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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要细丝是一台多工资调节机。正确调节心脏收缩需要单个薄丝蛋白的交流和控制运动之间的交流。该应用程序的目标是要了解翻译后修饰（PTM）和人类心肌病突变如何薄丝亚基之间的保守界面，影响蛋白质 - 蛋白质关联，调节肌肉功能和/或导致疾病。果蝇Melanogaster受益于允许的强大实验工具对细丝扰动最近端后果的高效但全面的审查。这动物模型将继续帮助我们辨别收缩法规的机理基础，重要的是对分子侮辱的肌病反应。然而，小鼠在遗传和生理上与人类。使用独特的技术组合，包括高速视频和冷冻电子显微镜，在计算机建模和机械测定中，我们将首次定义的结构和功能效应特定的PTM和心肌病突变，位于薄丝亚基之间的界面接缝处分子到组织水平。因此，将采用高度综合的方法，部分依赖于一种先驱策略来表达果蝇中的人类肌动蛋白变体用于纯化和生物物理分析，并在基于肌动蛋白和肌钙蛋白T（TNT）基于肌动蛋白T（TNT）的心肌病模型上。后者将是补充鼠模型。 AIM 1将专注于确定肌动蛋白乙酰化对肌动蛋白的影响（TM）使用重组人蛋白，苍蝇和小鼠定位和心脏性能。我们将测试假设K326和K328在肌动蛋白上的乙酰化，我们先前显示的残基与结合并帮助方向 TM使其可防止肌动球蛋白循环，抑制抑制性TM定位并促进心脏收缩。对于目标2，我们将描述某些肌动蛋白和TNT心肌病突变如何唯一影响心肌放松。我们将检验以下假设，即特定的肌动蛋白和TNT病变会干扰明显，关键与TM的界面接触，该接触差异改变了基于TM的基于TM的收缩和迫使产生的抑制作用启动离散的心脏病理。对于AIM 3，我们将确定是否在AIM 1中调查了同一肌动蛋白PTM 在鼠和苍蝇心肌病模型中改善或恶化心肌功能障碍。我们将检验假设通过肌动蛋白伪乙酰化赋予的增强的心脏收缩力将改善并加重扩张和肥厚性心肌病模型中的病理表型。总体而言，这项工作很重要，因为它将提供重要的结构和功能信息，以了解细丝机正常运行和疾病。此外，我们的努力将产生基因型 - 限制遗传修饰符和有助于建立涉及心脏重塑的疾病过程范式的环境因素。