Gi signaling in cardiomyopathy and cardioprotection

描述(由申请人提供)： 在人类中，通过GI偶联受体的慢性信号增加与由特发性扩张型心肌病或心肌梗死后的缺血性心肌病引起的充血性心力衰竭(CHF)有关。然而，对此负责的机制尚不清楚。我们的工作假设是，慢性增加的GI信号导致兴奋-收缩(EC)偶联受损。为了验证这一假设，我们将结合心肌功能的生理测量和一种新的转基因小鼠模型，在该模型中，修改的Gi偶联受体(RO1)被靶向心脏。RO1的表达受四环素控制的表达系统(TET系统)的调控。我们最近发现，慢性RO1的表达会导致CHF以及严重的钙瞬变和收缩异常。相反，急性RO1的表达对缺血/再灌注损伤有显著的保护作用，提示GI信号的增加在心脏保护和疾病中具有双重作用。 对于这项提议，我们将确定涉及充血性心力衰竭和心脏保护的EC偶联机制和GI信号机制。利用单个心肌细胞、心脏小梁和朗宁多夫灌流的小鼠心脏，我们将确定RO1表达对钙瞬变的影响，并通过将异常定位于特定的钙处理过程来确定其机制。我们将确定RO1表达对钙离子反应的影响，并通过定位特定收缩和调节蛋白的异常来确定其机制。使用TET系统在CHF诱导后关闭RO1的表达，我们将确定EC偶联异常可逆的程度。为了阐明信号机制，我们将确定心脏中哪些主要的GI通路(GI2和GI3)参与其中，以及是否通过G蛋白α亚基和/或β-淀粉二聚体参与信号传递。使用3个模型系统，我们将研究GI信号效应(有害的和有益的)和EC耦合-以及涉及的信号机制： 目的1.RO1基因表达所致的心力衰竭及终止RO1基因表达后的恢复。 目的2.RO1基因表达引起的急性心肌保护。 目的3.缺血性心肌病所致的心力衰竭。 这项研究将提供有关GI信号在心力衰竭和心脏保护中的双重作用的新信息，这可能有助于确定治疗心脏病的新策略。

DESCRIPTION (provided by applicant): In humans, chronically increased signaling through Gi-coupled receptors is associated with congestive heart failure (CHF) caused by idiopathic dilated cardiomyopathy or ischemic cardiomyopathy following myocardial infarction. However, the mechanisms responsible are unclear. Our working hypothesis is that chronically increased Gi signaling causes impaired excitation-contraction (ec) coupling. To test this hypothesis we will combine physiological measurements of cardiac muscle function with a novel transgenic mouse model in which a modified Gi-coupled receptor (Ro1) is targeted to the heart. Expression of Ro1 is regulated by a tetracycline-controlled expression system (tet-system). We have recently shown that chronic Ro1 expression causes CHF and major abnormalities of Ca2+ transients and contraction. In contrast, acute Ro1 expression causes significant protection against ischemia/reperfusion injury, suggesting a dual role for increased Gi signaling in cardioprotection and disease. For this proposal we will determine the ec-coupling mechanisms and Gi signaling mechanisms involved in CHF and cardioprotection. Using single myocytes, cardiac trabeculae, and Langendorff perfused mouse hearts, we will determine the effect of Ro1 expression on Ca2+ transients and determine the mechanisms responsible by localizing abnormalities to specific Ca2+ handling processes. We will determine the effect of Ro1 expression on Ca2+-responsiveness and determine the mechanisms responsible by localizing abnormalities to specific contractile and regulatory proteins. Using the tet-system to turn off Ro1 expression after induction of CHF, we will determine the extent to which ec-coupling abnormalities are reversible. To elucidate signaling mechanisms, we will determine which of the major Gi pathways in the heart (Gi2 and Gi3) are involved; and whether signaling via the G protein alpha subunit and/or the betagamma dimer is involved. Using 3 model systems we will investigate Gi signaling effects (both deleterious and beneficial) and the ec-coupling- and signaling mechanisms involved in: Aim 1. CHF caused by Ro1 expression; and recovery after terminating Ro1 expression. Aim 2. Acute Cardioprotection caused by Ro1 expression. Aim 3. CHF caused by ischemic cardiomyopathy. This research will provide new information on the dual role of Gi signaling in both heart failure and cardioprotection which may help identify new strategies to treat heart disease.