Exploring the Molecular Physiology of Atrial Fibrillation

探索心房颤动的分子生理学

基本信息

批准号：
10366410
负责人：
ANDREW Robert MARKS
金额：
$ 76.76万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-17 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10366410
关键词：
Action Potentials Adrenergic Agents Adrenergic Agonists Adrenergic beta-Agonists Affect Age-Years Alanine American Amino Acids Anterior Arrhythmia Arteries Atrial Fibrillation Binding Sites Biotin Cardiac Myocytes Cardiomyopathies Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Congestive Heart Failure Coupling Cryoelectron Microscopy Cyclic AMP-Dependent Protein Kinases Cysteine Development Disinhibition Dissection Electrophysiology (science)FKBP1B gene Forskolin Funding Goals Heart Heart Atrium Heart failure Human In Situ Inherited Ion Channel Knock-in Knock-in Mouse Label Lead Left Ligase Ligation Lipid Bilayers Macromolecular Complexes Maps Mass Spectrum Analysis Mediating Membrane Potentials Methods Mission Mitochondria Molecular Monomeric GTP-Binding Proteins Morbidity - disease rate Mus Mutate Neighborhoods Operative Surgical Procedures Oxidative Stress Oxides Pathogenesis Pathologic Patients Peroxidases Pharmacologic Substance Phosphorylation Phosphorylation Site Physiological Physiology Protein Isoforms Public Health Radiofrequency Interstitial Ablation Rattus Reactive Oxygen Species Recombinants Recurrence Regulation Research Resolution Risk Role RyR1 Ryanodine Receptors Sampling Sarcoplasmic Reticulum Signal Pathway Signal Transduction Skeletal Muscle Specificity Structure Tacrolimus Binding Protein 1A Testing Toxic effect Transgenic Mice Transgenic Organisms United States National Institutes of Health ascorbate base catalase high resolution imaging human disease in vivo inhibitor innovation insight mortality mouse model mutant novel novel therapeutics oxidation prevent structural biology three dimensional structure voltage

项目摘要

Atrial fibrillation (AF) is the most common cardiac arrhythmia and accounts for substantial morbidity and mortality. In atrial cardiomyocytes, excitation-contraction (E-C) coupling is initiated by activation of voltage-gated Na+ channels, NaV1.5. Depolarization of the membrane potential, by Na+ channels, leads to activation of voltage- gated Ca2+ channels, thereby triggering Ca2+-induced Ca2+ release in atria cardiomyocytes. During the prior funding period, we developed innovative methods to probe determinants triggering persistent Na+ current- induced spontaneous AF in mice. We identified heterogeneously prolonged action potential duration, abnormal Ca2+ handling, increased reactive oxygen species and oxidation of ryanodine receptors (RyR2) as drivers of atrial cardiomyopathy and arrhythmias. In this renewal, we will expand these studies, now applying innovative proximity labeling, novel mouse models, and groundbreaking atomic resolution structural studies of the human recombinant RyR2 channel. Three Aims are proposed: (1) Determine the role of adrenergic regulation of Ca2+ channels in atrial E-C coupling and arrhythmogenesis. Recently, we identified the mechanism by which β- adrenergic agonists stimulate voltage-gated Ca2+ channels. We observed that the Ca2+ channel inhibitor Rad, a monomeric G-protein, is enriched in the CaV1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific residues on Rad relieves constitutive inhibition of CaV1.2. To determine the role of PKA-induced stimulation of Ca2+ currents in the atria, we will use knock-in mice with the four PKA phosphorylation sites of Rad mutated to alanine, mice lacking the Rad-β subunit interaction as well mice expressing RyR2 channels that cannot be phosphorylated by PKA. Using these mice, we will determine whether phosphorylation of Rad and/or phosphorylation of RyR2 are required for adrenergic agonist-induced AF. (2) To define the atrial NaV1.5, CaV1.2 and RyR2 interactomes and “neighborhoods” in atrial cardiomyocytes under physiological and pathological conditions. We propose to use proximity labeling approaches to compare the neighborhoods of Ca2+, Na+ and RyR2 channels in the atria, and determine how these neighborhoods change in pathological conditions, such as HF, which predisposes patients to AF. (3) To elucidate the role of oxidation of RyR2 in the pathogenesis of AF. We speculate that oxidation of RyR2 and the resultant SR Ca2+ leak is an essential downstream effector leading to atrial arrhythmias. We will identify which cysteine residues are oxidized in atrial RyR2 in both mice and humans with AF. The structural effects of the identified oxidized cysteine residues will be investigated using an atomic-resolution structure of human recombinant RyR2 determined by cryo-EM, and by electrophysiological studies of heterologously expressed mutant RyR2 channels. Taken together, these highly innovative and novel studies will elucidate the intersecting signaling pathways that affect atrial cardiomyocyte contraction and arrhythmogenesis, and hopefully lead to the development of new therapeutics.

