Regulation of cellular calcium by cardiac sodium-calcium exchange

Goldhaber和Ottolia实验室之间的MPI提案的目的是改善我们的 钠钙交换(NCX)--主要钙离子(Ca)外流机制的理解 心肌细胞的机制，功能是调节细胞内钙离子，而细胞内钙离子又控制着收缩和起搏器 活动。我们的实验室已经研究了20多年的交换器，尽管有许多 挑战：1)没有已证实的NCX特异性阻滞剂可以细胞外给药；2)NCX 电流有时很难解释，因为传输器不仅受电压的影响，还受 温度、pH和细胞内外Na、Ca浓度；3)细胞内钙不是 不仅由交易所运输，而且还起到调节作用，可以影响运输活动。在……里面 除了我们隔离和研究NCX的长期专业知识外，我们还为这项提议产生了新的 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 研究钙调节如何随时间适应成年小鼠对NCX的急性清除；一种独特的pH- 研究不敏感的NCX敲击小鼠对NCX pH调节的生理影响； 测定KO小鼠脑室特异性质膜钙泵1(PMCA1)的相对贡献 NCX和PMCA对钙稳态和EC偶联的影响。我们的三个具体目标是研究：1.心房特异性 NCX-KO对结节节律和心房内皮细胞偶联的影响2.急性和慢性适应 脑室内皮细胞偶联和钙对Ncx基因改变的调节；3.Ncx pH 依赖性--对EC偶联和心律失常的影响。这些目标将检验NCX的假设 是房室结(AVN)传导和脉冲产生的重要组成部分，即急性 在成年小鼠中消融NCX激活一种不同的钙调节和EC偶联适应程序 从慢性适应来看，PMCA1是作为钙外流机制的NCX的关键替代品，而且pH 在低pH条件下，NCX的敏感性是维持钙调节的关键。我们的方法是使用 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 电生理学结合高速亚细胞钙成像技术，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 加州这些研究完成后，将提高我们对NCX和相关角色的机械性理解 钙处理蛋白在细胞钙调节、EC偶联和心脏起搏器(SAN和AVN)功能中的作用。 这些信息对于开发有效和安全的方法来改善收缩能力和细胞 心脏疾病的起搏器功能，如心力衰竭，射血分数降低，高度 房室结病引起的心脏传导阻滞。

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.