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

家族性肥厚型心肌病是一种常染色体显性遗传疾病 起源于编码血管主要收缩蛋白的基因突变 心脏，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，是导致FHC的主要原因。 年轻人心脏性猝死的原因本研究的目的是 阐明了参与触发FHC的分子机制， 单个肌球蛋白跨桥。我们建议检验FHC是由 由于肌球蛋白的改变，心肌对ATP的利用效率低下 转基因小鼠心脏中的跨桥动力学 肌球蛋白RLC和ELC的突变。我们将在一个单独的实验中检验这个假设。 转基因小鼠心脏的乳头肌纤维中的分子水平， 肌球蛋白的调节和/或必需轻链中的致病突变。我们 我坚信肌球蛋白跨桥动力学的明确测定 必须在一个单一的跨桥的水平和结果相比， 从皮肤和完整肌肉上的测量导出的跨桥力学 纤维单分子方法的优点是它能够避免平均化 具有不同动力学的分子集合，如WT和FHC的混合物 分子，以及明确确定“健康”和“健康”的动力学的能力。 “病态”肌肉由于人类患者对于FHC突变是杂合的，并且其 粗丝含有散置的WT和HCM突变头， 为了将单分子信息与FHC的表型相关联， 肌纤维水平。 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的心脏和 健康的转基因对照。从这个单分子中得到的信息 这项技术将被力量发展、ATP酶对 剥离乳头肌纤维以及完整肌肉上的力和钙瞬变 来自转基因小鼠的纤维（Aim 2A）。最终目标是将单个 分子衍生的数据与细胞的发现，以充分了解机制， 导致FHC的单个RLC和ELC突变的作用（目的2B）。的 一个基本的问题是，为什么以及如何这些个别的突变 在RLC和/或ELC中引起人类可变疾病表型， 相对轻度至恶性的临床FHC表型。 我们相信分子生物学方法与高分辨率光学的结合 纳米荧光光谱学将使我们能够成功地回答重要的问题， 关于心脏中FHC介导的病理学的分子基础的问题， RLC和ELC在心肌收缩中的作用。

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.