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

项目概要/摘要 Na+和Ca 2+离子稳态对于心脏兴奋性和收缩性是必不可少的。在细胞水平上， 膜蛋白Na+-Ca 2+交换器（NCX）在调节Na+和Ca 2+的离子稳态中起着重要作用。 Ca2+。它通过将一个Ca 2+挤出细胞外来交换三个胞外Na+离子来实现。此外 这两种离子都能变构地调节NCX的活性。细胞内Ca 2+增加NCX 细胞质Na+通过Na+依赖性失活过程使NCX失活。尽管 这种调节的潜在生理和病理生理相关性，是否Na+依赖 体内发生的失活是未知的，其影响尚未确定。既然这是如此精致的 控制系统，但迄今尚未调查，研究人员假设，细胞的微小变化， Na+浓度可能通过直接影响NCX活性对Ca 2+稳态产生显著影响， 从而影响心脏的兴奋性和收缩性。因此，本申请的目标是调查 NCX Na+调节的生理影响，并确定其最终如何塑造心脏收缩性。这些 研究一直受到在受控条件下研究完整心肌细胞中的这一过程的困难的阻碍。 条件然而，随着通过CRISPR技术进行基因组修饰的发展， 范式，迄今为止遥不可及，现在可以解决。使用CRISPR，研究人员插入了一个 在小鼠的天然心脏NCX基因中的单位点突变（K229 Q），其将专门消除Na+- 依赖性失活通过结合电生理学和钙成像技术， 初步数据表明，细胞质Na+对NCX的抑制改变了细胞的电和机械特性， 单细胞和完整心脏的特性。 这里提出的工作分为两个目标。目的1将研究如何缺乏Na+依赖性 失活改变小鼠成年心室肌细胞兴奋-收缩偶联 从分离的成人心室肌细胞测量的电位、Ca 2+瞬变和离子电流， 对照（WT）或遗传改变的小鼠（K229 Q）。Aim 2将进行类似的记录，但在完整的灌注 心中此外，将使用超声心动图评估活K229 Q小鼠的心脏功能。 这些研究是开创性的，因为它们将详细说明NCX变构Na+调节的潜在功能 心脏功能。这项工作也可能有病理生理学的应用，通过定义Na+的调节， 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.