The Role of the Sodium Channel Beta Subunit in Cardiac Conduction

钠通道β亚基在心脏传导中的作用

• 批准号: 9923757
• 负责人: ROBERT G GOURDIE
• 金额: $ 43.92万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9923757
• 关键词: Abbreviations; Action Potentials; Acute myocardial infarction; Adhesions; Agonist; Alleles; Amino Acids; Anti-Arrhythmia Agents; Antibodies; Arrhythmia; Biological Assay; Cardiac; Cavia; Cells; Chemistry; Collaborations; Complex; Computing Methodologies; Connexin 43; Coupling; Data; Defect; Docking; Dominant-Negative Mutation; Dose; Dysplasia; Electrons; Elements; Extracellular Domain; Funding; Gap Junctions; Generations; Green Fluorescent Proteins; Heart; Image; Immunoelectron Microscopy; Immunoglobulins; In Vitro; Incidence; Infarction; Infusion procedures; Intercalated disc; Ligands; London; Manuscripts; Mechanics; Mediating; Methods; Microscopy; Modeling; Mus; Muscle Cells; Myocardial Infarction; Neonatal; Optics; Peer Review; Peptides; Pre-Clinical Model; Property; Publishing; Rattus; Reporting; Research Personnel; Resolution; Role; Site; Sodium Channel; Spectrum Analysis; Structure; Surface Plasmon Resonance; Testing; Tissues; United States National Institutes of Health; Ventricular; Ventricular Arrhythmia; Virginia; Western Blotting; Width; acute coronary syndrome; base; college; design; desmoglein 2; drug discovery; efficacy study; electric impedance; extracellular; in silico; inhibitor/antagonist; insight; mimetics; monolayer; multidisciplinary; mutant; novel; novel strategies; novel therapeutics; patch clamp; reconstruction; response; tool

ABSTRACT: This project seeks to characterize a novel mechanism of cardiac action potential (AP) conduction and develop new approaches to treat arrhythmias based on the insights gained. We have reported that the Nav1.5 subunit of the Na+ channel is concentrated at the Cx43 (Gja1) gap junction (GJ) edge in an intercalated disk nanodomain called the perinexus. The high concentration of Na+ channels and narrow inter- membrane width at the perinexus are consistent with theoretical predictions of a structural unit that may support ephaptic conduction. The hypothesis tested is that the β subunit of the Na+ channel complex is the central organizing element of this unit: The ephapse. Our premise is that the adhesion function of β1 (Scn1b) promotes the formation of specialized regions of inter-membrane contact within intercalated disks. This arrangement is envisaged as enabling trans-interacting Na+ channels to form between myocytes, facilitating cell-to-cell conduction of AP. Furthermore, we propose that agonists that maintain ephapse adhesion could constitute new therapies to mitigate arrhythmogenic conduction defects following myocardial infarction. In support of the hypothesis, data is provided from super-resolution, electron, and immuno-electron microscopy and smart patch clamp (SPC) studies that Nav1.5 and β1 subunits, as well as active Na+ channels, are concentrated in the perinexus. Moreover, we have developed a novel β1 inhibitor - a 19 amino acid mimetic of the adhesion domain of β1 (βadp1). In electric cell-substrate impedance sensing assays, βadp1 caused loss of adhesion between cells heterologously over-expressing β1. Infusion of βadp1 into hearts resulted in dose-dependent: (1) Widening of the perinexal cleft consistent with β1 de-adhesion; (2) Conduction slowing; and (3) Increased ventricular arrhythmia incidence. Importantly, while inhibition of β1 adhesion appeared to have no effect on whole-cell Na+ currents, SPC revealed a selective decrease in GJ-associated Na+ channel activity. Finally, preliminary data is shown for a rationally designed agonist of β1-mediated trans-interaction (dbl-βadp) that promotes adhesion between β1 over-expressing cells. To test the hypotheses that: (1) β1 is required for stabilizing trans-interacting Na+ channels at the ephapse; and (2) β1-mediated adhesion is an anti-arrhythmic target: Aim 1 will determine the requirement of β1 and Cx43 GJs for the hypothesized ephaptic mechanism. Mice encoding Scn1b and Gja1 null alleles, together with the β1 trans-adhesion inhibitor βadp1, will be used in studies of the structure and autonomous function of the hypothesized ephapse. Aim 2 will determine the role of β1 adhesion on Na+ channel activity and remodeling and AP conduction in myocyte monolayers. A GJ-plaque-forming, but functionally incompetent Cx43 mutant (L90V) will be used in a strategy to study the contribution of the ephaptic mechanism to conduction independent of GJ coupling. Aim 3 will develop efficient molecules that stabilize β1 adhesion based on our first-generation agonist dbl-βadp and test the anti-arrhythmic efficacy of these compounds in the pro-arrhythmic setting of acute myocardial infarction.

