CaMKII-dependent regulation of cardiac excitability

描述(申请人提供)：可兴奋的细胞功能依赖于高度进化的局部信号域，这些信号域对离子通道、转运体和受体的翻译后修饰(如磷酸化、氧化)施加严格的空间和时间控制。这些局部信号域的破坏和/或膜蛋白翻译后修饰的改变与先天性和获得性心脏病(包括心力衰竭)心律失常的易感性增加有关。我们的研究试图了解负责特定亚细胞域中膜蛋白局部调节的细胞通路，总体目标是对人类心律失常和猝死产生新的见解。CaMKII是一种多功能的丝氨酸/苏氨酸激酶，调节心脏中一系列重要的细胞功能。尽管CaMKII对心脏功能很重要，但对控制CaMKII信号转导的局部结构域的生物发生知之甚少。我们最近证实，肌动蛋白相关多肽BetaIV-Spectrin是一种新的CaMKII锚定蛋白(CaMKAP)，它靶向CaMKII到间盘，调节电压门控钠通道(Nav)功能和心脏的兴奋性。然而，BetaIV-Spectrin/CaMKII连接到插入盘中NaV1.5的分子途径以及CaMKII改变NaV1.5功能的机制尚不清楚。此外，CaMKII依赖的NaV1.5调节和细胞兴奋性在心脏病和潜在的致命性电性心律失常(心律失常)中的作用尚未被探索。我们的初步数据表明，BetaIV-Spectrin作为组织CaMKII的支架，在插入的盘中结合适配蛋白ankyrin-G和NaV1.5。我们还在NaV1.5(Ser571)上确定了一个潜在的位点，负责CaMKII依赖的NAV功能的调节，并开发了新的试剂来研究该位点在原代心肌细胞中的作用。此外，我们还确定了CaMKII磷酸化基序附近的一系列人类变异的机制，这些变异通过扰乱正常的通道调节来增加对心律失常的易感性。最后，我们的初步结果表明，依赖于CaMKII的NaV1.5磷酸化的失调发生在心脏病的小鼠和犬模型中，以及在衰竭的人类心脏中。总之，这些初步数据支持我们的中心假设，即BetaIV-Spectrin在心肌细胞间盘组织局部膜域来控制依赖CaMKII的NaV1.5的磷酸化，并且NaV1.5中的CaMKII调节基序是调节与心律失常和猝死相关的各种形式的心脏病的通道功能的关键节点。我们期望，靶向阻断CaMKII/Spectrin相互作用将阻止CaMKII依赖的NaV1.5磷酸化，减少心律失常负担，并在心肌损伤的背景下改善心功能。我们预计，这些研究将对CaMKII信号域的组织产生新的见解，定义调节NaV1.5和细胞兴奋性的分子途径，并确定先天性和获得性心脏病心律失常的新机制。

DESCRIPTION (provided by applicant): Excitable cell function depends on highly evolved local signaling domains that exert tight spatial and temporal control over post-translational modification (e.g. phosphorylation, oxidation) of ion channels, transporters and receptors. Disruption of these local signaling domains and/or alterations in post-translational modification of membrane proteins are associated with increased susceptibility to arrhythmia in congenital and acquired forms of heart disease, including heart failure. Our research seeks to understand the cellular pathways responsible for local regulation of membrane proteins in specific subcellular domains with the overall objective of generating new insight into human cardiac arrhythmia and sudden death. CaMKII is a multifunctional serine/threonine kinase that regulates a broad spectrum of critical cellular functions in heart. Despite the importance of CaMKII for heart function, little is known regarding the biogenesis of local domains to control CaMKII signaling. We recently demonstrated that the actin-associated polypeptide betaIV-spectrin serves as a novel CaMKII-anchoring protein (CaMKAP), which targets CaMKII to the intercalated disc for regulation of voltage-gated Na+ channel (Nav) function and cardiac excitability. However, the molecular pathway linking betaIV-spectrin/CaMKII to Nav1.5 at the intercalated disc, and mechanisms by which CaMKII alters Nav1.5 function remain unknown. Moreover, the role for CaMKII-dependent regulation of Nav1.5 and cell excitability in heart disease and potentially fatal electrical rhythm disturbances (arrhythmias) is unexplored. Our preliminary data indicate that betaIV-spectrin acts as a scaffold for organizing CaMKII with the adapter protein ankyrin-G and Nav1.5 at the intercalated disc. We have also identified a potential site on Nav1.5 (Ser571) responsible for CaMKII-dependent regulation of Nav function and have developed new reagents to study the role of this site in primary myocytes. Furthermore, we have identified the mechanism for a cluster of human variants adjacent to the CaMKII phosphorylation motif that confer susceptibility to arrhythmia by disrupting normal channel regulation. Finally, our preliminary results indicate that dysregulation of CaMKII-dependent phosphorylation of Nav1.5 occurs in murine and canine models of heart disease, and in failing human hearts. Collectively, these preliminary data support our central hypothesis that betaIV-spectrin organizes a local membrane domain at the cardiomyocyte intercalated disc to control CaMKII-dependent phosphorylation of Nav1.5, and that the CaMKII regulatory motif in Nav1.5 is a critical nodal point for regulating channel function in diverse forms of cardiac disease associated with arrhythmias and sudden death. We expect that targeted disruption of CaMKII/spectrin interaction will prevent CaMKII-dependent phosphorylation of Nav1.5, decrease arrhythmia burden and improve heart function in the setting of myocardial insult. We anticipate that these studies will generate new insight into organization of CaMKII signaling domains, define molecular pathways for regulation of Nav1.5 and cell excitability, and identify novel mechanisms for arrhythmias in both congenital and acquired heart disease.