Exploring the Molecular Physiology of Atrial Fibrillation

心房颤动（AF）是最常见的心律失常，并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）偶联是由电压门控Na+激活启动的。 通道，Nav1.5。Na+通道对膜电位的去极化导致电压激活， 门控Ca 2+通道，从而触发心房心肌细胞中Ca 2+诱导的Ca 2+释放。前一 在资助期间，我们开发了创新的方法来探测触发持续Na+电流的决定因素， 诱发小鼠自发性房颤。我们发现了不均匀延长的动作电位时程，异常 Ca 2+处理、活性氧增加和兰尼碱受体（RyR 2）的氧化作为 心房心肌病和心律失常。在这次更新中，我们将扩大这些研究，现在应用创新的 邻近标记，新的小鼠模型，以及突破性的人类原子分辨率结构研究 重组RyR 2通道。本研究的目的有三：（1）确定肾上腺素能对Ca ~（2+）的调节作用 心房E-C耦合和心房肌发生中的通道。最近，我们确定了β- 肾上腺素能激动剂刺激电压门控Ca 2+通道。我们观察到钙通道抑制剂Rad， 单体G蛋白，在CaV1.2微环境中富集，但在β-肾上腺素能 刺激. Rad上特定残基的PKA催化磷酸化缓解CaV1.2的组成性抑制。 为了确定PKA诱导的Ca 2+电流刺激在心房中的作用，我们将使用具有 Rad的四个PKA磷酸化位点突变为丙氨酸，同样缺乏Rad-β亚基相互作用的小鼠 表达RyR 2通道的小鼠不能被PKA磷酸化。利用这些老鼠，我们将确定 肾上腺素能激动剂诱导的肾上腺素受体激动剂诱导的肾上腺素受体激动剂 AF. (2)确定心房NaV1.5、CaV1.2和RyR 2相互作用体和心房心肌细胞中的“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Ca 2+、Na+和RyR 2通道的邻近区域，并确定这些邻近区域如何变化 在病理条件下，如HF，这使患者容易发生AF。（3）阐明氧化的作用 我们推测RyR 2的氧化和由此产生的SR Ca 2+渗漏是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物。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RyR 2中。所鉴定的氧化半胱氨酸残基的结构效应 将使用通过冷冻EM确定的人重组RyR 2的原子分辨率结构进行研究， 和异源表达的突变体RyR 2通道的电生理学研究。综上所述各项 高度创新和新颖的研究将阐明影响心房肌的交叉信号通路， 心肌细胞收缩和心肌细胞生成，并有望导致新疗法的发展。

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.