CaMKII Regulation of Cardiac Ryanodine Receptors in Atrial Fibrillation

描述(申请人提供)：在遗传性心律失常综合征，如儿茶酚胺能多形性室性心动过速(CPVT)中，已证实肌浆网(SR)储备物释放的细胞内钙离子调节缺陷，但也可能在获得性心律失常(如房颤)中提供致心律失常的触发因素。最近的研究发现房颤患者兰尼定受体(RyR2)钙释放通道的调节存在缺陷。房颤是最常见的心律失常，每年影响200多万美国人，导致死亡率增加两倍。由于RyR2的活性受到钙/钙调蛋白依赖的激酶(CaMKII)的强烈调控，CaMKII与RyR2结合并在心率加快时被激活，因此有观点认为CaMKII调节异常在房颤的发生中起作用。 该项目的长期目标是通过研究人类心房活检和RyR2活性改变或RyR2上CaMKII磷酸化位点失活的转基因小鼠，确定RyR2丝氨酸2814位CaMKII异常磷酸化导致房颤的细胞/分子机制。我们的假设是，CaMKII与RyR2的直接结合使酶能够感知并放大SR钙泄漏，这可能会增加由于RyR2(R176Q)的遗传突变或RyR2稳定亚单位FKBP12.6的缺失而更容易发生心律失常的心脏发生房颤的可能性。这些研究的具体目的是：1)证明在房颤患者的心房活检组织中，RyR2的S2814位的CaMKII磷酸化水平增加；2)确定CaMKII调控RyR2的分子机制；3)确定RyR2的CaMKII磷酸化是否会增加RyR2-R176Q突变或FKBP12.6缺陷小鼠患房颤的可能性；4)确定在FKBP12.6缺陷小鼠中，阻止RyR2的CaMKII磷酸化是否会降低房颤的易感性。 我们建议进行翻译研究，从RyR2的单通道测量，分离的心肌细胞中的钙成像，以及在基因改变的小鼠中对心律失常的诱导研究，以阐明依赖RyR2启动房颤的分子机制。预计这些研究的结果将促进我们对房颤发生的CaMKII依赖机制以及其他与钙稳态异常相关的心律失常的理解。此外，为该项目开发的试剂和动物模型可能被用于开发治疗房颤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.