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

表达 FHC 突变的转基因小鼠心脏中的单桥动力学

• 批准号: 8055012
• 负责人: JULIAN BOREJDO
• 金额: $ 40.04万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055012
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Abbreviations; Actins; Address; Age; Animal Model; Applications Grants; Asses; Binding; Biological; Calcium; Calmodulin; Cardiac; Cardiac Muscle Contraction; Cardiovascular Diseases; Clinical; Contractile Proteins; Contracts; Data; Detection; Development; Disease; Dissociation; Dyspnea; Electrocardiogram; Environment; Familial Hypertrophic Cardiomyopathy; Fatigue; Fiber; Fluorescence; Fluorescence Spectroscopy; Gene Transfer Techniques; Genes; Goals; Head; Health; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Human; Hypertrophic Cardiomyopathy; Hypertrophy; Individual; Isometric Contraction; Kinetics; Label; Lead; Left; Light; Link; Malignant - descriptor; Measurement; Measures; Mechanics; Mediating; Microscopic; Mind; Modality; Molecular; Molecular Biology; Monitor; Muscle; Muscle Contraction; Muscle Fibers; Mutant Strains Mice; Mutation; Myocardium; Myofibrils; Myopathy; Myosin ATPase; Myosin Alkali Light Chains; Myosin Light Chain Kinase; Myosin Light Chains; Myosin Regulatory Light Chains; Nanotechnology; Optics; Organ; Pathology; Patients; Performance; Phenotype; Physiological; Physiology; Point Mutation; Preparation; Principal Investigator; Process; Proteins; Public Health; Recombinants; Research; Research Personnel; Resolution; Role; Rotation; Sarcomeres; Site; Skin; Solutions; Solvents; Spectrum Analysis; Structure; Techniques; Technology; Testing; Thick Filament; Thin Filament; Time; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Ventricular; Ventricular Myosins; Work; aqueous; base; blood pump; cost; disease phenotype; disease-causing mutation; experience; fluorescence microscope; fluorophore; innovation; mortality; multidisciplinary; mutant; nano; nanomechanics; papillary muscle; premature; public health relevance; single molecule; sudden cardiac death; ventricular hypertrophy

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.

描述（由申请人提供）：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突变，包括心室肌球蛋白调节（RLC）和必需（ELC）轻链。FHC导致室间隔肥厚、肌纤维紊乱，是年轻人心源性猝死的主要原因。本研究旨在阐明在单个肌球蛋白交叉桥水平上触发FHC的分子机制。我们提出在表达肌球蛋白RLC和ELC致病突变的转基因小鼠心脏中，验证FHC是由于肌球蛋白过桥动力学改变导致心肌对ATP的低效利用而引起的假设。我们将在来自转基因小鼠心脏的乳头状肌纤维的单分子水平上检验这一假设，这些小鼠心脏在肌球蛋白的调节和/或基本轻链中携带致病突变。我们坚信，肌球蛋白跨桥动力学的明确测定必须在单个跨桥水平上进行，并将结果与来自皮肤和完整肌纤维测量的跨桥力学进行比较。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”和“患病”肌肉的动力学。由于人类患者的FHC突变是杂合的，并且他们的粗纤维中含有散布的WT和HCM突变头，因此在肌纤维水平上将单分子信息与FHC表型相关联是非常重要的。具体来说，我们想知道在FHC心脏和健康转基因对照中，分离状态和强结合状态的持续时间（Aim 1A）和寿命（Aim 1B）是否相同。使用这种单分子技术获得的信息将与转基因小鼠皮肤乳头状肌纤维上的力发展、atp酶以及完整肌纤维上的力和钙瞬态的功能研究相平行（Aim 2A）。最终目标是将单分子衍生数据与细胞发现联系起来，以充分了解单个RLC和ELC突变导致FHC的作用机制（Aim 2B）。正在解决的基本问题是RLC和/或ELC中的这些个体突变为什么以及如何导致人类从相对轻度到恶性临床FHC表型的可变疾病表型。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的重要问题。公共卫生相关性：本研究旨在揭示家族性肥厚性心肌病的机制，这是一个主要的公共卫生问题。本研究的目的是了解肌球蛋白轻链突变导致人类心脏肥厚的分子基础。这一目标的成功实现可能会带来治疗严重心脏病的新方法。该应用的优势在于其结合了分子生物学和纳米荧光显微方法，在单分子水平上研究致病突变。此外，将单分子方法与患病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。