Molecular choreography of CaV1.2 channels in the aging myocardium

项目摘要 本申请中概述的实验提出了一种新的范例，我们在其中添加了一层新的复杂性 就目前对β肾上腺素能受体(βAR)介导的CaV1.2通道和EC- 耦合。我们认为，刺激βARs启动了CaV1.2通道丰度的动态增加， 增强CaV1.2通道的协同门控，并在Young肌膜上形成从头偶联 在需求旺盛的情况下，增强钙离子流入这些细胞并调节EC偶联。我们的 初步数据表明，一个预先合成的亚肌膜池，CaV1.2通道-包含 囊泡/内体，存在于心肌细胞中，在高峰期可被动员到肌膜上 代谢或血液动力学需求。我们假设这些“新”通道插入肌膜 以PKA和CaMKII介导的磷酸化依赖的方式发生，而随后的内化 当需求减少时，流动池中的钙离子以CaN介导的去磷酸化依赖的方式发生。 进一步的初步数据表明，含有杆状结构域的蛋白BIN1，编排了这种反应， 在微管和肌动蛋白介导的CaV1.2靶向肌膜和转运通道中的作用 并将它们循环回到细胞表面。引人注目的是，这种动态的监管过程 从老年小鼠分离的心室肌细胞中没有BIN1，我们发现心脏BIN1蛋白水平几乎 翻倍。BIN1在大脑中随年龄增长的表达增加与阿尔茨海默病和缺陷有关 在细胞内的运输中被称为“内体交通堵塞”。我们假设BIN1水平的增加与 衰老可引起类似的心室肌细胞内膜交通堵塞，导致基础CaV1.2增强 在肌膜上的表达和易于插入的通道池的耗竭。我们在文中提出了一个模型 在心室肌细胞中，哪种BIN1作为CaV1.2通道传递到肌膜的枢纽，并提示 这改变了CaV1.2通道的分布和活性，并随着年龄的增长而降低了对βAR刺激的反应性 是通过BIN1表达的变化来调节的。特定目标1验证了βAR激活刺激 依赖年龄的肌膜CaV1.2通道丰度和聚集的动态增强。特定目标 2检验了假设，即BIN1表达的变化是CaV1.2动态改变和保留的基础 频道随着年龄的增长。最后，特定目标3验证了CaV1.2通道中年龄相关差异的假设 在“战斗还是逃跑”中，心室肌细胞的动力学和运输导致EC偶联受损 回应。为了达到这些目标，我们采用了多方面的方法，使用了最先进的方法和 分析包括超分辨率成像、膜片钳电生理、TIRF和共聚焦成像

Project Summary Experiments outlined in this application, suggest a novel paradigm, in which we add a new layer of complexity to the current understanding of β-adrenergic receptor (βAR)-mediated regulation of CaV1.2 channels and EC- coupling. We propose that stimulation of βARs initiates dynamic augmention of CaV1.2 channel abundance, enhanced cooperative gating of CaV1.2 channels, and de novo couplon formation in the sarcolemma of young ventricular myocytes, to amplify Ca2+ influx into these cells and tune EC-coupling in times of high demand. Our preliminary data suggest that a pre-synthesized pool of sub-sarcolemmal, CaV1.2 channels-containing vesicles/endosomes, resides in cardiomyocytes and can be mobilized to the sarcolemma in times of high metabolic or hemodynamic demand. We hypothesize that insertion of these ‘new’ channels into the sarcolemma occurs in a PKA- and CaMKII-mediated phosphyorylation dependent fashion, while subsequent internalization of the mobile pool when demand decreases, occurs in a CaN-mediated dephosphorylation dependent manner. Further preliminary data suggests that the BAR-domain containing protein BIN1, choreographs this response, with roles in microtubule and actin mediated CaV1.2 targeting to the sarcolemma and in trafficking channels out of early endosomes and recycling them back to the cell surface. Strikingly, this dynamic regulatory process is absent in ventricular myocytes isolated from aged mice where we find cardiac BIN1 protein levels are almost doubled. Increased expression of BIN1 in the brain with aging is associated with Alzheimer’s Disease and defects in intracellular trafficking termed ‘endosomal traffic jams’. We hypothesize that increased levels of BIN1 with aging, could cause analogous endosomal traffic jams in ventricular myocytes, leading to enhanced basal CaV1.2 expression at the sarcolemma and depletion of the readily insertable pool of channels. We propose a model in which BIN1 acts as a hub for CaV1.2 channel delivery to the sarcolemma in ventricular myocytes, and suggest that altered distribution and activity of CaV1.2 channels, and reduced responsivity to βAR stimulation with aging is mediated by changes in BIN1 expression. Specific Aim 1 tests the hypothesis that βAR activation stimulates age-dependent dynamic augmentation of sarcolemmal CaV1.2 channel abundance and clustering. Specific Aim 2 tests the hypothesis that changes in BIN1 expression underlie the altered dynamics and retention of CaV1.2 channels with aging. Finally, specific Aim 3 tests the hypothesis that age-related differences in CaV1.2 channel dynamics and trafficking in ventricular myocytes leads to impaired EC-coupling during the ‘fight or flight’ response. To achieve these aims, we employ a multi-faceted approach using state-of-the-art methods and analyses including super-resolution imaging, patch clamp electrophysiology, TIRF and confocal imaging