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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

基本信息

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
项目类别：
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

项目摘要

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.

摘要