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）是家族性肥厚型心肌病（FHC）的主要原因之一，FHC是人类中相对广泛的心脏病。 尽管经过几十年的研究，一个单一的点突变的影响？- MHC对肌球蛋白分子、肌丝和复合肌细胞的功能和结构特性的影响还不清楚。 小鼠一直是FHC最受欢迎的动物模型，因为其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方法已经导致了这样的假设，即小鼠中的FH突变引起了“功能的获得”，而早期对人类组织的研究表明了“功能的丧失”。然而，转基因小鼠模型在蛋白质组成方面与兔和人有显著差异，就？- MHC是小鼠心脏中主要的肌球蛋白同种型，而？- MHC在所有大型哺乳动物的脑室中占主导地位。 因此，我们产生了一个转基因兔模型，其中严重的FHC突变，R403Q在？- MHC在心室过度表达。为了便于分离和定量，将His-标签与R403 Q突变一起沿着克隆在兔转基因的N-末端。 我们假设突变的功能效应依赖于异构体骨架，R403Q将导致“功能丧失”？- MHC。目的1：探讨FHC基因突变在兔心肌中的作用. - MHC影响跨桥循环的停流动力学和体外运动特性。目的2将使用力钳/激光陷阱测定法确定载荷对肌动蛋白丝速度和R403Q肌球蛋白产生的力的影响。 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球蛋白丝松弛状态的结构后果。这些研究应该有助于增加我们对这种疾病的基本分子机制的知识，通过检查FHC突变的影响，从分子到细丝的复杂程度不断增加。 公共卫生相关性：家族性肥厚型心肌病（FHC）是一种可引起年轻人猝死的遗传性心肌疾病。肌肉蛋白肌球蛋白重链的突变几乎占与FHC相关的数百个突变的一半。 希望从基因工程兔子模型中获得的分子见解将导致更好地了解疾病，最终目标是开发新的治疗干预靶点。

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. PUBLIC HEALTH RELEVANCE: Familial hypertrophic cardiomyopathy (FHC) is an inherited disease of heart muscle that can cause sudden death in young adults. Mutations in the heavy chain of the muscle protein, myosin, account for almost half of the several hundred mutations linked to FHC. It is hoped that molecular insights gained from a genetically engineered rabbit model will lead to a better understanding of the disease with the ultimate goal of developing new targets for therapeutic intervention.