Exploring the Molecular Physiology of Atrial Fibrillation

心房颤动(房颤)是最常见的心律失常，占相当大的发病率和死亡率。 在心房肌细胞中，兴奋-收缩(E-C)偶联是通过激活电压门控性钠离子来启动的 频道，NAV1.5。钠通道对膜电位的去极化导致电压激活。 门控钙通道，从而触发钙诱导的心房肌细胞钙释放。在上一次 在融资期间，我们开发了创新的方法来探索触发持续钠电流的决定因素- 诱发小鼠自发性房颤。我们发现动作电位时程异质性延长，异常 CA2的处理，增加的活性氧和兰尼定受体(RyR2)的氧化作为驱动因素 房性心肌病和心律失常。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、新的小鼠模型和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)确定肾上腺素能调节钙离子的作用 房性E-C偶联的通道与心律失常的发生最近，我们确定了β- 肾上腺素能激动剂刺激电压门控钙通道。我们观察到钙通道抑制剂Rad，a 单体G蛋白在CaV1.2微环境中富含，但在β-肾上腺素能期间被耗尽 刺激。PKA催化的Rad上特定残基的磷酸化解除了CaV1.2的结构性抑制。 为了确定PKA诱导的心房钙电流刺激的作用，我们将使用具有 Rad的四个pKa磷酸化位点突变为丙氨酸，缺乏Rad-β亚单位相互作用的小鼠也是如此 表达不能被PKA磷酸化的RyR2通道的小鼠。利用这些老鼠，我们将确定 肾上腺素能激动剂诱导是否需要Rad和/或RyR2的磷酸化 自动对焦。(2)确定心房肌细胞内NaV1.5、CaV1.2和RyR2的相互作用和“邻域” 在生理和病理条件下。我们建议使用邻近标记方法来比较 心房中钙、钠和RyR2通道的邻域，并确定这些邻域如何变化 在病理条件下，如心衰，容易使患者发生房颤。(3)阐明氧化的作用 RyR2在房颤发病机制中的作用我们推测RyR2的氧化和由此产生的SR钙泄漏是一种 导致房性心律失常的重要下游效应因子。我们将确定哪些半胱氨酸残基被氧化 在房颤小鼠和人类的心房RyR2。已鉴定的氧化半胱氨酸残基的结构效应 将使用冷冻-EM确定的人重组RyR2的原子分辨结构进行研究， 并对异源表达的突变型RyR2通道进行电生理学研究。这些加在一起， 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的发生，有望导致新的治疗方法的发展。

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.