Molecular choreography of CaV1.2 channels in the aging myocardium

衰老心肌CaV1.2通道的分子编排

基本信息

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
项目类别：
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&apos 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

项目摘要

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

项目概要本应用中概述的实验提出了一种新颖的范例，其中我们添加了新的复杂性层目前对 β-肾上腺素能受体 (βAR) 介导的 CaV1.2 通道和 EC- 调节的了解我们认为刺激 βAR 会启动 CaV1.2 通道丰度的动态增强，增强 CaV1.2 通道的协同门控，以及年轻人肌膜中从头形成耦合子心室肌细胞，以放大 Ca2+ 流入这些细胞并在高需求时调整 EC 耦合。初步数据表明，肌膜下、CaV1.2 通道的预合成库囊泡/内体，驻留在心肌细胞中，在高浓度时可以动员到肌膜我们反对将这些“新”通道插入肌膜。以 PKA 和 CaMKII 介导的磷酸化依赖性方式发生，而随后的内化当需求减少时，移动池的变化以 CaN 介导的去磷酸化依赖性方式发生。进一步的初步数据表明，含有 BIN1 蛋白的 BAR 结构域精心设计了这种反应，在微管和肌动蛋白介导的 CaV1.2 靶向肌膜和运输通道中发挥作用引人注目的是，这种动态调节过程是从老年小鼠分离的心室肌细胞中不存在，我们发现心脏 BIN1 蛋白水平几乎随着年龄的增长，大脑中 BIN1 的表达增加一倍，这与阿尔茨海默病和缺陷有关。我们发现 BIN1 水平升高。衰老，可能会导致心室肌细胞中类似的内体交通堵塞，导致基础 CaV1.2 增强肌膜处的表达和易于插入的通道池的耗尽我们提出了一个模型。其中 BIN1 充当 CaV1.2 通道传递至心室肌细胞肌膜的枢纽，并表明改变 CaV1.2 通道的分布和活性，并随着衰老降低对 βAR 刺激的反应性由 BIN1 表达的变化介导。具体目标 1 检验 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。 2 测试了以下假设：BIN1 表达的变化是 CaV1.2 动力学和保留改变的基础最后，具体目标 3 检验了 CaV1.2 通道与年龄相关的差异的假设。心室肌细胞的动力学和运输导致“战斗或逃跑”期间 EC 耦合受损为了实现这些目标，我们采用最先进的方法和多方面的方法。分析包括超分辨率成像、膜片钳电生理学、TIRF 和共焦成像