Functional analysis of point mutants of cardiac actin involved in dilative and hypertrophic cardiomyopathies

将研究引起扩张型和肥厚型心肌病的心脏肌动蛋白野生型和一些点突变的生化特性。突变的心肌肌动蛋白将利用杆状病毒/Sf9昆虫细胞系统进行表达、纯化，并测定其聚合和刺激心肌肌球蛋白ATPase的能力。将产生允许其在新生儿(NRC)和成人心肌细胞(ARC)中异位表达的载体。它们在肌节细丝中的掺入以及它们对弧线机械输出的影响将被确定。在对机械输出的影响减弱的情况下，将测试小分子化合物，以确定对转基因电弧具有正电离作用的试剂。此外，我们还将分析肌动蛋白结合小肽胸腺球蛋白4对对照组和转染组的存活率以及转染组前后机械输出量的影响。冷冻电子显微镜将被用来获得在没有和存在钙离子和/或肌球蛋白运动域的情况下，用原肌球蛋白和心肌肌钙蛋白复合体的野生型或突变体修饰的心脏F-肌动蛋白的高分辨率图像。

The biochemical properties of wild-type and a number of point mutants of cardiac actin causing dilative and hypertrophic cardiomyopathy will be investigated. The mutated cardiac actins will be expressed by using the bacculovirus/Sf9 insect cell system, purified and their ablity to polymerize and to stimulate the cardiac myosin ATPase will be determined. Vectors will be generated allowing their ectopic expression in neonatal (NRC) and adult cardiomyocytes (ARC). Their incorporation into sarcomeric thin filaments and their effect on the mechanical output of ARCs will be determined. In case of diminishing effects on the mechanical output small compounds will be tested in order to identify reagents with a positive ionotropic effect on the transfected ARCs. In addition, we will analyse the effect of the small actin binding peptide thymosin ß4 on the survival of control and transfected ARCs and their mechanical output before and after transfection. Cryo-electron microscopy will be used to obtain high resolution images of cardiac F-actin decorated with tropomyosin and wild-type or mutants of the cardiac troponin complex in the absence and presence of Ca2+-ions and/or myosin motor domains.