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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 possible mechanism of HCT caused by new human cardiac troponin I gene mutation (eTnI R145W) in hypertrophic cardiomyopathy (HCM). Methods Recombinant adenoviral vector containing wild-type and mutant cTnI cDNA was constructed by site-directed mutagenesis in which R146W (equivalent to human R145W) and enhanced green fluorescent protein (EGFP) were introduced into rat cTnI cDNA. Langendoff countercurrent perfusion system isolated adult rat cardiomyocytes, serum-free culture after transfection of recombinant adenovirus. The expression of recombinant protein was detected by Western blot. L-type calcium current was recorded by whole-patch-clamp technique. The intracellular free calcium concentration and caffeine-induced sarcoplasmic reticulum calcium release were measured after incubation with Fura-2 / AM. Results DNA sequencing confirmed that the R146W mutation successfully introduced rat cTnI cDNA. Freshly isolated adult rat cardiomyocytes survival rate of 70% to 80%, most of the serum-free culture after 7 days to maintain the long rod-shaped morphology. 48h after transfection, the green fluorescence was observed under the fluorescence microscope, and the recombinant protein could be detected by both cTnI and the green fluorescent protein monoclonal antibody. Compared with wild-type cTnI and normal control group, the peak of L-type calcium current in myocardial cell membrane was significantly decreased in mutant cTnI group. Fura-2 / AM determination of intracellular free calcium concentration and caffeine-induced sarcoplasmic reticulum calcium release, the difference between the three groups was not statistically significant. CONCLUSION: The cTnI protein with R146W mutation may cause electrophysiological remodeling of cardiomyocytes. Further study may explore the sensitivity of myofilament to calcium and the change of calcium regulatory protein expression.