The Role of Junctophilin Type 2 in Cardiac Node Automaticity

2 型亲结蛋白在心脏结自律性中的作用

• 批准号: 9294240
• 负责人: Andrew P. Landstrom
• 金额: $ 15.39万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-04 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9294240
• 关键词: Acute; Address; Affect; Arrhythmia; Artificial cardiac pacemaker; Biochemical; Biochemistry; Biological Assay; Calcium; Calcium Signaling; Cardiac; Cardiac Myocytes; Cardiac Surgery procedures; Cardiovascular Diseases; Cardiovascular system; Cell membrane; Cells; Chemicals; Child; Child Care; Childhood; Clinical; Coupled; Development; Disease; Ectopic Junctional Tachycardia; Electrophysiology (science); Enzymes; Etiology; Experimental Models; Follow-Up Studies; Foundations; Frequencies; Fright; Functional disorder; Funding; Grant; HCN4 gene; Heart; Heart Rate; Heart failure; Human; Image; Impairment; Investigation; Ion Channel; Knowledge; Learning; Life; Lipid Bilayers; Literature; Mediating; Medical; Modeling; Molecular; Molecular Target; Mus; Muscle Cells; Nodal; Nodal Arrhythmia; Operative Surgical Procedures; Pacemakers; Pathologic; Patient Care; Patients; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phosphorylation; Phosphotransferases; Physicians; Physiology; Play; Postoperative Period; Probability; Proteins; Regulation; Research Personnel; Rest; Role; RyR2; Scientist; Signal Transduction; Sinus Tachycardia; Sodium-Calcium Exchanger; Structural Protein; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; TimeLine; Tissues; Toxic effect; Training; atrioventricular node; base; calmodulin-dependent protein kinase II; career; clinically translatable; experience; in vitro testing; in vivo; inducible gene expression; inhibitor/antagonist; insight; junctophilin; knock-down; mouse model; novel therapeutics; patch clamp; single cell analysis; small molecule; targeted treatment; therapeutic evaluation; tool

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.

抽象的 心脏淋巴结组织的疾病可能危及生命，特别是在年轻人中。 自发去极化作为心脏收缩的起搏器，但尽管有这一核心作用 对于生存至关重要，但人们对淋巴结功能障碍的原因知之甚少。 理解损害了有效和选择性药物疗法的发展，并且作为 相关地，针对淋巴结疾病征收的药物对患者具有显着的毒性，并且仍然可以 传统上认为节点自动性是由离子控制的。 质膜上的通道，越来越多的证据表明，质膜上的钙信号传导 细胞可能调节自发去极化——之前的研究表明它具有自动性。 从内部钙释放通道 RyR2 的钙泄漏可能与结节增加有关 激发，从而了解这种所谓的“钙钟”可以为 我创建了节点特异性表达沉默的小鼠模型。 一种叫做 junctophilin-2 (JPH2) 的蛋白质，我之前已经证明它对于有效的钙- 我发现了收缩肌细胞的处理，作为研究功能失调的钙时钟的工具。 该小鼠静息时心率升高，房室结快速放电，导致 我提出了 3 个具体目标来测试我们的心律失常，称为加速交界节律 (AJR)。 中心假设是 JPH2 表达减少导致 CaMKII 介导的 RyR2 门控增加 这会导致储存钙泄漏增加并导致节点自动化和 AJR 增加。 1) 利用来自 HCN4:shJPH2 小鼠的分离淋巴结细胞的共聚焦钙成像，结合 使用 RyR2 单通道记录来确定 JPH2 表达减少是否导致 JPH2 表达增加 钙泄漏具有较高的 RyR2 通道开放概率；2) 应用已知的 RyR2 化学抑制剂 对分离的单细胞和 HCN4:shJPh2 小鼠进行评估，以评估钙渗漏是否可以正常化，以及 AJR 得到有效治疗；以及 3) 对分离的淋巴结组织进行生物化学测定，以确定其作用 我预计 CaMKII 信号传导，包括其下游磷酸化靶标，可调节节点放电。 完成这些目标将产生对“钙钟”的临床可转化机制的见解 通过探索第一个孤立的心脏结节疾病的小鼠模型。 文献中，这些目标将为专门测试新型治疗剂提供基础 针对淋巴结组织中钙信号传导的干扰，完成这项为期 5 年的培训资助将允许。 我将我在儿科电生理学方面的临床训练与分子生物学的探索结合起来 淋巴结疾病的机制，并成为一名独立资助的医师科学家，致力于 帮助患有心律失常的儿童。