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）是影响心肌细胞功能的疾病。 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 肌肉性能关键取决于心肌细丝的协同激活， 对Ca 2+结合肌钙蛋白和肌球蛋白结合肌动蛋白和原肌球蛋白（Tm）作出反应; 由于细丝中这些位点的突变而导致的有缺陷的协同相互作用导致心肌病。 在这里，使用跨学科的方法，我们测试的假设，合作互动需要（1） 细丝上相邻Tm分子之间特异性和稳定的头-尾相互作用，以及（2） 肌动蛋白和Tm之间的特定相互作用和肌球蛋白头在休息和在肌动蛋白的过桥周期。 具体目标1是确定适当组装薄膜所需的结构和功能相互作用， 并测试心肌病相关的细丝Tm突变导致错误的细丝的假设。 纤维组织和对磷酸化的反应。我们将：（一）确定模式和突变- 诱导的Tm与肌动蛋白丝结合的破坏，决定了Tm突变的效果， 使用TIRFM对Tm-肌动蛋白结合和Tm-Tm头-尾连接的磷酸化进行分析，并确定改变的 使用表面等离子体共振（SPR）测定突变原肌球蛋白和肌钙蛋白之间的相互作用;（ii）确定 如果通过电子显微镜（EM）发现分离的突变Tms中的特定位点显示构象改变， 分子动力学（MD）;（iii）通过比较野生型、突变体和磷酸化Tm的头-尾Tm连接， 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组 光谱（XL-MS）技术;并确定心肌病突变引起的变化，在薄 丝相互作用体具体目标2是检验心肌病相关细丝 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 Tm与肌动蛋白和肌球蛋白头（和肌钙蛋白），反过来，干扰合作细丝激活 和放松.我们将：（i）确定突变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和计算化学确定Tm突变体;（ii）确定Tm突变是否导致缺陷的或 使用体外运动性测定和激光捕获方法的缺乏细丝协同性;和（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，残留物 正常和突变心肌细丝的特定图片，将有助于设计新的 针对特定突变的疗法。

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.