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: 10320449
• 负责人: ARIEL L ESCOBAR
• 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/10320449
• 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; Data; Development; Drug Targeting; Echocardiography; Electrophysiology (science); Equilibrium; Event; Excision; Genes; Genomics; Goals; Heart; Heart Contractilities; Heart failure; Homeostasis; Hypertrophy; Imaging Techniques; Investigation; Ions; Knowledge; Light; Link; Measures; Membrane Potentials; Membrane Proteins; Modification; Mus; Muscle Cells; Mutate; Mutation; Myocardial Ischemia; Organ; Pathologic; Perception; Pharmacology; 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; 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.

项目摘要/摘要 Na+和Ca2+离子稳态对于心脏的兴奋性和收缩力至关重要。在细胞水平上血浆 膜蛋白Na+ -ca2+交换器（NCX）在调节这两种Na+的离子稳态中起着至关重要的作用 和Ca2+。它通过将一个Ca2+从细胞中挤出来换取三个细胞外Na+离子来做到这一点。此外 要运输，这两种离子都会变构调节NCX的活性。细胞内Ca2+增加了NCX 活性虽然细胞质Na+通过称为Na+依赖性失活的过程使NCX失活。尽管有 该调节的潜在生理和病理生理相关性，无论NA+依赖性是否依赖 灭活发生在体内尚不清楚，其影响尚未确定。因为这是一个精致的 调查人员的控制系统，但迄今为止未经研究，研究人员假设细胞的小变化 Na+浓度可能通过直接影响NCX活性和 从而影响心脏的兴奋性和收缩力。因此，此应用的目的是调查 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 在控制的完整肌细胞中研究这一过程的困难使研究受到了阻碍。 状况。但是，随着通过CRISPR技术进行基因组修饰的发展，该实验 迄今为止无法触及的范式现在可以解决。使用CRISPR，调查人员插入了 小鼠天然心脏NCX基因中的单位位点突变（K229Q），该基因将仅取消Na+ - 依赖灭活。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，细胞质Na+对NCX的抑制作用改变了电和机械 单细胞和完整心脏的特性。 这里提出的工作分为两个目标。 AIM 1将研究不存在Na+依赖性的 通过比较动作来改变小鼠成人心室心肌细胞中的激发触发耦合 电势，Ca2+瞬态和由从任何一个分离的成年心室心肌细胞测得的电流和离子电流 对照（WT）或遗传改变的小鼠（K229Q）。 AIM 2将进行类似的录音，但完好无损 心。此外，将使用超声心动图评估活K229Q小鼠的心脏功能。 这些研究是开创性的，因为它们将详细介绍NCX变构NA+调节的潜在功能 在心脏功能中。这项工作还可以通过定义Na+的调节为病理生理应用 控制NCX活性的潜在目标。