction system, was constructed and used to transfect the neonatal rat ventricular cardiomyocytes. Untransfected cardiomyocytes were used as controls. RT-PCR and immunofluorescence cytochemistry were used to detect the HCN4 mRNA and protein expression. The eletrophysiological characteristics of infected cardiomyocytes were studied by patch clamp. Results The mRNA and protein expression levels of HCN4 in AdHCN4 infected cardiomyocytes were both markedly higher than those of the control cardiomyocytes. AdHCN4 caused a significant increase in The spontaneous rate in the transfected cardiomyocytes was 92. 5 ＋ 7.4 bpm, significantly higher than that of the control ceils （68.9 ＋ 6.2 bpm, P〈0.05）. Patch clamp experiments showed that the pacemaker current density in the AdHCN4 infected cardiomyocytes was 115.7 ＋ 7.8 pA/pF, significantly higher than that of the untransfected ceils （7.2 ＋ 0.6 pA/pF, P〈0.05 ）. Conclusion Overexpression of HCN4 can enhance the autorhythmicity of neonatal cardiomyocytes and significantly increase the spontaneous beat rate. HCN4 channel gene may represent a candidate gene in gene therapy for bradycardia diseases.

关 键 词：离子通道 电生理学 动作电位 心肌细胞 

分 类 号：R33[医药卫生—人体生理学]