Regulation of cellular calcium by cardiac sodium-calcium exchange

通过心脏钠钙交换调节细胞钙

基本信息

批准号：
9906764
负责人：
Joshua I Goldhaber
金额：
$ 65.01万
依托单位：
CEDARS-SINAI MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-15 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9906764
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

项目摘要

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 实验室之间的 MPI 提案的目的是改进我们的对钠钙交换 (NCX)、主要钙 (Ca) 流出的机制的理解心肌细胞中的机制，具有调节细胞 Ca 的功能，进而控制收缩力和起搏器我们的实验室对交换器的研究已有二十多年，尽管存在许多问题。挑战：1) 没有经过证实的可在细胞外给药的 NCX 特异性阻断剂；2) NCX 电流有时很难解释，因为转运蛋白不仅受到电压的影响，还受到温度、pH 值以及细胞内和细胞外的 Na 和 Ca 浓度 3) 细胞内 Ca 不受影响；不仅通过交换器进行运输，而且还具有可以影响运输活动的调节功能。除了我们长期分离和研究 NCX 的专业知识之外，我们还为该提案生成了新的高度创新的 NCX 小鼠模型：他莫昔芬诱导的 NCX 心脏敲除，这使我们能够研究成年小鼠中 Ca2+ 调节如何随着时间的推移适应 NCX 的急性去除；不敏感的 NCX 敲入小鼠研究了 NCX pH 调节的生理影响；心室特异性质膜钙泵 1 (PMCA1) KO 小鼠以确定 NCX 和 PMCA 与 Ca 稳态和 EC 耦合我们的三个具体目标是研究： 1. 心房特异性。 NCX KO – 对结节律和心房 EC 耦合的影响；2. 急性和慢性适应心室 EC 耦合和 Ca 调节对 NCX 基因改变水平的影响；3. NCX pH 值；依赖性——对 EC 耦合和心律失常的影响这些目标将检验 NCX 的假设。是房室结 (AVN) 传导和冲动产生的重要组成部分，急性成年小鼠 NCX 的消融激活了独特的 Ca 调节和 EC 耦合适应程序从慢性适应来看，PMCA1 是 NCX 作为 Ca 流出机制的关键替代品，并且 pH NCX 的敏感性对于在低 pH 条件下维持 Ca 调节至关重要。我们现有的和新的创新小鼠模型，以及最先进的单细胞和组织电生理学与高速亚细胞 Ca 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 Ca. 完成后，这些研究将提高我们对 NCX 及相关作用的机制理解。细胞 Ca 调节、EC 耦合和心脏起搏器（SAN 和 AVN）功能中的 Ca 处理蛋白。这些信息对于开发有效和安全的方法来改善收缩性和细胞至关重要心脏起搏器在心脏疾病中的作用，例如射血分数降低的心力衰竭和高度严重的心力衰竭 AVN 疾病引起的心脏传导阻滞。