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遗传疾病，源自编码心脏主要收缩蛋白的基因突变，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，是年轻人心源性猝死的主要原因。本研究旨在阐明在单个肌球蛋白跨桥水平上触发 FHC 的分子机制。我们建议检验以下假设：FHC 是由于表达肌球蛋白 RLC 和 ELC 致病突变的转基因小鼠心脏中肌球蛋白跨桥动力学的改变而导致心肌对 ATP 的低效利用而引起的。我们将在来自转基因小鼠心脏的乳头肌纤维的单分子水平上检验这一假设，这些纤维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，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​​来自带皮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能够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）进行平均，并且能够明确确定“健康”和“患病”肌肉的动力学。由于人类患者是 FHC 突变的杂合子，并且他们的粗肌丝包含散布的 WT 和 HCM 突变头，因此将单分子信息与在肌纤维水平评估的 FHC 表型关联起来极其重要。具体来说，我们询问 FHC 心脏的单个跨桥和健康转基因对照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。使用这种单分子技术获得的信息将与力发展、剥皮乳头肌纤维上的 ATP 酶以及转基因小鼠完整肌纤维上的力和钙瞬变的功能研究并行（目标 2A）。最终目标是将单分子衍生数据与细胞研究结果联系起来，以充分了解导致 FHC 的个体 RLC 和 ELC 突变的作用机制（目标 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在人类中引起不同的疾病表型，从相对轻微的临床 FHC 表型到恶性的临床 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 介导的心脏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。公共健康相关性：这项研究旨在揭示家族性肥厚型心肌病这一主要公共健康问题的机制。该提案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带来治疗严重心脏病的新方法。该应用的优势在于将分子生物学和纳米荧光显微方法相结合，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回答有关 FHC 介导的心脏病理学分子基础的重要问题。