PATHOGENESIS AND IN VIVO SUPPRESSION OF THIN FILAMENT-BASED CARDIOMYOPATHIES

细丝型心肌病的发病机制和体内抑制

• 批准号: 10366554
• 负责人: Anthony Cammarato
• 金额: $ 59.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366554
• 关键词: Acetylation; Actins; Actomyosin; Affect; Amino Acids; Animal Model; Automobile Driving; Binding; Binding Sites; Biological Assay; Biological Models; Cardiac; Cardiomyopathies; Characteristics; Charge; Communication; Complement; Complex; Cryoelectron Microscopy; Defect; Development; Dilated Cardiomyopathy; Disease; Disease model; Drosophila genus; Drosophila melanogaster; Electrostatics; Elements; Ensure; Environmental Risk Factor; F-Actin; Funding; Generations; Genetic; Genotype; Goals; Growth; Heart; Human; Hypertrophic Cardiomyopathy; In Vitro; Individual; Laboratories; Lead; Lesion; Location; Lysine; Mechanics; Mediating; Modeling; Modification; Molecular; Movement; Mus; Muscle; Muscle function; Mutation; Myocardial; Myocardial dysfunction; Myosin ATPase; Organ; Pathogenesis; Pathologic; Pathology; Performance; Phenotype; Physiological; Positioning Attribute; Post-Translational Protein Processing; Process; Production; Property; Proteins; Recombinants; Regulation; Relaxation; Rest; Running; Severities; Signal Transduction; Slide; Speed; Structure; Surface; Techniques; Testing; Thick; Thin Filament; Time; Tissues; Transgenic Organisms; Tropomyosin; Troponin; Troponin I; Troponin T; Variant; Ventricular Remodeling; Work; alpha Tropomyosin; base; biophysical analysis; cardiac muscle disease; cell motility; disease-causing mutation; fly; heart function; image reconstruction; improved; in silico; in vivo; insight; interfacial; mimetics; mouse model; novel; prevent; response; tool

Project Summary The thin filament is a multi-subunit regulatory machine. Proper regulation of cardiac contraction requires communication among, and controlled movement of, individual thin filament proteins. The goal of this application is to understand how post-translational modifications (PTMs) and human cardiomyopathy mutations, located at conserved interfaces between thin filament subunits, affect protein-protein associations, modulate muscle function, and/or lead to disease. Drosophila melanogaster benefits from robust experimental tools that permit efficient, yet comprehensive, scrutiny of the most proximal consequences of thin filament perturbations. This animal model will continue to help us discern the mechanistic basis of contractile regulation and, importantly, of myopathic responses to molecular insults. Mice, however, are more genetically and physiologically similar to humans. Using a unique combination of techniques including high-speed video and cryo-electron microscopy, in silico modeling, and mechanical assays we will define, for the first time, the structural and functional effects of specific PTMs and cardiomyopathy mutations, located at interfacial seams between thin filament subunits, from the molecular to the tissue level. Therefore, a highly integrative approach will be employed that relies, in part, on a pioneering strategy to express human actin variants in Drosophila for purification and biophysical analysis, and upon several new fly models of actin and troponin T (TnT)-based cardiomyopathies. The latter will be complemented by murine models. Aim 1 will focus on determining the effects of actin acetylation on tropomyosin (Tm) positioning and cardiac performance using recombinant human proteins, flies, and mice. We will test the hypothesis that acetylation of K326 and K328 on actin, residues we previously showed bind to and help orient Tm such that it prevents actomyosin cycling, discourages inhibitory Tm positioning and promotes cardiac contraction. For Aim 2 we will delineate how certain actin and TnT cardiomyopathy mutations uniquely affect myocardial relaxation. We will test the hypothesis that particular actin and TnT lesions disturb distinct, critical interfacial contacts with Tm, which differentially alters Tm-based inhibition of contraction and force production to initiate discrete cardiac pathologies. For Aim 3, we will ascertain if the same actin PTMs investigated in Aim 1, improve or worsen myocardial dysfunction in murine and fly cardiomyopathy models. We will test the hypothesis that enhanced cardiac contractility, conferred by actin pseudo-acetylation, will improve and aggravate the pathological phenotypes in models of dilated and hypertrophic cardiomyopathy, respectively. Overall, this work is significant since it will provide critical structural and functional information necessary to understand how the thin filament machine operates normally and during disease. Additionally, our efforts will yield genotype- phenotype information in a less complex model system (Drosophila) that limits genetic modifiers and environmental factors to help establish paradigms for disease processes involved in cardiac remodeling.

项目摘要 细丝是一个多亚基调节机器。正确调节心脏收缩需要 个体细丝蛋白之间的通讯和受控运动。这个应用程序的目标是 是了解翻译后修饰（PTM）和人类心肌病突变（位于 细丝亚基之间的保守界面，影响蛋白质-蛋白质缔合，调节肌肉 功能和/或导致疾病。果蝇从强大的实验工具中受益， 有效的，但全面的，细灯丝扰动的最近端的后果的审查。这 动物模型将继续帮助我们识别收缩调节的机械基础，重要的是， 对分子损伤的肌病反应然而，小鼠在遗传和生理上更类似于 人类使用包括高速视频和低温电子显微镜在内的独特技术组合， 通过计算机模拟和机械分析，我们将首次定义 特定的PTM和心肌病突变，位于细纤维亚基之间的界面缝， 从分子水平到组织水平。因此，将采用高度综合的方法，部分依赖于 在果蝇中表达人类肌动蛋白变体用于纯化和生物物理分析的开创性策略，以及 几种新的基于肌动蛋白和肌钙蛋白T（TnT）的心肌病苍蝇模型。后者将 补充了鼠模型。目的1将集中于确定肌动蛋白乙酰化对原肌球蛋白的影响 (Tm)定位和心脏性能使用重组人类蛋白质，苍蝇，和小鼠。我们将测试 假设肌动蛋白上K326和K328乙酰化，我们先前显示的残基结合并帮助定向 Tm，使得其阻止肌动球蛋白循环，阻止抑制性Tm定位并促进心脏收缩。 收缩。对于目标2，我们将描述某些肌动蛋白和TnT心肌病突变如何独特地影响 心肌舒张我们将检验这样一个假设，即特定的肌动蛋白和TnT病变干扰了不同的、关键的 与Tm的界面接触，其差异性地改变了基于Tm的收缩和力产生的抑制， 引发离散的心脏病对于目标3，我们将确定目标1中研究的相同肌动蛋白PTM， 改善或恶化小鼠和苍蝇心肌病模型心肌功能障碍。我们将检验这个假设 增强的心肌收缩力，由肌动蛋白假乙酰化赋予，将改善和加重 扩张型和肥厚型心肌病模型中的病理表型。总的来说，这项工作 是重要的，因为它将提供关键的结构和功能信息，了解如何 细丝机正常运行和疾病期间。此外，我们的努力将产生基因型- 表型信息在一个不太复杂的模型系统（果蝇），限制遗传修饰剂， 环境因素，以帮助建立涉及心脏重塑的疾病过程的范例。