Cooperativity in the Cardiac Myofilament Interactome in Health and Disease

心脏肌丝相互作用组在健康和疾病中的协同作用

• 批准号: 9330241
• 负责人: JEFFREY R MOORE
• 金额: $ 39.02万
• 依托单位: UNIVERSITY OF MASSACHUSETTS LOWELL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-11 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9330241
• 关键词: Actins; Affect; Behavior; Binding; Biological Assay; Cardiac; Cardiac Myosins; Cardiomyopathies; Cells; Complex; Computational Biology; Contracts; Dilated Cardiomyopathy; Disease; Drug Design; Drug Targeting; Electron Microscopy; Engineering; Environment; Familial Hypertrophic Cardiomyopathy; Filament; Fluorescence Microscopy; Functional disorder; Future; Goals; Head; Health; Heart; Heart Diseases; Heart failure; Human; Hypertrophic Cardiomyopathy; In Vitro; Induced Mutation; Lasers; Lead; Length; Link; Mass Spectrum Analysis; Mechanics; Methods; Microfilaments; Molecular; Mutation; Myocardium; Myosin ATPase; Pathway interactions; Performance; Phase; Phenotype; Phosphorylation; Physiological; Point Mutation; Positioning Attribute; Process; Proteins; Relaxation; Research Personnel; Resolution; Rest; Sedimentation process; Site; Slide; Specimen; Structure; Surface Plasmon Resonance; Tail; Technology; Testing; Thick Filament; Thin Filament; Time; Tissues; Translating; Tropomyosin; Troponin; Work; blood pump; cell motility; computational chemistry; conformational alteration; crosslink; design; genetic regulatory protein; innovation; interdisciplinary approach; mechanical properties; molecular dynamics; mutant; novel therapeutics; personalized approach; response; structural biology

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.

抽象的 家族性肥厚型心肌病（FHC）和扩张型心肌病（DC）是影响心脏功能的疾病。 心脏有效泵血的能力，可能导致心力衰竭。我们的主要假设是心脏 肌肉性能关键取决于心肌细丝的协同激活，因为它们 对 Ca2+ 与肌钙蛋白和肌球蛋白与肌动蛋白和原肌球蛋白 (Tm) 的结合作出反应；以及那个不足或 由于细丝中这些位点的突变导致协作相互作用缺陷，导致心肌病。 在这里，我们使用跨学科方法检验合作互动需要的假设 (1) 细丝上相邻 Tm 分子之间特异性且稳定的头尾相互作用以及 (2) 肌动蛋白与 Tm 和肌球蛋白头在静止时以及肌动蛋白上的横桥循环期间之间的特定相互作用。 具体目标 1 是确定正确组装薄层所需的结构和功能相互作用 并检验了与心肌病相关的细丝 Tm 突变导致细丝缺陷的假设 丝组织和对磷酸化的反应。我们将：（i）确定模式和突变- Tm 与肌动蛋白丝结合的诱导破坏决定了 Tm 突变的影响， 使用 TIRFM 对 Tm-肌动蛋白结合和 Tm-Tm 头尾连接进行磷酸化并确定改变 使用表面等离子共振 (SPR) 观察突变原肌球蛋白和肌钙蛋白之间的相互作用； (二) 确定 如果分离的突变体 Tms 中的特定位点通过电子显微镜 (EM) 显示构象改变，并且 分子动力学（MD）； (iii) 比较野生型、突变型和磷酸化 Tm 的头尾 Tm 连接 EM 和 MD； iv) 通过零长度交联和分层质量定义细丝相互作用组 光谱测定（XL-MS）技术；并确定心肌病突变引起的薄层改变 丝相互作用组。具体目标 2 是检验以下假设：心肌病与细丝相关 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 肌动蛋白和肌球蛋白头（和肌钙蛋白）的 Tm 反过来会干扰细丝的协同激活 和放松。我们将：（i）确定突变体 Tm 在细丝上的调节位置是否受到以下因素的影响： 使用 3D-EM 和计算化学的突变 Tms； (ii) 确定 Tm 突变是否导致缺陷或 使用体外运动测定和激光捕获方法的细丝协同性不足； (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同性 更复杂的细胞和组织环境。这些研究预计将产生高分辨率的残留物 正常和突变心肌细丝的具体图片，这将有助于设计新的心肌细丝 针对特定突变的特定疗法。