Pathogenesis and in vivo suppression of thin filament based cardiomyopathies

 描述(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沟通和相对运动。这项应用的目标是了解位于细丝亚基之间保守界面的人类心肌病突变是如何导致疾病的。果蝇黑腹果蝇受益于强大的实验工具，这些工具允许在心肌或骨骼肌中高效地组织特异性表达疾病等位基因，并相对快速地进行遗传相互作用筛选。苍蝇代表了一个强大的体内系统，可以仔细观察细丝损伤的最近端后果，以便于我们努力辨别收缩调节的分子基础，更重要的是，人类肌肉病变反应的分子基础。将采用一种非常综合的方法，依赖于几种基于肌动蛋白和肌钙蛋白T(TNT)的新的果蝇模型。在这里研究的六个突变中，有五个目前还不存在动物模型，这使得我们对这些疾病等位基因在肌肉生理背景下的病理影响的理解变得最少。利用一种独特的成像技术组合，包括高速现场视频、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。这些研究将涉及在体内评估果蝇收缩和舒张期分子力学的开创性策略。目的1将重点放在表达三个a-心脏肌动蛋白错义突变之一的多发性肥厚型心肌病(HCM)模型上。我们将测试这一假设，即HCM肌动蛋白变体在果蝇中引起类似的心脏和骨骼病理，这是由于同等干扰的基于原肌球蛋白(TM)的收缩调节导致过度收缩活动。对于目标2，将描述几种TNT心肌病突变的等级效应。我们将检验这样一个假设，即突变不同地影响TNT-TM相互作用，TNT-TM相互作用明显影响收缩抑制的程度，从而提示 苍蝇心脏重塑的多样性。对于AIM 3，肌动蛋白的“第二位点”突变将在体内和体外用于改善由异常TnT引起的心功能障碍。利用果蝇，我们确定了抑制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。总体而言，这项工作意义重大，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如何在正常和疾病期间发挥作用。此外，我们的努力将在一个不那么复杂的模型系统中产生基因-表型信息，该模型系统限制遗传修饰物和环境因素，以帮助建立涉及心脏重塑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.