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，特别是在年轻人中。结节组织 自发去极化作为心脏收缩的起搏器，然而，尽管这一中心作用 对于生存至关重要，但对淋巴结功能障碍的原因知之甚少。这种缺乏机械性的 这种理解损害了有效和选择性药物治疗的发展， 相关，针对淋巴结疾病征收的药物对患者具有显著的毒性，并且仍然可以被治疗。 完全无效。传统上认为结自律性受离子控制 细胞膜上的钙通道，有越来越多的证据表明，钙信号内的细胞膜上的钙通道， 细胞可以调节自发去极化-其自动性。此前的调查显示， 来自内部钙释放通道RyR 2的钙泄漏可能与淋巴结转移增加有关。 因此，了解这种所谓的“钙时钟”可以为我们提供额外的分子靶点。 尿道特异性新疗法。我建立了一个大脑特异性表达沉默的小鼠模型 一种叫做junctophilin-2（JPH 2）的蛋白质，我以前已经证明它对有效的钙离子至关重要， 在收缩肌细胞中的处理，作为研究功能失调的钙时钟的工具。我发现 这只老鼠在休息时心率加快，房室结快速放电， 一种称为加速性交界性心律（AJR）的心律失常。我提出了三个具体目标，以测试我们的 中心假设，JPH 2表达减少导致CaMK II介导的RyR 2门控增加 其导致增加的钙储存渗漏并驱动增加的结自律性和AJR。我的目标 1）利用来自HCN 4：shJPH 2小鼠的分离的结细胞的基于共焦的钙成像， 用RyR 2单通道记录来确定JPH 2表达的减少是否导致JPH 2表达的增加。 具有较高RyR 2通道开放概率的钙泄漏; 2）应用已知的RyR 2化学抑制剂 以评估钙渗漏是否可以正常化， AJR有效治疗;和3）从分离的结组织进行生物化学，以确定AJR的作用， CaMKII信号传导，包括其下游磷酸化靶点，在调节结放电中的作用。我预计 这些目标的完成将产生对“钙钟”的临床可翻译的机械见解， 的节点。通过对我国第一个孤立性心脏结节病小鼠模型的探索， 文献，这些目标将提供一种底物，从其中测试新的治疗剂，特别是 靶向于结节组织中的干扰钙信号传导。完成这5年的培训补助金将允许 我联合收割机结合我的临床培训，在儿科电生理学与探索的分子 淋巴结疾病的机制，并成为一个独立资助的医生，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.