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

描述（由申请人提供）：家族性肥厚型心肌病（FHC）是一种常染色体显性遗传疾病，起源于编码心脏主要收缩蛋白（包括心室肌球蛋白调节（RLC）和必需（ELC）轻链）的基因突变。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。本研究旨在阐明在单个肌球蛋白跨桥水平触发FHC的分子机制。我们建议测试的假设，FHC是由于心肌ATP的利用效率低下，由于改变肌球蛋白跨桥动力学在转基因小鼠心脏表达致病突变的肌球蛋白RLC和ELC。我们将在转基因小鼠心脏的乳头肌纤维的单分子水平上研究这一假设，转基因小鼠心脏在肌球蛋白的调节和/或必需轻链中携带致病突变。我们坚信，明确的肌球蛋白跨桥动力学的测定必须在一个单一的跨桥的水平进行，并与来自皮肤和完整的肌纤维测量的跨桥力学的结果进行比较。单分子方法的优点是其避免对具有不同动力学的分子集合（例如WT和FHC分子的混合物）求平均的能力，以及明确确定“健康”和“患病”肌肉的动力学的能力。由于人类患者对于FHC突变是杂合的，并且他们的粗肌丝含有散布的WT和HCM突变体头部，因此将单分子信息与在肌纤维水平评估的FHC表型相关联是极其重要的。具体来说，我们问是否持续时间（目标1A）和寿命（目标1B）的分离和强结合状态是相同的，在一个单一的跨桥从FHC心脏和健康的转基因对照。使用这种单分子技术获得的信息将与转基因小鼠（Aim 2A）的力发展、皮肤乳头肌纤维上的ATP酶以及完整肌纤维上的力和钙瞬变的功能研究相结合。最终目的是将单分子衍生数据与细胞结果联系起来，以充分了解导致FHC的单个RLC和ELC突变的作用机制（目的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温和到恶性临床FHC表型的可变疾病表型。我们相信，分子生物学方法与高分辨率光学和纳米荧光光谱的整合将使我们能够成功地回答有关心脏中FHC介导的病理学的分子基础以及RLC和ELC在心肌收缩中的作用的重要问题。公共卫生相关性：本研究旨在阐明家族性肥厚型心肌病的发病机制，这是一个主要的公共卫生问题。本研究的目的是了解肌节肌球蛋白轻链突变导致人类心脏肥大的分子基础。成功完成这一目标可能会导致严重心脏病治疗的新模式。这种应用的优势是通过其分子生物学和纳米荧光显微镜方法的结合形成的，在单分子水平上研究致病突变。此外，单分子方法与患病肌肉的生理评估的整合将使我们能够成功地回答有关心脏中FHC介导的病理学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.