Calsequestrin in Ventricular Arrhythmia and Sudden Death

描述(申请人提供)：心脏钙调蛋白(CASQ2)及其结合伙伴连接和三聚氰胺-1(TRDN)是肌浆网(SR)钙储存和释放的关键调节因子。在人类中，CASQ2突变会导致儿茶酚胺能多形性室性心动过速(CPVT)和心脏性猝死的综合征。在之前的资助期间，我们已经培育并研究了Casq2缺失(Casq2-/-)小鼠，以确定CASQ2突变导致电生理不稳定的机制。我们发现，尽管缺乏Casq2蛋白，这些小鼠仍保持着接近正常的SR钙储存、钙释放和收缩功能，这可能是由于SR体积的扩大和Casq2结合蛋白Triadin-1和Junctin的急剧减少所致。Casq2-/-小鼠表现出CPVT表型，即经儿茶酚胺输注儿茶酚胺或运动后出现多形性室性心动过速。我们现在的观点是，在高SR钙负荷条件下，钙释放不应性的丧失和SR Ca的过早释放，导致延迟的后除极，触发的心动过速和多形性室性心动过速。然而，如果触发物来自心室肌细胞或传导系统的特殊细胞，浦肯野细胞，则尚未解决。来自RyR2突变小鼠模型的实验数据和理论考虑支持浦肯野网络。此外，生化、电子显微镜和共聚焦研究表明，Casq2仅存在于与RyR2钙释放通道密切接触的连接SR的终池中。因此，我们推测，在心室肌细胞和/或浦肯野细胞中，RyR2附近Casq2的减少是导致SRCa过早释放和增加心律失常易感性的根本缺陷。这适用于遗传综合征(例如，Casq2连锁的CPVT；AIMS 1+2)和由于Casq2转运和/或在RyR2附近保留方面的缺陷而潜在的获得性心脏病(AIMS 3)。目的：验证Casq2基因修复可挽救Casq2-/-小鼠CPVT表型的假说。目的2：验证浦肯野细胞中Casq2缺失是引起CPVT的充要条件的假说。目的3：验证心肌梗死后存活心肌中Casq2在RyR2钙释放通道附近减少的假说。 公共卫生相关性：拟议的工作将研究某些遗传性心脏病的原因，这些疾病与心律失常(心律不齐)和猝死和过早死亡的高风险有关。这些研究还将在动物模型中检验基因治疗的好处，这将为遗传性心律失常患者的新疗法提供重要信息。

DESCRIPTION (provided by applicant): Cardiac calsequestrin (CASQ2), and its binding partners junction and triadin-1 (TRDN), are key regulators of sarcoplasmic reticulum (SR) Ca storage and release. In humans, CASQ2 mutations cause the syndrome of catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death. During the previous funding period, we have generated and studied Casq2 null (Casq2-/-) mice to determine the mechanisms whereby CASQ2 mutations cause electrophysiological instability. We found that despite a lack of Casq2 protein, these mice maintain near normal SR Ca storage, Ca release and contractile function, likely as a result of an expansion of SR volume and drastic reductions in the Casq2 binding proteins triadin-1 and junctin. Casq2-/- mice exhibit the CPVT phenotype, i.e. they develop polymorphic ventricular tachycardia with catecholamine infusion or exercise. Our current concept is that the loss of Ca release refractoriness and premature SR Ca release under conditions of high SR Ca load, results in delayed after-depolarizations, triggered beats and polymorphic ventricular tachycardia. However, if the trigger originates from ventricular myocytes or from specialized cells of the conduction system, the Purkinje cells, is unresolved. Experimental data from a RyR2 mutant mouse model and theoretical considerations favor the Purkinje network. Furthermore, biochemical, electron microscopy and confocal studies show that Casq2 is only found in the terminal cisternae of the junctional SR in close contact with RyR2 Ca release channels. Thus, we hypothesize that the reduced presence of Casq2 near the RyR2 in ventricular myocytes and/or Purkinje cells is a fundamental defect that causes premature SR Ca release and increases arrhythmia susceptibility. This is applicable to inherited syndromes (e.g., Casq2-linked CPVT; Aims 1+2) and potentially acquired heart disease due to defects in the trafficking to and/or retention of Casq2 near the RyR2 (Aim 3). Aim 1: To test the hypothesis that restoration of Casq2 rescues the CPVT phenotype of Casq2-/- mice. Aim 2: To test the hypothesis that loss of Casq2 in Purkinje cells is both necessary and sufficient to cause CPVT Aim 3: To test the hypothesis that Casq2 is reduced near RyR2 Ca release channels in cardiac muscle surviving after myocardial infarction. PUBLIC HEALTH RELEVANCE: The proposed work will study the causes for certain inherited heart diseases associated with arrhythmia (irregular heartbeats) and a high risk for sudden and premature deaths. The studies will also examine the benefit of gene therapy in an animal model, which will provide important information for new therapies for patients with inherited arrhythmia disorders.