Sodium Dependent Inactivation of the Na+-Ca2+ exchange: Relevance to Cardiac Function

项目摘要/摘要 钠和钙离子的稳态对心脏的兴奋性和收缩能力是必不可少的。在细胞水平上，血浆 膜蛋白Na-Ca~(2+)交换器(NCX)在调节两种钠离子动态平衡中起重要作用 和钙离子。它通过从细胞中挤出一个钙离子来换取三个细胞外的钠离子来实现这一点。此外 在转运过程中，这两种离子都以变构方式调节NCX的活性。细胞内钙离子增加NCX 而细胞质Na通过一种称为钠依赖失活的过程使NCX失活。尽管 这种调节的潜在生理学和病理生理学相关性，是否依赖于钠 灭活在体内发生的情况尚不清楚，其影响还有待确定。既然这是一个如此精致的 控制系统，但到目前为止还没有被研究过，研究人员假设细胞中的微小变化 Na浓度可能通过直接影响NCX活性而对钙稳态产生显著影响 从而影响心脏的兴奋性和收缩能力。因此，此应用程序的目标是调查 NCX钠调节的生理影响，并确定它最终如何塑造心脏的收缩能力。这些 在受控的完整心肌细胞中研究这一过程的困难阻碍了研究。 条件。然而，随着通过CRISPR技术进行基因组修饰的发展，本实验 迄今为止遥不可及的范式，现在可以解决了。使用CRISPR，调查人员插入了一个 小鼠天然心脏Ncx基因的单点突变(K229Q)，它将独占消除Na- 依赖失活。通过结合电生理学和钙成像技术，收集的小说 初步数据表明，胞质钠对NCX的抑制改变了NCX的电和机械特性 单细胞和完整心脏的特性。 这里提出的工作有两个目标。目标1将研究钠依赖的缺失如何 失活改变成年小鼠心室肌细胞的兴奋-收缩偶联 分离的成年心肌细胞的电位、钙瞬变和离子电流 对照组(WT)或转基因小鼠(K229Q)。Aim 2将进行类似的录音，但在完整的灌流中 红心。此外，还将使用超声心动图评估活K229Q小鼠的心功能。 这些研究具有开创性，因为它们将详细说明NCX变构钠调节的潜在功能 在心脏功能方面。这项工作还可能通过将钠的调节定义为 控制NCX活动的潜在目标。

PROJECT SUMMARY/ABSTRACT Na+ and Ca2+ ion homeostasis are essential for heart excitability and contractility. At the cellular level the plasma membrane protein Na+-Ca2+ exchanger (NCX) plays a vital role in regulating the ionic homeostasis of both Na+ and Ca2+. It does so by extruding one Ca2+ out of the cell in exchange for three extracellular Na+ ions. In addition to being transported, both these ions allosterically regulate the activity of NCX. Intracellular Ca2+ increases NCX activity while cytoplasmic Na+ inactivates NCX via a process known as Na+-dependent inactivation. Despite the potential physiological and pathophysiological relevance of this regulation, whether the Na+-dependent inactivation occurs in vivo is unknown and its impact has yet to be determined. Since this is such an exquisite controlling system, but heretofore uninvestigated, the investigators hypothesize that small changes in cellular Na+ concentrations may have significant effects on Ca2+ homeostasis by directly affecting NCX activity and thereby affect excitability and contractility of the heart. Therefore, the goal of this application is to investigate the physiological impact of NCX Na+ modulation and determine how it ultimately shapes heart contractility. These studies have been hampered by the difficulties of studying this process in intact myocytes under controlled conditions. However, with the development of genomic modification via CRISPR technology, this experimental paradigm, heretofore out of reach, can now be addressed. Using CRISPR, the investigators have inserted a single site mutation (K229Q) in the native cardiac NCX gene of mice, which will exclusively abolish Na+- dependent inactivation. By combining electrophysiology and calcium imaging techniques, the collected novel preliminary data demonstrating that the inhibition of NCX by cytoplasmic Na+ alters the electrical and mechanical properties of both single cells and intact hearts. The work proposed here is organized into two aims. Aim 1 will investigate how the absence of Na+-dependent inactivation alters excitation-contraction coupling in mouse adult ventricular myocytes by comparing, action potentials, Ca2+ transients and ionic currents measured from adult ventricular myocytes isolated from either control (WT) or the genetically altered mice (K229Q). Aim 2 will conduct similar recordings but in intact perfused hearts. Additionally, the cardiac function of live K229Q mice will be assessed using echocardiography. These investigations are groundbreaking as they will detail the potential function of NCX allosteric Na+ regulation in cardiac function. This work may also have pathophysiological applications by defining the regulation of Na+ as a potential target for controlling NCX activity.