Regulation of cellular calcium by cardiac sodium-calcium exchange

Goldhaber实验室和Alfredolia实验室之间的MPI提案的目的是改善我们的 了解钠钙交换（NCX），主要的钙（Ca）流出 心肌细胞中的一种机制，其功能是调节细胞Ca，从而控制收缩性和起搏 活动我们的实验室已经研究了二十多年的交换器，尽管有一些问题， 挑战：1）没有经过证实的可以在细胞外给药的NCX特异性阻滞剂; 2）NCX 电流有时很难解释，因为转运蛋白不仅受电压的影响， 温度、pH以及细胞内和细胞外Na和Ca的浓度; 3）细胞内Ca不是 它不仅由交换器输送，而且还具有调节功能，可以影响输送活性。在 除了我们长期以来隔离和研究NCX的专业知识外，我们还为该提案产生了新的 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Ca调节如何随着时间的推移适应成年小鼠中NCX的急性去除;一种独特的pH- 不敏感的NCX敲入小鼠，以研究NCX pH调节的生理影响; 心室特异性质膜Ca泵1（PMCA 1）KO小鼠，以确定 NCX和PMCA对Ca稳态和EC偶联。我们的三个具体目标是研究：1。心房特异性 NCX KO对结性节律和心房EC耦合的影响; 2.急性和慢性适应 心室EC偶联和Ca对遗传改变的NCX水平的调节; 3. NCX pH值 依赖-对EC偶联和心律失常的影响。这些目标将测试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 在成年小鼠中，NCX的消融激活了Ca调节和EC偶联适应程序， 从慢性适应来看，PMCA 1是NCX作为Ca流出机制的关键替代物，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。我们的方法是使用 我们现有的和新的创新小鼠模型，沿着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，高质量的蛋白质组学， 下一代RNA测序，以确定NCX如何通过调节 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。 细胞钙调节、EC偶联和心脏起搏器（SAN和AVN）功能中的钙处理蛋白。 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增殖至关重要。 心脏起搏器在心脏疾病中的功能，如射血分数降低的心力衰竭，以及高度 房室结疾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.