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

Na-Ca2 交换的钠依赖性失活：与心脏功能的相关性

• 批准号: 10531590
• 负责人: Riccardo Olcese
• 金额: $ 54.61万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-18 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10531590
• 关键词: Action Potentials; Address; Adult; Affect; Allosteric Regulation; Animals; Anti-Arrhythmia Agents; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cardiac health; Cell membrane; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Congestive Heart Failure; Coupling; Cytoplasm; Darkness; Data; Development; Drug Targeting; Echocardiography; Electrophysiology (science); Equilibrium; Event; Excision; Genes; Genomics; Goals; Heart; Heart Contractilities; Heart failure; Homeostasis; Hypertrophy; Imaging Techniques; Investigation; Ions; Ischemia; Knowledge; Link; Measures; Membrane Potentials; Membrane Proteins; Modification; Mus; Muscle Cells; Mutate; Mutation; Myocardial Ischemia; Organ; Pathologic; Perception; Perfusion; Phase; Physiological; Physiology; Play; Process; Property; Proteins; Regulation; Reperfusion Injury; Reperfusion Therapy; Research; Research Personnel; Role; Route; Shapes; Site; Sodium; Sodium-Calcium Exchanger; Spottings; System; Ventricular; Work; design; electrical property; extracellular; heart function; in vivo; inhibitor; mechanical properties; novel; novel therapeutic intervention; novel therapeutics; operation; pharmacologic; response

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.

项目概要/摘要 Na+ 和 Ca2+ 离子稳态对于心脏兴奋性和收缩性至关重要。在细胞水平上，血浆 膜蛋白 Na+-Ca2+ 交换器 (NCX) 在调节 Na+ 离子稳态中起着至关重要的作用 和Ca2+。它通过将一种 Ca2+ 挤出细胞以交换三种细胞外 Na+ 离子来实现这一点。此外 为了被运输，这两种离子都变构调节 NCX 的活性。细胞内 Ca2+ 增加 NCX 活性，而细胞质 Na+ 通过称为 Na+ 依赖性失活的过程使 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Na+ 体内发生的失活尚不清楚，其影响也尚未确定。既然如此精致 控制系统，但迄今为止未经调查，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 活性和对 Ca2+ 稳态产生显着影响。 从而影响心脏的兴奋性和收缩性。因此，本应用程序的目标是调查 NCX Na+ 调节的生理影响，并确定它最终如何塑造心脏收缩力。这些 由于在受控的完整心肌细胞中研究这一过程的困难，研究受到了阻碍 状况。然而，随着通过 CRISPR 技术进行基因组修饰的发展，这一实验 迄今为止无法实现的范式现在可以得到解决。研究人员利用 CRISPR 插入了 小鼠天然心脏 NCX 基因中的单点突变 (K229Q) 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，收集的新颖 初步数据表明，细胞质 Na+ 对 NCX 的抑制会改变电气和机械性能 单细胞和完整心脏的特性。 这里提出的工作分为两个目标。目标 1 将研究 Na+ 依赖性缺失如何 失活通过比较作用改变小鼠成年心室肌细胞的兴奋-收缩耦合 从成年心室肌细胞中测量的电位、Ca2+瞬变和离子电流 对照（WT）或基因改造小鼠（K229Q）。目标 2 将进行类似的记录，但灌注完好 心。此外，还将使用超声心动图评估活体 K229Q 小鼠的心脏功能。 这些研究具有开创性，因为它们将详细说明 NCX 变构 Na+ 调节的潜在功能 在心脏功能方面。这项工作也可能具有病理生理学应用，将 Na+ 的调节定义为 控制 NCX 活动的潜在目标。