Pathogenesis and in vivo suppression of thin filament based cardiomyopathies

 描述（由申请人提供）：横纹肌收缩涉及高度动态的过程，需要各个细丝成分之间的协调通信和相对运动。本申请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。果蝇，果蝇，受益于强大的实验工具，允许有效的组织特异性表达的疾病等位基因在心脏或骨骼肌和相对快速的遗传相互作用的屏幕。苍蝇代表了一个强大的体内系统，以审查最近端的细丝病变的后果，以促进我们的努力，以辨别收缩调节的分子基础，重要的是，在人类的肌病反应。一个显着的综合方法将采用依赖于几个新的果蝇模型的肌动蛋白和肌钙蛋白T（TnT）为基础的心肌病。动物模型目前还不存在的六个突变中的五个在这里调查，最大限度地减少了我们的理解这些疾病等位基因在肌肉的生理背景下的病理影响。使用独特的成像技术组合，包括高速实时视频，共聚焦，原子力和电子显微镜，我们将首次定义心肌病突变从组织到分子水平的结构和功能影响。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。目的1将集中在多发性肥厚型心肌病（HCM）模型，表达三个α-心脏肌动蛋白错义突变之一。我们将测试的假设，即HCM肌动蛋白变体诱导类似的心脏和骨骼病理学在苍蝇中，由于同等干扰原肌球蛋白（Tm）为基础的收缩调节，导致过度的收缩活动。对于目标2，将描述几种TnT心肌病突变的分层效应。我们将检验突变差异影响TnT-Tm相互作用的假设，TnT-Tm相互作用明显影响收缩抑制的程度，从而提示TnT-Tm相互作用的发生。 不同的心脏重塑。对于目标3，“第二位点”肌动蛋白突变将用于改善由异常TnT引发的体内和体外心功能障碍。使用果蝇，我们确定了特定的肌动蛋白病变，抑制TnT介导的骨骼肌病变。我们现在将测试的假设，当共表达，这些第二个网站肌动蛋白突变可以改善我们的苍蝇模型中的肌钙蛋白为基础的心肌病。总的来说，这项工作是重要的，因为它将提供必要的关键结构功能信息，以更好地理解细丝机器如何正常工作和疾病期间。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所涉及的病理过程的范例和治疗策略。

 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.