心房颤动 (AF) 是最常见的心律失常，导致较高的发病率和死亡率。在心房心肌细胞中，兴奋-收缩 (E-C) 耦合是通过电压门控 Na+ 的激活来启动的通道，NaV1.5 通过 Na+ 通道使膜电位去极化，导致电压-的激活。门控 Ca2+ 通道，从而在之前的心房心肌细胞中触发 Ca2+ 诱导的 Ca2+ 释放。资助期间，我们开发了创新方法来探测触发持续Na+电流的决定因素- 我们在小鼠中发现了自发性房颤的异常延长的动作电位持续时间。 Ca2+ 处理、活性氧增加和兰尼碱受体 (RyR2) 氧化作为驱动因素在这次更新中，我们将扩大这些研究，现在应用创新。邻近标记、新颖的小鼠模型以及突破性的人类原子分辨率结构研究重组 RyR2 通道提出了三个目标：（1）确定 Ca2+ 的肾上腺素调节作用。最近，我们确定了 β- 心房 E-C 耦合和心律失常发生的机制。肾上腺素能激动剂刺激电压门控 Ca2+ 通道我们观察到 Ca2+ 通道抑制剂 Rad（一种）。单体 G 蛋白，在 CaV1.2 微环境中富集，但在 β-肾上腺素能期间耗尽 PKA 催化的 Rad 上特定残基的磷酸化可缓解 CaV1.2 的组成型抑制。为了确定 PKA 诱导的心房 Ca2+ 电流刺激的作用，我们将使用具有 Rad 的四个 PKA 磷酸化位点突变为丙氨酸，小鼠也缺乏 Rad-β 亚基相互作用表达不能被 PKA 磷酸化的 RyR2 通道的小鼠使用这些小鼠，我们将确定。肾上腺素能激动剂诱导是否需要 Rad 磷酸化和/或 RyR2 磷酸化 (2) 定义心房 NaV1.5、CaV1.2 和 RyR2 相互作用组和心房心肌细胞中的“邻域” 我们建议使用邻近标记方法来比较生理和病理条件下的情况。心房中 Ca2+、Na+ 和 RyR2 通道的邻域，并确定这些邻域如何变化在病理条件下，例如心力衰竭，患者容易发生房颤 (3) 阐明氧化的作用。我们推测 RyR2 的氧化和由此产生的 SR Ca2+ 泄漏是 AF 发病机制中的一个重要因素。导致房性心律失常的重要下游效应子，我们将确定哪些半胱氨酸残基被氧化。在患有 AF 的小鼠和人类的心房 RyR2 中，鉴定出氧化半胱氨酸残基的结构效应。将使用冷冻电镜测定的人重组 RyR2 的原子分辨率结构进行研究，以及通过异源表达突变 RyR2 通道的电生理学研究，将这些结合起来。高度创新和新颖的研究将阐明影响心房的交叉信号通路心肌细胞收缩和心律失常，并有望导致新疗法的开发。