Regulation of cellular calcium by cardiac sodium-calcium exchange

通过心脏钠钙交换调节细胞钙

• 批准号: 10376807
• 负责人: Joshua I Goldhaber
• 金额: $ 63.31万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376807
• 关键词: Ablation; Acidosis; Acute; Address; Adrenergic beta-Agonists; Adult; Affect; Affinity; Arrhythmia; CRISPR/Cas technology; Ca(2+)-Transporting ATPase; Calcium; Cardiac; Cardiac Myocytes; Cardiac pacemaker; Cell Death; Cell membrane; Cells; Chronic; Coupling; Data; Dependence; Development; Digoxin; Disease; EFRAC; Electrophysiology (science); Excision; Functional disorder; Gene Expression; Generations; Goals; Grant; Heart; Heart Atrium; Heart Block; Heart Diseases; Heart failure; Homeostasis; Hospitalization; Imaging Techniques; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Mus; Muscle Cells; Myocardial Infarction; Myocardial dysfunction; Myocardium; Nodal; Optics; Pacemakers; Patch-Clamp Techniques; Physiological; Physiology; Prevention; Proteins; Proteomics; Pump; Regulation; Rest; Role; Sick Sinus Syndrome; Sinoatrial Node; Sodium; Sodium-Calcium Exchanger; Speed; Structure; Systole; Tamoxifen; Temperature; Testing; Therapeutic; Time; Tissues; Ventricular; Work; atrioventricular node; cell growth regulation; cell injury; confocal imaging; experimental study; extracellular; heart cell; heart function; implantation; improved; innovation; insight; mouse model; next generation; novel; programs; release of sequestered calcium ion into cytoplasm; sensor; tissue preparation; transcriptome sequencing; voltage

The purpose of this MPI proposal between the Goldhaber and Ottolia laboratories is to improve our mechanistic understanding of how sodium-calcium exchange (NCX), the dominant calcium (Ca) efflux mechanism in cardiac cells, functions to regulate cellular Ca, which in turn controls contractility and pacemaker activity. Our labs have been studying the exchanger for more than two decades, despite a number of challenges: 1) there is no proven specific blocker of NCX that can be administered extracellularly; 2) NCX current is sometimes difficult to interpret because the transporter is influenced not only by voltage, but also by temperature, pH and the intracellular and extracellular concentrations of Na and Ca; 3) intracellular Ca is not only transported by the exchanger, but also serves a regulatory function that can influence transport activity. In addition to our longstanding expertise isolating and studying NCX, for this proposal we have generated new highly innovative NCX mouse models: a tamoxifen-inducible cardiac knockout of NCX, which allows us to investigate how Ca regulation adapts over time to the acute removal of NCX in the adult mouse; a unique pH- insensitive NCX knockin mouse to investigate the physiological impact of NCX pH regulation; and the first ventricular-specific plasma membrane Ca pump 1 (PMCA1) KO mouse to determine the relative contribution of NCX and PMCA to Ca homeostasis and EC coupling. Our three specific aims are to study: 1. Atrial-Specific NCX KO–Effects on Nodal Rhythm and Atrial EC Coupling; 2. Acute and Chronic Adaptations of Ventricular EC Coupling and Ca regulation to Genetically Altered Levels of NCX; 3. NCX pH dependence – implications for EC coupling and arrhythmia. These aims will test the hypotheses that NCX is an essential component of atrioventricular node (AVN) conduction and impulse generation, that acute ablation of NCX in adult mice activates a Ca regulatory and EC coupling adaptation program that is distinct from chronic adaptation, that PMCA1 is a critical alternative to NCX as a Ca efflux mechanism, and that pH sensitivity of NCX is critical for maintaining Ca regulation under conditions of low pH. Our approach is to use our existing and new innovative mouse models, along with state-of-the-art single cell and tissue electrophysiology combined with high speed subcellular Ca imaging techniques, high-quality proteomics and next generation RNA sequencing, to determine how NCX contributes to cardiac function through regulation of Ca. When completed, these studies will improve our mechanistic understanding of the role of NCX and related Ca handling proteins in cellular Ca regulation, EC coupling, and cardiac pacemaker (SAN and AVN) function. Such information is critical to develop effective and safe approaches to improving contractility and cellular pacemaker function in cardiac diseases such as heart failure with reduced ejection fraction, and high degree heart block from AVN disease.

Goldhaber和Ottolia实验室之间此MPI提案的目的是改善我们的 对钠钙交换（NCX）的机械理解，主要钙（CA）外排 心脏细胞中的机理，调节细胞CA的功能，进而控制收缩力和起搏器 活动。我们的实验室一直在研究交换器已有二十年了， 挑战：1）没有可靠的NCX特定阻滞剂可以细胞外施用； 2）NCX 电流有时很难解释，因为运输蛋白不仅受电压的影响，而且受到 Na和Ca的温度，pH以及细胞内和细胞外浓度； 3）细胞内CA不是 仅由交换器运输，但也具有可能影响运输活动的调节功能。在 除了我们长期以来的专业知识隔离和研究NCX，对于此建议，我们已经产生了新的 高度创新的NCX小鼠模型：NCX的他莫昔芬诱导的心脏敲除，这使我们能够 研究CA随着时间的流逝如何适应成年小鼠中NCX的急性去除；独特的pH- 不敏感的NCX敲击小鼠研究NCX pH调节的物理影响；和第一个 心室特异性质膜Ca泵1（PMCA1）KO小鼠以确定相对贡献 NCX和PMCA到CA稳态和EC耦合。我们的三个具体目的是研究：1。心房特异性 NCX KO效应对淋巴结和心房EC耦合； 2。急性和慢性适应 心室EC耦合和CA调节NCX水平的遗传变化； 3。NCXph 依赖性 - 对EC耦合和心律不齐的影响。这些目标将检验NCX的假设 是房屋节点（AVN）传导和冲动产生的重要组成部分， 成年小鼠中NCX的消融会激活CA调节和EC耦合适应程序，该计划是不同的 从慢性适应中，PMCA1是NCX作为CA外排机制的关键替代品，而该pH NCX的敏感性对于在低pH的条件下维持CA调节至关重要。我们的方法是使用 我们现有和新的创新鼠标模型以及最先进的单细胞和组织 电生理学结合高速亚细胞成像技术，高质量蛋白质组学和 下一代RNA测序，以确定NCX如何通过调节来促进心脏功能 大约完成后，这些研究将提高我们对NCX和相关作用的机械理解 CA处理细胞CA调节，EC耦合和心脏起搏器（SAN和AVN）功能中的蛋白质。 这样的信息对于开发有效且安全的方法至关重要 心脏病的起搏器功能，例如心力衰竭，射血分数减少和高度 AVN病的心脏障碍。