Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle
Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle
复制标题
DOI：
10.1161/circheartfailure.108.818237
复制
发表时间：
2009-09-01
影响因子：
9.7
通讯作者：
Marston, Steven B.
中科院分区：
文献类型：
--
作者：
Dyer, Emma C.;Jacques, Adam M.;Marston, Steven B.
背景--家族性扩张型心肌病可由肌肉细丝的蛋白质突变引起。在体外，这些突变降低了对钙的敏感性和跨桥转换率，但这些突变尚未在人类组织中进行研究。我们研究了一例因cTNC G159D突变导致的遗传性扩张型心肌病患者心脏中提取的肌钙蛋白和心肌细胞的钙调节特性。方法和结果-质谱仪显示突变的cTNC与野生型cTNC的表达接近等摩尔。比较了cTNC G159D患者和正常供者心脏的心肌细胞收缩情况。CTNC G159D和供体心肌细胞的最大钙激活力相似，但cTNC G159D心肌细胞对钙的敏感性更高(EC50G159D/供体=0.60)。对骨骼肌肌动蛋白与人心肌原肌球蛋白和肌钙蛋白重组的细丝进行了体外运动实验研究。含有突变的细丝具有更高的钙敏感性(EC(50)G159D/供体=0.55+/-0.13)，而最大激活滑动速度没有改变。此外，当TnI去磷酸化时，cTNC G159D突变减弱了钙敏感性的变化。野生型肌钙蛋白使钙敏感性增加(EC50P/UNP=4.7+/-1.9)，而cTNC G159D不增加钙敏感性(EC50P/UNP=1.2+/-0.1)。结论：磷酸化与钙敏感性关系的解偶联可能是扩张型心肌病表型的原因。这些数据与之前的体外实验结果之间的差异表明，肌钙蛋白I和肌钙蛋白T的天然磷酸化以及肌节蛋白的其他翻译后修饰强烈影响突变的功能效应。(中国保监会心力衰竭。2009年；2：456-464。)
Background-Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca2+ sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca2+-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation.Methods and Results-Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca2+-activated force was similar in cTnC G159D and donor myocytes, but the Ca2+ sensitivity of cTnC G159D myocytes was higher (EC50 G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca2+ sensitivity (EC(50)G159D/donor=0.55 +/- 0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca2+ sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca2+ sensitivity was increased (EC50 P/unP=4.7 +/- 1.9) but not with cTnC G159D troponin (EC50 P/unP=1.2 +/- 0.1).Conclusions-We propose that uncoupling of the relationship between phosphorylation and Ca2+ sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation. (Circ Heart Fail. 2009;2:456-464.)