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：
10544556
负责人：
ANDREW Robert MARKS
金额：
$ 77.23万
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/10544556
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 Crossbreeding 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 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 Receptor Calcium Release Channel 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 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，并且是大量的发病率和死亡率。在心房心肌细胞中，通过电压门控的Na+激活引发兴奋 - 收缩（E-C）耦合频道，NAV1.5。 Na+通道的膜电位去极化导致电压激活门控Ca2+通道，从而在心房心肌细胞中触发Ca2+诱导的Ca2+释放。在先验期间资金期，我们开发了创新方法来探测确定词，从而触发持续的Na+电流 - 诱导小鼠的赞助AF。我们确定了异质延长动作电位持续时间，异常 Ca2+处理，增加的活性氧和Ryanodine受体的氧化（RYR2）作为驱动因素心房心肌病和心律不齐。在此续约中，我们将扩大这些研究，现在采用创新接近标记，新型小鼠模型和人类的突破性原子分辨率研究重组RYR2通道。提出了三个目的：（1）确定CA2+的肾上腺素调节的作用心房E-C耦合和心律失常发生中的通道。最近，我们确定了β-的机制肾上腺激动剂刺激电压门控Ca2+通道。我们观察到Ca2+通道抑制剂RAD，A 单体G蛋白富集在Cav1.2微环境中，但在β-肾上腺素期间耗尽刺激。 PKA催化的磷酸化对RAD救援的特定救援的磷酸化对CAV1.2的本构抑制作用。为了确定PKA诱导的Ca2+电流在心房中的作用，我们将使用敲入小鼠与 RAD突变为丙氨酸的四个PKA磷酸化位点，也缺乏RAD-β亚基相互作用的小鼠表达RYR2通道的小鼠不能被PKA磷酸化。使用这些老鼠，我们将确定是否需要肾上腺激动剂诱导的RAD和/或RyR2的磷酸化和/或磷酸化 AF。（2）定义心房nav1.5，cav1.2和ryr2相互作用以及心房心肌细胞中的“邻域” 在身体和病理状况下。我们建议使用接近标签方法比较心房中CA2+，Na+和RYR2通道的社区，并确定这些社区如何改变在病理条件下，例如HF，使患者患有AF。（3）阐明氧化的作用 RYR2在AF的发病机理中的我们推测RYR2的氧化和结果SR Ca2+泄漏是必需的下游效应子导致心律不齐。我们将确定哪些半胱氨酸保留被氧化了在小鼠和人类中的心房里2中。已鉴定的氧化半胱氨酸残基的结构效应将使用由Cryo-Em确定的人重组RYR2的原子分辨率结构进行研究以及通过异源表达的突变RYR2通道的电生理研究。总的来说，这些高度创新和新颖的研究将阐明影响心房的相交信号通路心肌细胞收缩和心律失常，并希望导致新疗法的发展。