Impact of dilated cardiomyopathy mutations on cardiac myosin structure and function

项目摘要/摘要 扩张型心肌病(DCM)是世界范围内导致心力衰竭的第二大常见原因，而且是遗传性的 形式的扩张型心肌病占非缺血性病例的30%。编码β-心肌肌球蛋白(M2β)的MYH7就是其中之一 是更常见的突变基因之一，是推动心室收缩的分子马达 心肌细胞。这项建议的重点是检查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 这是治疗策略的基础。肌肉的力量、速度和发电能力是相关的。 能够在粗丝中招募肌球蛋白分子与细丝中的肌动蛋白相互作用 肌肉肌节。募集的肌球蛋白分子通过利用保守的ATPase循环在 肌球蛋白在与肌动蛋白相互作用时会产生有力的中风。心肌肌球蛋白可存在于自体抑制 ATP周转缓慢的状态(超级放松状态，SRX)，在这种状态下，头-头和头-尾的相互作用阻止 它来自于与肌动蛋白的相互作用(相互作用的头部基序，IHM)或不受抑制的状态(无序放松状态， (DRX)，这是容易获得的产生武力的工具。募集的肌球蛋白也影响细胞的钙敏感性 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 DCM突变通过改变产生的内在力量损害心脏收缩的假说 单个心肌肌球蛋白分子、稳定SRX/IHM状态和/或改变协同作用的能力 激活肌动蛋白细丝。在第一个目标中，我们将检查DCM突变对 肌球蛋白ATPase周期、占空比和SRX状态的形成。突变的结构性影响将是 通过使用FRET生物传感器来监测肌球蛋白的功率冲程，以及使用另一种FRET传感器来进行检查 检查IHM状态的形成。电子显微镜也将被用来评估IHM态的形成 这将直接与荧光光谱分析和生化分析进行比较。Aim 2将检查 DCM突变对人M2-β单分子力学性质包括步长的影响 以及依赖于负载的分离，使用负载夹持式光学陷阱。在目标3中，我们将使用计算性的 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量， 速度和力量。然后，我们将直接检查突变对力生成属性的影响 通过将人类M2β结构掺入基于dna的“设计者”粗丝中，并检测它们的能力 以钙依赖的方式与受调控的细丝相互作用。力、速度和力量 测量将在“设计者”粗丝中进行，这些粗丝包含天然的粗丝状 肌球蛋白分子的几何间距。总体而言，完成这项提案的具体目标将加强 我们对扩张型心肌病发病机制的分子机制的认识，为 开发治疗扩张型心肌炎的治疗方法。

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.