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：该项目旨在表征心脏动作电位的新型机制（AP） 根据获得的见解，传导并开发了治疗心律不齐的新方法。我们报告了 Na+通道的NAV1.5亚基集中在CX43（GJA1）间隙连接（GJ）边缘 插入的磁盘纳米域称为Perinexus。高浓度的Na+通道和狭窄的间 Perinexus处的膜宽度与可能的结构单位的理论预测一致 支持ephaptic传导。测试的假设是Na+通道复合物的β亚基是 该单元的中央组织元素：ephapse。我们的前提是β1（SCN1B）的粘合剂功能 促进插入式磁盘内膜间接触的专业区域的形成。这 安排被设想为促进跨性别Na+通道在肌细胞之间形成，并支持 AP的细胞间传导。此外，我们提出，维持浮球粘合剂的激动剂可以 构成新疗法，以减轻心肌梗塞后心律失常传导缺陷。在 对假设的支持，数据来自超分辨率，电子和免疫电子显微镜 NAV1.5和β1亚基以及主动Na+通道的智能贴片夹（SPC）研究 集中在阴部。此外，我们已经开发了一种新型的β1抑制剂 - 19个氨基酸模拟物 β1的粘附结构域（βadp1）。在电细胞基底阻抗传感测定中，βadp1引起 细胞之间异源表达β1的粘附丧失。将βadp1输注到心脏中导致 剂量依赖性：（1）与β1去粘附一致的周期裂裂； （2）传导放慢； （3）增加心室心律不齐。重要的是，虽然抑制β1粘合剂似乎 SPC对全细胞Na+电流没有影响，揭示了与GJ相关的Na+通道的选择性下降 活动。最后，显示了针对β1介导的反互动的合理设计的激动剂的初步数据 （DBL-βADP）促进β1过表达细胞之间的粘附。测试以下假设：（1）β1为 稳定在ephapse上稳定反互动的Na+通道所必需的； （2）β1介导的广告是 抗心律失常目标：AIM 1将确定β1和CX43 GJ的要求 机制。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的小鼠 将用于研究假设的ephapse的结构和自主功能。 AIM 2意志 确定β1粘附在心肌细胞中Na+通道活性以及重塑和AP传导中的作用 单层。策略中将使用GJ型平原形成，但功能无能的CX43突变体（L90V）将使用 研究边缘机制对独立于GJ耦合的传导的贡献。目标3意志 开发有效的分子，这些分子基于我们的第一代动力学DBL-βADP稳定β1粘合剂并测试 这些化合物在急性心肌梗死的促性心律失常环境中的抗心律失常效率。