CaMKII Regulation of Cardiac Ryanodine Receptors in Atrial Fibrillation

描述（由申请人提供）：在遗传性心律失常综合征（如儿茶酚胺能性多形性室性心动过速（CPVT））中，已证实了肌浆网（SR）储存释放的细胞内Ca 2+调节缺陷，但也可能在获得性心律失常（如房颤（AF））中提供促心律失常触发因素。最近的研究表明，在AF患者中Ryanodine受体（RyR 2）Ca 2+释放通道的调节存在缺陷。AF是最常见的心律失常，每年影响超过200万美国人，并导致死亡率增加一倍。由于RyR 2的活性受到Ca 2 +/钙调蛋白依赖性激酶（CaMKII）的强烈调节，CaMKII结合RyR 2并响应于更快的心率而被激活，因此已经提出异常的CaMKII调节在AF的发病中起作用。 该项目的长期目标是通过研究人类心房活检和RyR 2活性改变或RyR 2上的CaMKII磷酸化位点失活的遗传改变小鼠，确定RyR 2在丝氨酸2814（S2814）处的异常CaMKII磷酸化导致AF的细胞/分子机制。我们的假设是，CaMK II与RyR 2的直接结合使酶能够感知和放大SR Ca 2+泄漏，这可能会增加由于RyR 2（R176 Q）遗传突变或RyR 2稳定亚基FKBP 12.6缺失而更易发生心律失常的心脏中AF的可能性。具体目标是：1）证明在来自AF患者的人类心房活检中RyR 2的S2814处的CaMK II磷酸化增加; 2）定义CaMK II调节RyR 2的分子机制; 3）确定RyR 2的CaMK II磷酸化是否增加RyR 2-R176 Q突变或FKBP 12.6缺陷小鼠中AF的可能性; 4）确定FKBP 12.6缺陷小鼠中RyR 2的CaMK II磷酸化的预防是否降低对AF的易感性。 我们建议进行转译研究，范围从RyR 2的单通道测量，分离的心肌细胞中的Ca 2+成像和遗传改变小鼠中的心肌炎诱导研究，阐明RyR 2依赖的AF启动的分子机制。预计这些研究的结果将促进我们对CaMKII的理解。AF以及与异常Ca 2+稳态相关的其他心律失常的启动依赖性机制。此外，为该项目开发的试剂和动物模型可用于创建治疗AF和其他常见心脏疾病的新药。

DESCRIPTION (provided by applicant): Defects in the regulation of intracellular Ca2+ released from sarcoplasmic reticulum (SR) stores have been demonstrated in inherited arrhythmia syndromes such as catecholaminergic polymorphic ventricular tachycardia (CPVT), but may also provide an arrhythmogenic trigger in acquired arrhythmias like atrial fibrillation (AF). Recent studies have revealed defects in the regulation of ryanodine receptor (RyR2) Ca2+ release channels in patients with AF. AF is the most prevalent arrhythmia, affecting more than 2 million Americans each year and causing a twofold increase in mortality. Since the activity of RyR2 is strongly regulated by Ca2+/calmodulin-dependent kinase (CaMKII), which binds to RyR2 and is activated in response to faster heart rates, it has been proposed that abnormal CaMKII regulation plays/a role in the onset of AF. The long-term goal of this project is to define the cellular/ molecular mechanisms by which abnormal CaMKII phosphorylation of RyR2 at serine 2814 (S2814) leads to AF, by studying human atrial biopsies and genetically-altered mice in which RyR2 activity has been altered, or the CaMKII phosphorylation site on RyR2 has been inactivated. Our hypothesis is that direct binding of CaMKII to RyR2 enables the enzyme to sense and amplify SR Ca2+ leak, which may increase the likelihood of AF in hearts more susceptible to arrhythmias due to an inherited mutation in RyR2 (R176Q) or the absence of the RyR2- stabilizing subunit FKBP12.6. The specific aims are to: 1) Demonstrate that CaMKII phosphorylation at S2814 of RyR2 is increased in human atrial biopsies from patients with AF; 2) Define the molecular mechanisms by which CaMKII regulates RyR2; 3) Determine if CaMKII phosphorylation of RyR2 increases the probability of AF in RyR2-R176Q mutant or FKBP12.6-deficient mice; 4) Determine if prevention of CaMKII phosphorylation of RyR2 in FKBP12.6-deficient mice decreases susceptibility to AF. We propose to conduct translational studies ranging from single channel measurements of RyR2, Ca2+ imaging in isolated cardiomyocytes and arrhythmia-inducibility studies in genetically-altered mice, to elucidate the molecular mechanisms underlying RyR2-dependent initiation of AF. It is anticipated that the results of these studies will advance our understanding of CaMKII-dependent mechanisms underlying the initiation of AF as well as other cardiac arrhythmias associated with abnormal Ca2+ homeostasis. Furthermore, the reagents and animal models developed for this project may be utilized to create new drugs for AF and other common diseases of the heart.