Mechanisms of Long-term Cardiac Ion Channel Regulation

描述(申请人提供)：心力衰竭(HF)和房颤(AF)是美国最常见的两种心脏疾病。在中到重度HF和房颤中，会出现许多表型变化，包括电压门控钙通道电流密度降低。钙通道表达的改变可能会对心脏功能产生深远的影响，因为它们在兴奋收缩偶联中起着中心作用。此外，钙通道也参与了激发-转录偶联。因此，心脏中钙通道表达的改变也可以影响疾病的进展。心脏主要的电压门控钙通道(CaV1.2)在其生物物理特性和第二信使调节方面得到了广泛的研究。然而，关于钙通道表达的长期调控方面的信息非常稀少。在这项提议中，我们引入了一个新的机制假说，该假说控制了心肌细胞肌膜上心肌钙通道的功能表达。在我们的初步数据中，我们显示了与ras相关的单体G蛋白与CaV1.2辅助亚基CaVb2相互作用的证据。我们建议检验G蛋白与CaVb亚基相互作用通过竞争CaV1.2结合而降低钙通道电流密度的全球假说。此外，我们的初步数据显示，这是一个受慢性PKA活动调节的动态过程。有四个具体的目标指导我们的实验设计：目标1将描述新生G蛋白和CaVb复合体的时间过程和亚细胞定位。目的2研究细胞内第二信使G蛋白CaVb对钙通道电流表达的调节作用。目的3研究天然细胞内钙通道电流和G蛋白的调节。目标4将描述这些G蛋白在心脏功能中的功能。这些目标包括从纯分子性质的研究(蛋白质-蛋白质相互作用)，到体外模型系统(重组蛋白质的异源表达)，到天然细胞系统(心脏细胞的原代分离和培养)，再到单一类别的G蛋白对组织水平心脏功能的影响。这一建议可能确定一类新的治疗靶点，以缓解心衰和房颤患者的心功能障碍。

DESCRIPTION (provided by applicant): Heart failure (HF) and atrial fibrillation (AF) represent two of the most prevalent heart diseases in the U.S. In moderate to severe HF and in AF numerous phenotypic changes occur including a reduction of voltage-gated calcium channel current density. Changes in Ca channel expression may have far-reaching effects on heart function due to their central role in excitation contraction coupling. Also, Ca channels have been implicated in excitation-transcription coupling. Thus alterations of Ca channel expression in the heart can also impact on disease progression. The principal voltage-gated Ca channel in the heart (CaV1.2) is extensively studied in the realm of its biophysical characteristics and second messenger modulation. However, very sparse information is available regarding longer term regulation of Ca channel expression. In this proposal we introduce a novel mechanistic hypothesis governing the functional expression of cardiac Ca channels in the sarcolemma of cardiac myocytes. In our preliminary data we show evidence for a ras-related monomeric G-protein interaction with the CaV1.2 accessory subunit CaVb2. We propose to test the global hypothesis that G-protein interaction with CaVb subunits reduces Ca channel current density by competing for CaV1.2 binding. Moreover, our preliminary data shows strong evidence that this is a dynamic process that is modulated by chronic PKA activity. There are four specific aims that guide our experimental design: Aim 1 will characterize the time course and sub-cellular localization of nascent G-protein and CaVb complexes. Aim 2 will characterize the intracellular second messenger modulation of G-protein-CaVb regulation of Ca channel current expression. Aim 3 will characterize the modulation of Ca channel current and G-protein modulation in native cells. Aim 4 will characterize the function of these G-proteins in heart function. These aims encompass approaches ranging from investigations of a purely molecular nature (protein-protein interactions), to in vitro model systems (heterologous expression of recombinant proteins), to native cell systems (primary isolates and cultures of heart cells), to the impact of a single class of G-proteins on tissue level heart function. This proposal may identify a novel class of therapeutic targets for alleviation of heart dysfunction in HF and AF.