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.

项目摘要/摘要 扩张的心肌病（DCM）是全球心力衰竭的第二大最常见原因，并继承了 DCM的形式占非缺血病例的30％。编码β-心肌球蛋白（M2β）的MyH7是一个 更常见的突变基因，是分子运动，它可以在心室中供电 心肌细胞。该提案的重点是检查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并发展 理论策略的基础。肌肉的力，速度和发电能力是相关的 具有在厚细丝中募集肌球蛋白分子与肌动蛋白相互作用的能力 肌肉肌膜。招募的肌球蛋白分子通过使用构成的ATPase循环产生力 肌球蛋白在与肌动蛋白互动时会产生动力中风。心脏肌球蛋白可以在自动抑制中存在 具有缓慢的ATP营业额（超放松状态，SRX）的状态，其中头部和头尾相互作用可防止 它来自与肌动蛋白（相互作用的基序，IHM）或未抑制状态（无序的松弛状态， DRX）很容易产生力。招募的肌球蛋白还影响 肌膜丝是因为肌球蛋白结合会协同激活肌动蛋白细丝。我们将测试中央 假设DCM突变通过改变内在力的产生来损害心脏的收缩收缩 单个心脏肌球蛋白分子的能力，稳定SRX/IHM状态和/或改变合作 肌动蛋白细丝的激活。在第一个目的中，我们将研究DCM突变对 肌球蛋白ATPase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fret生物传感器来检查肌球蛋白动力中风和另一个fret传感器 检查IH​​M状态形成。电子显微镜也将用于评估IHM状态的形成 AIM 2将检查哪些将直接与荧光光谱和生化分析进行比较。 DCM突变对人M2β的单分子机械性能的影响，包括步长 使用负载夹紧的光学陷阱和载荷依赖性脱离。在AIM 3中，我们将使用计算 肌肉合同的模型，以预测目标1和2中测量的参数将如何影响合奏力， 速度和力量。然后，我们将直接检查突变对产生力特性的影响 通过将人类M2β构建体纳入基于DNA的“设计师”厚细丝中，并检查其能力 以钙依赖的方式与受调节的细丝型相互作用。力量，速度和力量 测量将在“设计师”厚的细丝中进行，其中包含像天然厚细丝一样 肌球蛋白分子的几何间距。总体而言，该提案的具体目的的完成将增强 我们对DCM中疾病发病机理的分子机制的理解，为 开发用于治疗DCM的疗法。