Beta-arrestin Signaling and Sarcomere Calcium-Response in Familial Dilated Cardiomyopathy

 描述(申请人提供)：家族性扩张型心肌病(DCM)是一种导致心力衰竭的疾病，也是心脏移植最常见的原因。疾病进展的分子机制还没有得到很好的描述，目前还没有治疗这种疾病的方法。扩张型心肌病的一个典型的分子变化是肌丝对钙离子的反应减弱。因此，我们推测，通过增加肌丝对钙离子的反应，我们可能能够防止DCM的发展。作为原理的证明，我们将一个带有肌瘤蛋白原肌球蛋白(TG-TM-E54K)错义突变的转基因模型与一只表现出与人类DCM相似的表型的转基因小鼠与一只具有结构性钙敏感性增加特征的转基因小鼠进行杂交。在这些双转基因小鼠中，没有发生DCM。我们的初步研究结果表明，新的β-arrestin信号通路直接参与对肌丝的信号传递，传递钙敏化的翻译后修饰，并改善心功能。在这个方案中，我们假设β-arrestin信号通过改善肌丝的钙反应来阻止和逆转DCM的进展。我们的方法是使用TG-TmE54K小鼠并用有偏见的配体TRV120023治疗它们，TRV120023作为血管紧张素受体阻滞剂，但能够激活β-arrestin信号，或氯沙坦，血管紧张素受体阻滞剂。具体而言 目的1利用系列超声心动图评价活体治疗前、中、后的病情进展情况。我们还将分别使用血流动力学研究和组织学方法评估活体左心功能和形态。为了达到特定的目标2，我们将使用洗涤剂提取的纤维束来测量处理动物和生理盐水对照组的稳态力和动力学。我们还将使用生化方法来确定这些变化背后的机制，在那里我们将阐明激活的信号转导途径。在具体目标3中，我们将使用生化方法和显微镜来检测β-arrestin的定位，以充分表征这些新的信号转导途径。我们预计，激活β-arrestin信号，同时阻断G-蛋白介导的信号，将通过增加肌丝对钙的反应性来阻止和逆转DCM的进展。因此，这项提议的结果将为扩张型心肌病的潜在治疗提供临床前数据，以及对心力衰竭的分子机制的洞察。

 DESCRIPTION (provided by applicant): Familial dilated cardiomyopathy (DCM) is a condition that leads to heart failure and is the most common cause of heart transplantation. The molecular mechanisms of disease progression are not well characterized, and there are no current therapies available for this condition. A characteristic molecular change in DCM is decreased myofilament response to Ca2+. Thus, we hypothesized that by increasing the myofilament response to Ca2+ we may be able to prevent the development of DCM. As proof of principle, we crossed a transgenic model with a missense mutation on sarcomeric protein, tropomyosin (TG-Tm-E54K), which displays a phenotype similar to human DCM, with a transgenic mouse characterized by a constitutive increase in Ca2+-sensitivity. In these double transgenic mice, DCM does not develop. Results from our preliminary studies indicate that novel beta-arrestin signaling pathways are directly involved in signaling to myofilaments, imparting Ca2+-sensitizing post-translational modification, and improving heart function. In this proposal, we hypothesize that beta-arrestin signaling prevents the progression of and reverses DCM by improving the Ca2+ responsiveness of the myofilament. Our approach is to employ TG-TmE54K mice and treat them with the biased ligand, TRV120023, which acts as an angiotensin receptor blocker but is able to activate beta-arrestin signaling, or Losartan, an angiotensin receptor blocker. In Specific Aim 1 we will evaluate the progression of the disease before, during and after treatment in vivo utilizing serial echocardiography. We will also assess left ventricular function in vivo and morphology using hemodynamic studies and histological methods, respectively. To approach Specific Aim 2, we will employ detergent extracted fiber bundles to measure steady-state force and kinetics of treated animals and saline controls. We will also determine the mechanism behind these changes using biochemical methods where we will elucidate the signal transduction pathways activated. In Specific Aim 3 we will use biochemical methods and microscopy to examine beta-arrestin localization to fully characterize these novel signal transduction pathways. We expect that activation of beta-arrestin signaling, while blocking G- protein mediated signaling, will prevent the progression of and reverse DCM by increasing Ca2+-responsiveness of the myofilament. Therefore, results from this proposal will yield pre-clinical data for a potential treatment for DCM, as well as insight into the molecular mechanisms of heart failure.