Molecular choreography of CaV1.2 channels in the aging myocardium

衰老心肌CaV1.2通道的分子编排

• 批准号: 9980760
• 负责人: Rose Ellen Dixon
• 金额: $ 32.19万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980760
• 关键词: Actins; Acute; Age; Aging; Alzheimer' s Disease; Animals; Back; Brain; Cardiac; Cardiac Myocytes; Cell surface; Cells; Chronic stress; Coupling; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Data; Defect; Diabetes Mellitus; Distributional Activity; Early Endosome; Electrocardiogram; Electrophysiology (science); Endocytosis; Endosomes; Heart; Heart failure; Hypertension; Image; Immobilization; Impairment; Isoproterenol; Left; Link; Mediating; Membrane; Metabolic; Methods; Microtubules; Modeling; Molecular; Mus; Muscle Cells; Myocardial; Myocardium; Phenotype; Phosphorylation; Play; Process; Protein Dephosphorylation; Proteins; Receptor Activation; Recycling; Refractory; Regulation; Resolution; Role; RyR2; Sarcolemma; Scaffolding Protein; Surface; System; Testing; Time; Up-Regulation; Ventricular; Vesicle; Western Blotting; acute stress; age related; aged; beta-adrenergic receptor; calmodulin-dependent protein kinase II; confocal imaging; experimental study; fighting; hemodynamics; nanoscale; novel; overexpression; patch clamp; response; stressor; theories; trafficking; voltage

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

项目摘要 在此应用程序中概述的实验建议了一种新型范式，其中我们添加了新的复杂性层 目前了解β-肾上腺素受体（βAR）介导的CAV1.2通道和EC-的调节 耦合。我们提出，刺激β会引发CAV1.2通道丰度的动态增强， 增强了Cav1.2频道的教练，以及在Young的肌肉中的Nevo夫妇编队 心室心肌细胞，以扩大Ca2+影响到这些细胞中，并在需求较高时调整EC偶联。我们的 初步数据表明，次级和cav1.2含通道的前合成的池 蔬菜/内体，居住在心肌细胞中，可以在较高的时期动员到肌膜 代谢或血流动力学需求。我们假设将这些“新”频道插入到肌肉中 以PKA和CAMKII介导的磷酸化方式发生，而随后内在化 当需求减少时，移动池的依赖性依赖性的方式发生。 进一步的初步数据表明，含有蛋白质BIN1的条形域，编舞这种反应， 在微管和肌动蛋白介导的CAV1.2靶向肌膜和贩运通道中的作用 早期内体并将其回收回细胞表面。令人惊讶的是，这个动态的调节过程是 在我们发现心脏BIN1蛋白水平的年龄小鼠中分离出的心室心肌细胞中不存在 加倍。衰老中BIN1在大脑中的表达增加与阿尔茨海默氏病和缺陷有关 在细胞内交通中，称为“内体交通拥堵”。我们假设BIN1的水平增加了 衰老，可能导致心室肌细胞中类似的内体交通堵塞，从而增强基本CAV1.2 在肌膜上的表达和容易插入的通道池的耗竭。我们提出了一个模型 哪个BIN1充当cav1.2通道在心室心肌细胞中传递到肌膜的枢纽，并建议 改变了Cav1.2通道的分布和活性，并降低了对βAR刺激的反应性 由BIN1表达的变化介导。特定的目标1检验了βAR激活刺激的假设 肌膜CAV1.2通道丰度和聚类的年龄依赖性动态增强。具体目标 2检验了以下假设：BIN1表达的变化是动力学改变和保留CAV1.2的基础的。 衰老的通道。最后，特定的目标3检验了以下假设：CAV1.2通道中与年龄相关的差异 心室肌细胞的动态和贩运导致“战斗或飞行”期间的EC偶联受损 回复。为了实现这些目标，我们使用最先进的方法采用多方面的方法 分析包括超分辨率成像，斑块夹电生理学，TIRF和共聚焦成像