Pathogenesis and in vivo suppression of thin filament based cardiomyopathies

基于细丝的心肌病的发病机制和体内抑制

• 批准号: 8884895
• 负责人: Anthony Cammarato
• 金额: $ 36.67万
• 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 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8884895
• 关键词: Actins; Actomyosin; Affect; Alleles; Amino Acids; Animal Model; Animals; Atomic Force Microscopy; Binding; Binding Sites; Biological Models; Cardiac; Cardiomyopathies; Charge; Clinical; Communication; Complex; Comprehension; Computer Simulation; Data; Defect; Development; Disease; Drosophila genus; Drosophila melanogaster; Electron Microscopy; Elements; Environmental Risk Factor; F-Actin; Face; Figs - dietary; Functional disorder; Genetic; Genetic Suppression; Genotype; Goals; Heart Diseases; Human; Hypertrophic Cardiomyopathy; Image; Imaging Techniques; In Vitro; Individual; Inherited; Investigation; Laboratories; Lead; Lesion; Life; Light; Location; Mechanics; Mediating; Missense Mutation; Modality; Modeling; Modification; Molecular; Molecular Genetics; Movement; Muscle; Muscle Contraction; Mutation; Myocardial dysfunction; Myocardium; Myopathy; Myosin ATPase; N-terminal; Pathogenesis; Pathologic Processes; Pathology; Performance; Phenotype; Physiological; Positioning Attribute; Process; Production; Proteins; Regulation; Relative (related person); Restrictive Cardiomyopathy; Role; Running; Signal Transduction; Site; Skeletal Muscle; Speed; Striated Muscles; Structure; Suppressor Mutations; Surface; System; Testing; Therapeutic; Thin Filament; Time; Tissues; Transgenic Animals; Tropomyosin; Troponin; Troponin T; Variant; Work; base; design; disease-causing mutation; fly; improved; in vivo; innovation; insight; molecular mechanics; novel; prevent; public health relevance; research study; response; skeletal; tool; treatment strategy

 DESCRIPTION (provided by applicant): Striated muscle contraction involves highly dynamic processes that require coordinated communication among, and relative movement of, individual thin filament components. The goal of this application is to understand how human cardiomyopathy mutations located at conserved interfaces between thin filament subunits lead to disease. Drosophila melanogaster, the fruit fly, benefits from robust experimental tools that permit efficient tissue-specific expression of disease alleles in cardiac or skeletal muscle and relatively rapid genetic interaction screens. The fly represents a powerful in vivo system to scrutinize the most proximal consequences of thin filament lesions to facilitate our effort to discern the molecular basis of contractile regulation and, importantly, of myopathic responses in humans. A remarkably integrative approach will be employed that relies upon several new Drosophila models of actin and troponin T (TnT)-based cardiomyopathies. Animal models do not currently exist for five of the six mutations under investigation here, minimizing our comprehension of the pathological effects of these disease alleles in the physiological context of muscle. Using a unique combination of imaging techniques that includes high-speed live video, confocal, atomic force and electron microscopy we will define, for the first time, the structural and functional effects of the cardiomyopathy mutations from the tissue to the molecular level. The studies will involve pioneering strategies to evaluate Drosophila systolic and diastolic molecular mechanics in vivo. Aim 1 will focus on multiple hypertrophic cardiomyopathy (HCM) models that express one of three a-cardiac actin missense mutations. We will test the hypothesis that the HCM actin variants induce similar cardiac and skeletal pathology in flies due to equivalently disturbed tropomyosin (Tm)-based contractile regulation that leads to excessive contractile activity. For Aim 2 the hierarchical effects of several TnT cardiomyopathy mutations will be delineated. We will test the hypothesis that the mutations differentially influence TnT-Tm interaction, which distinctly affects the extent of contractile inhibition and consequently prompts diverse cardiac remodeling in flies. For Aim 3 "second-site" actin mutations will be used to improve cardiac dysfunction initiated by aberrant TnT, in vivo and in vitro. Using Drosophila we identified specific actin lesions that suppress TnT-mediated skeletal myopathy. We will now test the hypothesis that, when co-expressed, these second-site actin mutations can ameliorate TnT-based cardiomyopathies in our fly models. Overall this work is significant since it will provide critical structural-functional information necessary to better comprehend how the thin filament machine functions normally and during disease. Additionally, our efforts will yield genotype-phenotype information in a less complex model system that limits genetic modifiers and environmental factors to help establish paradigms and treatment strategies for pathological processes involved in cardiac remodeling.

 描述（由申请人提供）：横纹肌收缩涉及高度动态的过程，需要各个细丝组件之间的协调通信和相对运动。该应用的目的是了解位于细丝亚基之间保守界面的人类心肌病突变如何导致疾病。黑腹果蝇受益于强大的实验工具，这些工具允许疾病等位基因在心肌或骨骼肌中有效的组织特异性表达以及相对快速的遗传相互作用筛选。果蝇代表了一个强大的体内系统，可以仔细检查细丝病变的最近后果，以促进我们努力辨别收缩调节的分子基础，更重要的是，辨别人类肌病反应的分子基础。将采用一种非常综合的方法，该方法依赖于几种新的基于肌动蛋白和肌钙蛋白 T (TnT) 的心肌病的果蝇模型。目前正在研究的六种突变中的五种目前尚不存在动物模型，这最大限度地减少了我们对这些疾病等位基因在肌肉生理背景下病理影响的理解。通过使用高速实时视频、共聚焦、原子力和电子显微镜等成像技术的独特组合，我们将首次从组织到分子水平定义心肌病突变的结构和功能影响。这些研究将涉及评估果蝇体内收缩和舒张分子力学的开创性策略。目标 1 将重点关注表达三种α-心脏肌动蛋白错义突变之一的多种肥厚型心肌病 (HCM) 模型。我们将测试以下假设：HCM 肌动蛋白变体在果蝇中诱导类似的心脏和骨骼病理学，因为基于原肌球蛋白 (Tm) 的收缩调节受到同等干扰，导致过度收缩活动。对于目标 2，将描述几种 TnT 心肌病突变的分层效应。我们将检验这样的假设：突变对 TnT-Tm 相互作用有不同的影响，这会明显影响收缩抑制的程度，从而提示 果蝇的不同心脏重塑。对于 Aim 3，“第二位点”肌动蛋白突变将用于改善体内和体外异常 TnT 引发的心脏功能障碍。利用果蝇，我们发现了抑制 TnT 介导的骨骼肌病的特定肌动蛋白损伤。我们现在将测试以下假设：当共表达时，这些第二位点肌动蛋白突变可以改善我们的果蝇模型中基于 TnT 的心肌病。总的来说，这项工作意义重大，因为它将提供更好地理解细丝机器正常和疾病期间如何运作所必需的关键结构功能信息。此外，我们的努力将在一个不太复杂的模型系统中产生基因型-表型信息，该模型系统限制遗传修饰剂和环境因素，以帮助建立心脏重塑涉及的病理过程的范例和治疗策略。