Impact of dilated cardiomyopathy mutations on cardiac myosin structure and function

扩张型心肌病突变对心肌肌球蛋白结构和功能的影响

• 批准号: 10595237
• 负责人: Sivaraj Sivaramakrishnan
• 金额: $ 75.05万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595237
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Actins; Adopted; Binding; Biochemical; Biomedical Engineering; Biophysics; Biosensor; Calcium; Cardiac; Cardiac Myocytes; Cardiac Myosins; Closure by clamp; Computer Models; Contracts; DNA; Data; Defect; Dilated Cardiomyopathy; Disease; Electron Microscopy; Enzymatic Biochemistry; Equilibrium; Filament; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Foundations; Generations; Genes; Genetic; Goals; Head; Heart; Heart failure; Human; Impairment; Individual; Inherited; Kinetics; Lasers; Left; Link; Measurement; Measures; Mechanics; Microfilaments; Modeling; Molecular; Molecular Conformation; Molecular Motors; Molecular Structure; Monitor; Motor; Muscle; Muscle Cells; Muscle Contraction; Muscle Fibers; Mutate; Mutation; Myocardium; Myosin ATPase; Nanotubes; Nonmuscle Myosin Type IIA; Pathogenesis; Point Mutation; Power stroke; Property; Regulation; Relaxation; Reporting; Sarcomeres; Slide; Striated Muscles; Structure; System; Systole; Systolic Pressure; Tail; Testing; Therapeutic; Thick Filament; Thin Filament; Ventricular; arm; beta-Myosin; blood pump; design; human disease; in silico; mechanical properties; molecular mechanics; motor impairment; muscle physiology; mutant; novel; optic trap; optical traps; prevent; recruit; sensor; single molecule; structural biology; therapy development

Project Summary/Abstract Dilated cardiomyopathy (DCM) is the second most common cause of heart failure world-wide, and inherited forms of DCM make up 30% of non-ischemic cases. MYH7, which encodes beta-cardiac myosin (M2β), is one of the more commonly mutated genes and is the molecular motor that powers contraction in ventricular cardiomyocytes. This proposal is focused on examining the structural and functional impact of DCM mutations in human M2β, with an overall goal of determining molecular mechanisms of contractile defects and developing a foundation for therapeutic strategies. The force, velocity, and power generating capacity of muscle is related to the ability to recruit myosin molecules in the thick filament to interact with actin in the thin filament of the muscle sarcomere. The recruited myosin molecules generate force by utilizing a conserved ATPase cycle in which myosin generates a power stroke while interacting with actin. Cardiac myosin can exist in the auto-inhibited state with slow ATP turnover (super relaxed state, SRX) in which head-head and head-tail interactions prevent it from interacting with actin (interacting heads motif, IHM) or the uninhibited state (disordered relaxed state, DRX) that is readily available to produce force. The recruited myosin also impacts the calcium sensitivity of the myofilaments because myosin binding cooperatively activates the actin thin filament. We will test the central hypothesis that DCM mutations impair systolic contraction in the heart by altering the intrinsic force producing ability of individual cardiac myosin molecules, stabilizing the SRX/IHM state, and/or altering cooperative activation of the actin thin filament. In the first Aim we will examine the impact of the DCM mutations on the myosin ATPase cycle, duty ratio, and formation of the SRX state. The structural impact of the mutations will be examined by using a FRET biosensor that monitors the myosin power stroke and another FRET sensor that examines IHM state formation. Electron microscopy will also be used to evaluate the formation of the IHM state which will be directly compared to the fluorescence spectroscopy and biochemical analysis. Aim 2 will examine the impact of DCM mutations on the single molecule mechanical properties of human M2β, including step size and load-dependent detachment, using a load clamped optical trap. In Aim 3 we will utilize a computational model of muscle contraction to predict how the parameters measured in Aims 1&2 will impact ensemble force, velocity, and power. We will then directly examine the impact of the mutations on the force generating properties by incorporating the human M2β constructs into DNA-based “designer” thick filaments, and examining their ability to interact with regulated thin filaments in a calcium dependent manner. The force, velocity, and power measurements will be performed in the “designer” thick filaments, which contain native thick filament-like geometric spacing of myosin molecules. Overall, the completion of the specific aims of this proposal will enhance our understanding of the molecular mechanisms of disease pathogenesis in DCM and provide a foundation for developing therapies for treating DCM.

项目总结/摘要 扩张型心肌病（DCM）是全球范围内心力衰竭的第二大常见原因， 扩张型心肌病占非缺血性病例的30%。MYH 7编码β-心肌肌球蛋白（M2β），是一种 它是一种分子马达，为心室肌细胞的收缩提供动力。 心肌细胞该提案的重点是研究DCM突变的结构和功能影响 在人类M2β中，总体目标是确定收缩缺陷的分子机制， 治疗策略的基础。肌肉的力、速度和发电能力是相关的 在粗肌丝中募集肌球蛋白分子与细肌丝中的肌动蛋白相互作用的能力， 肌节募集的肌球蛋白分子通过利用一个保守的ATP酶循环产生力， 其中肌球蛋白在与肌动蛋白相互作用时产生动力冲程。心肌肌球蛋白可以存在于自抑制的 ATP转换缓慢的状态（超松弛状态，SRX），其中头-头和头-尾相互作用阻止 它与肌动蛋白相互作用（相互作用头基序，IHM）或不受抑制的状态（无序松弛状态， DRX），其易于产生力。募集的肌球蛋白也影响心肌细胞的钙敏感性。 肌丝，因为肌球蛋白结合协同激活肌动蛋白细丝。我们将测试中央 假设DCM突变通过改变产生心肌收缩力的内在力来损害心脏收缩， 单个心肌肌球蛋白分子的能力，稳定SRX/IHM状态，和/或改变合作 激活肌动蛋白细丝。在第一个目标中，我们将研究DCM突变对 肌球蛋白ATP酶周期、占空比和SRX状态的形成。突变的结构影响将是 通过使用监测肌球蛋白动力冲程的FRET生物传感器和另一个 研究IHM状态形成。电子显微镜也将用于评估IHM状态的形成 其将直接与荧光光谱和生化分析进行比较。目标2将检查 DCM突变对人M2β单分子力学性质的影响，包括步长 和负载依赖性分离。在目标3中，我们将利用计算 肌肉收缩模型，以预测目标1和2中测量的参数将如何影响整体力， 速度和功率然后，我们将直接研究突变对力产生特性的影响 通过将人类M2β构建体整合到基于DNA的“设计师”粗丝中，并检查它们的能力， 以钙依赖的方式与受调节的细丝相互作用。力、速度和功率 测量将在“设计师”粗长丝中进行，该长丝含有天然的粗的类金刚石纤维。 肌球蛋白分子的几何间距。总的来说，完成这项建议的具体目标将提高 我们对扩张型心肌病发病机制的分子机制的理解，为 开发治疗DCM的疗法。