摘要：该项目旨在描述心脏动作电位 (AP) 的一种新机制 传导并根据我们所报告的见解开发治疗心律失常的新方法。 Na+ 通道的 Nav1.5 亚基集中在 Cx43 (Gja1) 间隙连接 (GJ) 边缘 嵌入的圆盘纳米域称为周缘，高浓度的 Na+ 通道和狭窄的间隙。 周缘处的膜宽度与结构单元的理论预测一致，该结构单元可能 支持触觉传导。所测试的假设是 Na+ 通道复合体的 β 亚基是 该单元的中心组织元素：ephapse 我们的前提是 β1 (Scn1b) 的粘附函数。 促进闰盘内膜间接触的特殊区域的形成。 这种排列被设想为能够在肌细胞之间形成反式相互作用的 Na+ 通道，从而促进 此外，我们提出维持 ephase 粘附的激动剂可以。 构成减轻心肌梗塞后致心律失常传导缺陷的新疗法。 为了支持该假设，数据由超分辨率、电子和免疫电子显微镜提供 和智能膜片钳 (SPC) 研究表明 Nav1.5 和 β1 亚基以及活性 Na+ 通道 此外，我们还开发了一种新型 β1 抑制剂 - 一种 19 个氨基酸的模拟物。 β1 (βadp1) 的粘附结构域在细胞-基质阻抗传感测定中，βadp1 引起。 将βadp1输注到心脏中会导致异源过度表达β1的细胞之间的粘附丧失。 剂量依赖性：(1) 会周裂增宽，与 β1 脱粘一致；(2) 传导减慢； (3) 室性心律失常的发生率增加，但重要的是，抑制β1 粘附似乎会增加室性心律失常的发生率。 对全细胞 Na+ 电流没有影响，SPC 显示 GJ 相关 Na+ 通道选择性减少 最后，显示了合理设计的 β1 介导的反式相互作用激动剂的初步数据。 (dbl-βadp) 促进 β1 过表达细胞之间的粘附 测试以下假设：(1) β1 是。 稳定 ephase 的反式相互作用 Na+ 通道所需；(2) β1 介导的粘附是一种 抗心律失常目标：目标 1 将确定敲击的上皮细胞对 β1 和 Cx43 GJ 的需求 编码 Scn1b 和 Gja1 无效等位基因的小鼠，以及 β1 反式粘附抑制剂 βadp1， 将用于研究捕获的 Ephase 2 will 的结构和自主功能。 确定 β1 粘附对肌细胞 Na+ 通道活性、重塑和 AP 传导的作用 单层细胞形成 GJ 斑块，但功能不全的 Cx43 突变体 (L90V) 将用于策略中。 研究触觉机制对独立于 GJ 耦合的传导的贡献 目标 3。 基于我们的第一代激动剂 dbl-βadp 开发稳定 β1 粘附的有效分子并进行测试 这些化合物在急性心肌梗死的促心律失常情况下的抗心律失常功效。