Single Cross-Bridge Kinetics in Transgenic Mouse Hearts Expressing FHC Mutations

描述(申请人提供)：家族性肥厚性心肌病(FHC)是一种常染色体显性遗传病，起源于心脏主要收缩蛋白编码基因的突变，包括心室肌球蛋白调节(RLC)和必需(ELC)轻链。FHC导致心室和间隔肥大，肌原纤维紊乱，是年轻人心脏性猝死的主要原因。本研究旨在从单个肌球蛋白跨桥的水平上阐明FHC的触发机制。我们建议验证这一假设，即FHC是由于在表达肌球蛋白RLC和ELC致病突变的转基因小鼠心脏中，由于肌球蛋白跨桥动力学的改变而导致心肌对ATP的低效利用所引起的。我们将在单分子水平上检验来自转基因小鼠心脏的乳头肌纤维中的这一假设，转基因小鼠心脏携带肌球蛋白调节和/或基本轻链的致病突变。我们强烈认为，肌球蛋白跨桥动力学的明确测定必须在单个跨桥的水平上进行，并将结果与通过测量皮肤和完整的肌肉纤维得出的跨桥力学结果进行比较。单分子方法的优点是它能够避免对具有不同动力学的分子集合进行平均，例如WT和FHC分子的混合物，并且能够明确地确定“健康的”和“患病的”肌肉的动力学。由于人类患者是FHC突变的杂合子，并且他们的粗丝含有穿插的WT和HCM突变头，因此在肌纤维水平上将单分子信息与FHC的表型相关联是极其重要的。具体地说，我们询问在FHC心脏和健康转基因对照组的单个交叉桥中，分离状态和强结合状态的持续时间(目标1A)和寿命(目标1B)是否相同。利用这一单分子技术获得的信息将与力发展、皮肤乳头肌纤维上的ATPase以及转基因小鼠完整肌肉纤维上的力和钙瞬变的功能研究平行(Aim 2A)。最终目标是将单分子得出的数据与细胞发现联系起来，以充分了解导致FHC的单个RLC和ELC突变的作用机制(目标2B)。正在解决的基本问题是，为什么以及如何在RLC和/或ELC中的这些个体突变导致人类从相对轻微的临床FHC表型到恶性临床FHC表型的各种疾病表型。我们相信，将分子生物学方法与高分辨率光学和纳米荧光光谱相结合，将使我们能够成功地回答有关FHC介导的心脏病理的分子基础以及RLC和ELC在心肌收缩中的作用等重要问题。公共卫生相关性：这项研究旨在揭示家族性肥厚型心肌病的机制，这是一个主要的公共卫生问题。这项建议的目标是了解肌节肌球蛋白轻链突变导致人类心肌肥大的分子基础。成功完成这一目标可能会导致一种严重心脏病的新治疗方式。这种应用的力量来自于它结合了分子生物学和纳米荧光显微镜方法在单个分子水平上研究致病突变。此外，将单分子方法与病变肌肉的生理评估相结合，将使我们能够成功地回答有关FHC介导的心脏病理的分子基础的重要问题。

DESCRIPTION (provided by applicant): Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease originating from mutations in genes that encode for the major contractile proteins of the heart, including the ventricular myosin regulatory (RLC) and essential (ELC) light chains. FHC results in ventricular and septal hypertrophy, myofibrillar disarray and is the leading cause of sudden cardiac death in young individuals. This research is aimed at elucidating the molecular mechanisms involved in triggering of FHC at the level of a single myosin cross-bridge. We propose to test the hypothesis that FHC is caused by inefficient utilization of ATP by cardiac muscle due to alteration of myosin cross-bridge kinetics in transgenic mouse hearts expressing disease-causing mutations in myosin RLC and ELC. We will examine this hypothesis at the single molecule level in papillary muscle fibers from transgenic mouse hearts which carry disease-causing mutations in the regulatory and/or essential light chains of myosin. We strongly believe that the unambiguous determination of myosin cross-bridge kinetics must be carried out at the level of a single cross-bridge and the results compared to cross-bridge mechanics derived from measurements on skinned and intact muscle fibers. The advantage of the single molecule approach is its ability to avoid averaging over ensembles of molecules with different kinetics such as a mixture of WT and FHC molecules, and the ability to unambiguously determine the kinetics of "healthy" and "diseased" muscle. Since human patients are heterozygous for FHC mutations and their thick filaments contain interspersed WT and HCM mutant heads it is extremely important to correlate the single molecule information with the phenotype of FHC assessed at the muscle fiber level. Specifically we ask whether the durations (Aim 1A) and lifetimes (Aim 1B) of detached and strongly-bound states are the same in a single cross-bridge from FHC hearts and in healthy transgenic controls. The information derived using this single molecule technology will be paralleled with functional studies of force development, ATPase on skinned papillary muscle fibers as well as force and calcium transients on intact muscle fibers from transgenic mice (Aim 2A). The ultimate objective is to link the single molecule derived data with the cellular findings to fully understand the mechanism of action of the individual RLC and ELC mutations causing FHC (Aim 2B). The fundamental question that is being addressed is why and how these individual mutations in RLC and/or ELC cause variable disease phenotypes in humans ranging from relatively mild to malignant clinical FHC phenotypes. We believe that integration of molecular biology approaches with high resolution optics and nano-fluorescence spectroscopy will enable us to successfully answer important questions regarding the molecular basis of FHC-mediated pathology in the heart and the role of RLC and ELC in cardiac muscle contraction. PUBLIC HEALTH RELEVANCE: This research is directed toward unraveling the mechanisms of familial hypertrophic cardiomyopathy, a major public health problem. The goal of this proposal is to understand the molecular bases by which mutations in the sarcomeric myosin light chains lead to cardiac hypertrophy in humans. Successful completion of this goal may lead to new modalities of treatment of a serious heart disease. The strength of this application is formed by its combination of molecular biological and nano-fluorescence microscopic approaches in the study disease-causing mutations at the level of a single molecule. Furthermore, the integration of single molecule approaches with the physiological assessment of the diseased muscle will enable us to successfully answer important questions regarding the molecular basis of FHC-mediated pathology in the heart.