The Role of Junctophilin Type 2 in Cardiac Node Automaticity

摘要 心脏结节组织的疾病可能危及生命，特别是在年轻人。结节组织 自发去极化作为心脏收缩的起搏器，尽管起着中心作用 结节功能障碍的原因对生存至关重要，但人们对此知之甚少。这种机械性的缺乏 了解损害了有效和选择性药物疗法的发展，作为一种 相关地，针对结节病征收的药物对患者具有显著的毒性，并且仍然可以 完全无效。而节点自动机传统上被认为是由离子控制的 在质膜上，有越来越多的证据表明钙信号在细胞内 细胞可能调节自发去极化--它的自动性。此前的调查显示， 体内钙释放通道RyR2的钙泄漏可能与结节增加有关 因此理解这个所谓的“钙钟”可以提供额外的分子靶点 针对结节的新疗法。我已经创建了一个节点特异性表达沉默的小鼠模型 一种名为JPH2(JPH2)的蛋白质，我之前已经证明它对有效的钙- 在收缩的心肌细胞中的处理，作为研究功能失调的钙钟的工具。我找到了 这只小鼠在休息时心率加快，房室结快速放电，导致 一种称为加速交界性心律失常(AJR)的心律失常。我提出了三个具体的目标来测试我们的 JPH2表达减少导致CaMKII介导的RyR2门控增加的中心假设 这会导致存储钙泄漏增加，并导致节点自动机和AJR增加。我的目标 1)利用基于共聚焦的钙成像技术对HCN4：shJPH2小鼠分离的结节细胞进行钙成像 用RyR2单通道记录来确定JPH2表达减少是否导致增加 钙泄漏具有更高的RyR2通道开放概率；2)应用已知的RyR2化学抑制剂 分离单个细胞和HCN4：shJPh2小鼠，以评估钙泄漏能否正常化和 有效治疗AJR；以及3)从分离的结节组织进行生化处理，以确定 CaMKII信号，包括其下游的磷酸化靶点，在结节放电的调节中。我希望 这些目标的完成将产生对“钙钟”的临床可翻译的机械论洞察。 节点的。通过对中国首只小鼠孤立性心脏结节病模型的探索 文献，这些目标将提供一个底物，从它专门测试新的治疗药物 针对结节组织中受干扰的钙信号。完成这笔为期5年的培训补助金将使 我将把我在儿科电生理学方面的临床训练与对分子的探索结合起来 结节疾病的机制，并成为一名独立资助的内科科学家致力于 帮助患有心律失常的儿童。

ABSTRACT Diseases of the nodal tissue of the heart can be life threatening, particularly in the young. Nodal tissue spontaneously depolarizes serving as a pacemaker for cardiac contraction, yet despite this central role critical for survival, the cause of nodal dysfunction is poorly understood. This lack of mechanistic understanding has impaired development of efficacious and selective pharmacotherapies, and as a correlate, drugs levied against nodal disease carry significant toxicity for the patient and can still be entirely ineffective. While the nodal automaticity was traditionally thought to be controlled by ion channels on the plasma membrane, there is a growing body of evidence that calcium-signaling within the cell may regulate spontaneous depolarization – its automaticity. Previous investigation has shown that calcium leak from the internal calcium release channel, RyR2, may be associated with increased nodal firing, thus understanding this so-called “calcium clock” can provide additional molecular targets for nodal-specific novel therapeutics. I have created a mouse model of nodal-specific expression silencing of a protein called junctophilin-2 (JPH2), which I have previously shown to be critical to effective calcium- handling in the contractile myocyte, as a tool for studying a dysfunctional calcium clock. I have found that this mouse has an elevated heart rate at rest and a rapidly firing atrioventricular node which causes an arrhythmia known as accelerated junctional rhythm (AJR). I propose 3 specific aims to test our central hypothesis that reduced JPH2 expression results in CaMKII-mediated increase in RyR2 gating which causes increased store calcium leak and drives increased nodal automaticity and AJR. My aims are to 1) utilize confocal-based calcium imaging of isolated nodal cells from HCN4:shJPH2 mice, coupled with RyR2 single channel recordings to determine whether reduced JPH2 expression causes increased calcium leak with higher RyR2 channel opening probability; 2) apply known chemical inhibitors of RyR2 to isolated single cells and HCN4:shJPh2 mice to assess whether calcium leak can be normalized and AJR effectively treated; and 3) conduct biochemistry from isolated nodal tissue to determine the role of CaMKII signaling, including its downstream phosphorylation targets, in regulation of nodal firing. I expect that completion of these aims will yield clinically translatable mechanistic insight into the “calcium clock” of the node. Through exploration of the first murine model of isolated cardiac nodal disease in the literature, these aims will provide a substrate from which to test novel therapeutic agents specifically targeted at perturbed calcium-signaling in nodal tissue. Completion of this 5-year training grant will allow me to combine my clinical training in pediatric electrophysiology with exploration of the molecular mechanisms of nodal disease and become an independently funded physician-scientist committed to helping children with arrhythmias.