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

 描述（由申请人提供）：家族性扩张型心肌病（DCM）是一种导致心力衰竭的疾病，是心脏移植的最常见原因。疾病进展的分子机制尚未得到很好的表征，目前尚无治疗这种疾病的方法。DCM的特征性分子变化是肌丝对Ca 2+的反应降低。因此，我们假设通过增加肌丝对Ca 2+的反应，我们可能能够防止DCM的发展。作为原理的证明，我们将肌节蛋白原肌球蛋白（TG-Tm-E54 K）上具有错义突变的转基因模型（其表现出与人DCM相似的表型）与转基因小鼠（其特征在于Ca 2+敏感性的组成性增加）杂交。在这些双转基因小鼠中，DCM不会发展。我们的初步研究结果表明，新的β-arrestin信号通路直接参与肌丝的信号传导，赋予Ca 2+敏感的翻译后修饰，并改善心脏功能。在这个提议中，我们假设β-抑制蛋白信号通过改善肌丝的Ca 2+反应性来阻止和逆转DCM的进展。我们的方法是采用TG-TmE 54 K小鼠，并用偏向配体TRV 120023治疗它们，TRV 120023作为血管紧张素受体阻滞剂，但能够激活β-arrestin信号传导，或氯沙坦，血管紧张素受体阻滞剂。在特定 目的1：我们将利用连续超声心动图在体内评价治疗前、治疗中和治疗后的疾病进展。我们还将评估左心室功能在体内和形态学的血流动力学研究和组织学方法，分别。为了达到特定目标2，我们将使用洗涤剂浸提的纤维束测量给药动物和生理盐水对照的稳态力和动力学。我们还将使用生物化学方法确定这些变化背后的机制，我们将阐明激活的信号转导途径。在具体目标3中，我们将使用生物化学方法和显微镜检查β-arrestin定位，以充分表征这些新的信号转导途径。我们预期β-抑制蛋白信号传导的激活，同时阻断G-蛋白介导的信号传导，将通过增加肌丝的Ca 2+反应性来防止DCM的进展并逆转DCM。因此，该提案的结果将为DCM的潜在治疗提供临床前数据，并深入了解心力衰竭的分子机制。

 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.