肥厚型心肌病心肌肌钙蛋白I R145W突变对大鼠心肌细胞钙调控的影响  

Reduced Ca^(2+) current in rat cardiomyocytes transfected with troponin I R145W mutation gene

作　　者：吴恒芳[1] 陈相健[1] 杨笛[1] 卞智萍[1] 徐晋妉[1] 张寄南[1] 

机构地区：[1]南京医科大学第一附属医院心内科 心血管病研究所临床生物学诊断与治疗实验室,210029

出　　处：《中华心血管病杂志》2007年第11期1000-1004,共5页Chinese Journal of Cardiology

基　　金：国家自然科学基金(30570745);江苏省高校重点实验室开放课题(K04003);江苏省临床生物学诊断与治疗重点实验室基金(SK200205)

摘　　要：目的探讨国人新的致肥厚型心肌病(HCM)心肌肌钙蛋白 I 基因突变(eTnI R145W)引起 HCM 发病的可能机制。方法采用定点突变技术在大鼠 cTnI cDNA 引入 R146W(相当于人R145W)突变,增强型绿色荧光蛋白(EGFP)做报告基因,构建含野生型和突变型大鼠 cTnI cDNA 的重组腺病毒载体。Langendoff 逆流灌注系统分离成年大鼠心肌细胞,无血清法培养后重组腺病毒转染。Western blot 检测重组蛋白的表达,全膜片钳技术记录心肌细胞膜 L 型钙电流,Fura-2/AM 孵育后测定细胞内游离钙离子浓度和咖啡因诱导的肌浆网钙释放。结果 DNA 测序证实 R146W 突变成功引入大鼠 cTnI cDNA。新鲜分离的成年大鼠心肌细胞存活率达70%～80%,无血清培养7天后绝大部分能保持长杆状形态。重组腺病毒转染48h 后荧光显微镜下可观察到绿色荧光,cTnI 和绿色荧光蛋白单抗均能检测到重组蛋白。与野生型 cTnI 和正常对照组比较,突变型 cTnI 组心肌细胞膜 L 型钙电流峰值明显降低。Fura-2/AM 法测定细胞内游离钙离子浓度和咖啡因诱导的肌浆网钙释放,三组之间差异无统计学意义。结论含 R146W 突变的 cTnI 蛋白可能引起心肌细胞的电生理学重构,进一步研究可探讨肌丝对钙的敏感性及钙调节蛋白表达的改变。Objective To investigate the effects of cardiac troponin Ⅰ R145W mutation, detected in Chinese patients with hypertrophic cardiomyopathy, on Ca^2＋ current modulation. Methods R146W mutation （resemble R145W in human） was introduced into rat cardiac tropenin Ⅰ cDNA by site-directed mutagenesis. With EGFP as a reporter gene, replication-defective adenovirus containing the wild or mutant cTnⅠ gene was constructed. Adult rat cardiomyocytes, were isolated by Langendoff perfusion and cultured with serum-free medium and transduced with the recombinant adenoviruses. Western blot was used to determine the recombinant proteins. Whole cell patch clamp was employed to record L-type Ca^2＋ currents on cultured myocytes. Intracellular free Ca^2＋ and caffeine-induced sareoplasmic reticulum （SR） Ca^2＋ release were determined after the cells incubated with Fura-2/AM. Results DNA sequencing confirmed that R146W mutation was generated in rat cTnⅠ cDNA. Bright green fluorescence was observed in the cultured cardiomyocytes at 48 h after transduction. The recombinant proteins could be identified with cTnⅠ or GFP monoclonal antibody. The peak current of L-type Ca^2＋ channel in cells transduced with cTnⅠ R146W was significantly decreased compared to control cells and cells transfected with wild cTnⅠ. Intracellular free Ca^2＋ concentrations and caffeine-induced SR Ca^2＋ release determined by Fura-2/AM were similar among various cells. Conclusion Reduced peak current of L-type Ca^2＋ channel in cells transduced with cTnⅠ R146W might contribute to the disease-causing mechanism of this mutation in patients with hypertrophic cardiomyopathy.

关 键 词：心肌病 肥厚性 肌钙蛋白Ⅰ 突变 腺病毒科 L型钙通道 

分 类 号：R542.2[医药卫生—心血管疾病]