A TG model for functional effects of FHC mutations in b-cardiac myosin

描述（由申请人提供）：？-肌球蛋白重链（MHC）是家族性肥厚性心肌病（FHC）的主要原因之一，FHC是一种相对普遍的人类心脏病。尽管经过了几十年的研究，基因单点突变的影响？-MHC对肌球蛋白分子、丝和复杂肌细胞的功能和结构特性还不太清楚。小鼠是FHC最受欢迎的动物模型，因为它具有先进的遗传学和相对容易产生突变株。从单分子力学到心肌生理学的各种方法都提出了一种假设，即小鼠的FH突变产生了“功能的获得”，而早期对人体组织的研究表明是“功能的丧失”。然而，转基因小鼠模型的蛋白质组成与兔子和人类有很大的不同。-MHC是小鼠心脏中肌球蛋白的主要亚型，而？-MHC在所有大型哺乳动物的心室中占主导地位。因此，我们产生了一种转基因兔子模型，在这种模型中，FHC基因的R403Q发生了严重突变。-MHC，在心室过度表达。为了便于分离和定量，在兔转基因的n端克隆了一个his标签，同时克隆了R403Q突变。我们假设突变的功能影响依赖于同工异构体主链，R403Q将导致基因中的“功能丧失”。- MHC。目的1将确定FHC突变如何在兔心脏？-MHC影响过桥循环的停流动力学和体外运动特性。目的2将使用力钳/激光诱捕法确定负载对肌动蛋白丝速度和R403Q肌球蛋白产生力的影响。目的3将通过先进的高分辨率冷冻电子显微镜和单粒子成像技术确定FHC突变对肌动球蛋白相互作用和肌动蛋白丝松弛状态的结构后果。这些研究应该通过检查FHC突变在从分子到丝的复杂程度不断增加的影响，有助于增加我们对这种疾病的基本分子机制的认识。

DESCRIPTION (provided by applicant): Mutations in the ?-myosin heavy chain (MHC) are among the major causes of familial hypertrophic cardiomyopathy (FHC), a relatively widespread heart disease in humans. Despite many decades of study, the effect of a single point mutation in the ? -MHC on the functional and structural properties of the myosin molecule, the filament and the complex muscle cell are not well understood. The mouse has been the most popular animal model for FHC because of its advanced genetics and the relative ease in generating mutant strains. Approaches ranging from single molecule mechanics to cardiac muscle physiology have led to the hypothesis that a "gain of function" arises from FH mutations in mice, in contrast to earlier studies on human tissue suggesting a "loss of function". However, the transgenic mouse model differs significantly in protein composition from rabbits and humans, insofar as ? -MHC is the major myosin isoform in the mouse heart, whereas ? -MHC predominates in the ventricles of all larger mammals. We have therefore generated a transgenic rabbit model in which a severe FHC mutation, R403Q in the ? -MHC, was over-expressed in the ventricles. To facilitate isolation and quantification, a His-tag was cloned at the N-terminus of the rabbit transgene along with the R403Q mutation. We hypothesize that the functional effects of a mutation are dependent on the isoform backbone, and R403Q will lead to a "loss of function" in ? - MHC. Aim 1 will determine how an FHC mutation in rabbit cardiac ? -MHC affects the stopped-flow kinetics and in vitro motility properties of the cross-bridge cycle. Aim 2 will determine the effect of load on actin filament velocity and force generation by R403Q myosin using a force clamp/laser trap assay. Aim 3 will determine the structural consequences of an FHC mutation on the actomyosin interaction and the relaxed state of the myosin filament by advanced high resolution cryo-electron microscopy and single particle imaging techniques. These studies should serve to increase our knowledge of the basic molecular mechanism of this disease by examining the effects of an FHC mutation at increasing levels of complexity from the molecule to the filament.