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: 7806533
• 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/7806533
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Abbreviations; Actins; Address; Age; Animal Model; Applications Grants; Arts; 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; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Human; Hypertrophy; Individual; Isometric Contraction; Kinetics; Label; Lead; Left; Light; Link; Malignant - descriptor; Measurement; Measures; Mechanics; Mediating; Microscopic; Mind; Modality; Molecular; Molecular Biology; Monitor; Mus; Muscle; Muscle Contraction; Muscle Fibers; 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疾病，源自基因中的突变，编码心脏的主要收缩蛋白，包括心室肌球蛋白调节（RLC）（RLC）和必需（ELC）轻链。 FHC导致心室和间隔肥大，肌原纤维混乱，是年轻人突然心脏死亡的主要原因。这项研究旨在阐明在单个肌球蛋白杂交桥的水平上触发FHC的分子机制。我们建议检验以下假设：FHC是由于心肌对ATP的效率低下而引起的，这是由于肌球蛋白跨桥动力学改变了转基因小鼠心脏中表达肌球蛋白RLC和ELC中引起疾病​​的突变的转基因小鼠心脏。我们将在转基因小鼠心脏的乳头肌纤维中的单分子水平上检查该假设，这些肌肉纤维在肌球蛋白的调节和/或必要的轻链中携带引起疾病的突变。我们坚信，必须在单个杂交桥的水平上进行肌球蛋白跨桥动力学的明确测定，并且与从皮肤和完整的肌肉纤维上的测量中得出的跨桥力学相比，结果。单分子方法的优点是它避免平均比具有不同动力学的分子（例如WT和FHC分子的混合物）以及明确确定“健康”和“疾病”肌肉动力学的能力。由于人类患者的FHC突变是杂合的，并且其厚细胞包含散布的WT和HCM突变型头，因此将单分子信息与在肌肉纤维水平上评估的FHC表型相关联非常重要。具体而言，我们询问持续时间（AIM 1A）和寿命（AIM 1B）在FHC心脏的单个杂交桥和健康的转基因控制中是否相同。使用该单分子技术得出的信息将与力发育的功能研究，皮肤乳头肌纤维的ATPase以及来自转基因小鼠完整肌肉纤维的力和钙瞬变（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，以充分了解引起FHC的单个RLC和ELC突变的作用机理（AIM 2B）。正在解决的基本问题是为什么这些在RLC和/或ELC中的个体突变如何引起人类中的疾病表型，从相对轻度到恶性的临床FHC表型。我们认为，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回答有关FHC介导病理学在心脏中的分子基础以及RLC和ELC在心脏肌肉收缩中的作用的重要问题。公共卫生相关性：这项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。该建议的目的是了解分子碱基的肌肉肌球蛋白光链中的突变导致人类心脏肥大。成功完成此目标可能会导致严重心脏病的新方式。该应用的强度是由单个分子水平的研究疾病的突变中分子生物学和纳米荧光显微镜方法组合而成的。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回答有关心脏中FHC介导的病理学分子基础的重要问题。