Cooperativity in the Cardiac Myofilament Interactome in Health and Disease

摘要 家族性肥厚型心肌病(FHC)和扩张型心肌病(DC)是影响 心脏有效泵血的能力，可导致心力衰竭。我们的主要假设是心脏 肌肉表现关键取决于心肌细丝的协同激活，因为它们 对钙离子与肌钙蛋白的结合以及肌球蛋白与肌动蛋白和原肌球蛋白(TM)的结合作出反应； 细丝中这些位点的突变导致的合作作用缺陷会导致心肌病。 在这里，使用跨学科的方法，我们检验了合作互动需要的假设(1) 细丝上相邻TM分子之间特定而稳定的头尾相互作用以及(2) 肌动蛋白与TM和肌球蛋白头部在静息状态和交叉桥循环中的特异性相互作用。 具体目标1是确定正确组装薄膜所需的结构和功能相互作用 并测试与心肌病相关的细丝TM突变会导致有缺陷的细丝的假设 细丝组织和对磷酸化的反应。我们将：(I)确定模式和突变- TM与肌动蛋白细丝结合的诱导中断决定了TM突变和 用TIRFM研究TM-肌动蛋白结合和TM-TM头尾连接的磷酸化及其改变 突变型原肌球蛋白与肌钙蛋白相互作用的表面等离子共振(SPR)研究 如果分离的突变型TMS中的特定位点通过电子显微镜(EM)和 分子动力学(MD)；(Iii)比较野生型、突变型和磷酸化TM的头尾连锁 EM和MD；以及iv)通过零长度交联和分级质量来定义细丝相互作用体 光谱(XL-MS)技术；并确定心肌病突变诱导的薄层改变 花丝互作组。具体目标2是检验与心肌病有关的细丝的假设 突变产生有缺陷的细丝结构并扰乱细丝之间的功能相互作用 带有肌动蛋白和肌球蛋白头部(和肌钙蛋白)的TM，进而干扰协同细丝激活 和放松。我们将：(I)确定突变TM对细丝的调控位置是否受到 使用3D-EM和计算化学的突变TM；(Ii)确定TM突变是否导致缺陷或 使用体外运动分析和激光捕获方法检测缺乏细丝的协作性；以及(Iii)建立 细丝分子相互作用的改变如何转化为有缺陷的细丝协作性 更复杂的细胞和组织环境。预计这些研究将产生高分辨率的残留物 特定的、正常和突变的心肌细丝的图片，这将有助于设计新的 针对特定突变的治疗方法。

Abstract Familial hypertrophic cardiomyopathy (FHC) and dilated cardiomyopathy (DC) are diseases that affect the ability of the heart to effectively pump blood and can result in heart failure. Our main hypothesis is that cardiac muscle performance depends critically on cooperative activation of cardiac muscle thin filaments as they respond to Ca2+ binding to troponin and myosin binding to actin and tropomyosin (Tm); and that deficient or defective cooperative interactions due to mutations at these sites in the thin filament lead to cardiomyopathies. Here, using interdisciplinary approaches we test the hypothesis that cooperative interaction requires (1) specific and stable head-to-tail interactions between adjacent Tm molecules on thin filaments as well as (2) specific interactions between actin and Tm and myosin heads at rest and during the crossbridge cycle on actin. Specific Aim 1 is to determine the structure and functional interactions required for proper assembly of thin filaments and test the hypothesis that cardiomyopathy-linked thin filament Tm mutations lead to faulty thin filament organization and responses to phosphorylation. We will: (i) determine the mode, and mutation- induced disruptions, of Tm binding to actin filaments, determine the effect of Tm mutations and phosphorylation on Tm-actin binding and Tm-Tm head-to-tail linkages using TIRFM and determine altered interactions between mutant tropomyosin and troponin using surface plasmon resonance (SPR); (ii) determine if specific sites in isolated mutant Tms show conformational alterations by electron microscopy (EM) and Molecular Dynamics (MD); (iii) compare head-to-tail Tm linkage of wild-type, mutant and phosphorylated Tm by EM and MD; and iv) define the thin filament interactome by zero-length cross-linking and hierarchical mass spectrometry (XL-MS) technology; and determine cardiomyopathy-mutation-induced alterations in the thin filament interactome. Specific Aim 2 is to test the hypothesis that cardiomyopathy-linked thin filament mutations generate defective thin filament structures and perturb functional interactions between thin filament Tm with actin and myosin heads (and troponin) that, in turn, interfere with cooperative thin filament activation and relaxation. We will: (i) determine if the regulatory position of mutant Tm on thin filaments is affected by mutant Tms using 3D-EM and computational chemistry; (ii) determine if Tm mutation leads to defective or deficient thin filament cooperativity using in vitro motility assays and laser trapping methods; and (iii) Establish how alterations in thin filament molecular interactions translate to defective thin filament cooperativity in the more complex cellular and tissue environment. The studies are expected to yield a high resolution, residue specific, picture of normal and mutant cardiac muscle thin filaments that will facilitate the design of new therapies specific for particular mutations.