The Effects of the SCN1B Mimetic Peptide Badp1 on the Regulated Intramembrane Proteolysis Pathway

SCN1B 模拟肽 Badp1 对调节膜内蛋白水解途径的影响

基本信息

批准号：
10676749
负责人：
Zachary Williams
金额：
$ 2.42万
依托单位：
VIRGINIA POLYTECHNIC INST AND ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-10 至 2023-12-25
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10676749
关键词：
Action Potentials Acute Adhesions Affect Anti-Arrhythmia Agents Area Arrhythmia Atrial Fibrillation Biological Assay Buffers C-terminal Cardiac Cardiac Myocytes Cause of Death Cavia Cell Nucleus Cell membrane Cell model Cells Chinese Hamster Clinical Communication Complex Data Development Drug Design Drug Targeting Electrophysiology (science)Enzyme Inhibitor Drugs Enzymes Extracellular Domain Fellowship Fibroblasts Gap Junctions Gene Expression Genes Genetic Transcription Goals Heart Hour Human Immunoglobulin Domain Immunoglobulins Implant In Vitro Incidence Intercalated disc Investigation Ion Channel Knowledge Label Ligands Lung Mannitol Maps Measures Mediating Mentors Messenger RNA Molecular Molecular Mechanisms of Action Monitor Names Neonatal Optics Pathology Pathway interactions Patients Peptide Hydrolases Peptides Perfusion Pharmaceutical Preparations Process Production Proteins Proteolysis Rattus Reporting Research Science Sodium Channel Sodium Chloride Structure Technical Expertise Techniques Testing Therapeutic Time Tissues Training Transmission Electron Microscopy Up-Regulation Ventricular Western Blotting Width Work beta-site APP cleaving enzyme 1 comparison control drug discovery drug production electric impedance established cell line experimental study extracellular gamma secretase gene product guinea pig model heart rhythm in vivo inhibitor insight knock-down mimetics nano novel novel strategies overexpression peptidomimetics prevent protective effect protein expression response sequential proteolysis skills stable cell line sudden cardiac death transcriptome sequencing treatment effect voltage

项目摘要

Project Summary Sudden cardiac death (SCD) caused by arrhythmia continues to be prevalent in the US and the world. Many drugs that aim to correct or prevent arrhythmias target ion channels, including the voltage-gated sodium channel (VGSC). The perinexus is a specialized nanodomain of the intercalated disc directly adjacent to gap junctions. It has been shown by our group that the VGSC subunit SCN1B/β1 is critical to perinexal adhesion. Loss of adhesion and widening of the perinexus leads to slowed conduction velocity and increased incidence of arrhythmia. As yet, no drug targeting β1 has yet been explored in preventing arrhythmias. My preliminary data indicates that >24 hour treatment with βadp1, a mimetic of the β1 extracellular domain, may result in upregulation of the VGSC β1 subunit, as well as increased levels of intercellular adhesion in β1-expressing 1610 cells, as measured by electric cell-substrate impedance sensing (ECIS). The proposed research aims to test the overarching hypothesis that targeting the adhesion function of the VGSC β1 subunit with βadp1 will result in increased abundance of β1 in the plasma membrane, increased β1-mediated adhesion, and a narrower perinexus over 24-48 hours of treatment. Furthermore, I will test the mechanistic hypothesis that βadp1 upregulates intramembrane proteolysis (RIP) of the β1 subunit, which was recently reported to alter gene transcription of many important electrogenic proteins, including VGSC subunits. In specific aim 1, an established cell line stably expressing the VGSC β1 subunit (Chinese hamster lung fibroblast 1610 cells) and isolated neonatal rat cardiomyocytes will be used to assay effects of βadp1 treatment in vitro over 48 hour time-courses in the presence and absence of inhibitors of RIP. Assessments will include ECIS assays of intercellular adhesion, and monitoring of protein and gene expression responses by Western blotting, quantitative IF, RNA-Seq and qPCR. In specific aim 2, effects of βadp1 treatment over 48 hours in vivo will be tested in guinea pigs, including studies of cardiac conduction using optical mapping, perinexal ultrastructure using transmission electron microscopy and monitoring of protein and gene expression responses using similar approaches to aim 1. The goal of my research is to gain further insight into βadp1 mode-of-action and its effects on heart structure and electrophysiological function, as well as to use this knowledge as a path to develop therapeutics for preventing fatal arrhythmias. In addition to completing the research aims, the purpose of this fellowship is to enable me to gain training in new techniques and areas of research, to undertake professional development, and also develop skills in mentoring and communicating science. The research will be performed under the guidance and expertise of Dr. Rob Gourdie. Training in optical mapping of electrical activation will be done with Dr. Steve Poelzing, training in RNA-Seq analyses will be under the expertise of Dr. Yassine Sassi, and training in ECIS will be done with Dr. Charles Keese.

项目概要由心律失常引起的心源性猝死（SCD）在美国和世界各地仍然很普遍。旨在纠正或预防心律失常的药物靶向离子通道，包括电压门控钠通道（VGSC）是与间隙直接相邻的闰盘的特殊纳米域。我们的研究小组已经证明，VGSC 亚基 SCN1B/β1 对于外周粘连至关重要。粘连丧失和会周增宽导致传导速度减慢和发病率增加迄今为止，尚未探索出针对β1的药物来预防心律失常。数据表明，用 βadp1（β1 细胞外结构域的模拟物）治疗 >24 小时可能会导致 VGSC β1 亚基上调，以及表达 β1 的细胞间粘附水平增加 1610 细胞，通过细胞-基底阻抗传感 (ECIS) 进行测量。测试总体假设，即用 βadp1 靶向 VGSC β1 亚基的粘附功能将导致质膜中 β1 丰度增加，β1 介导的粘附增加，以及治疗 24-48 小时后会阴连接变窄此外，我将检验以下机制假设。 βadp1 上调 β1 亚基的膜内蛋白水解 (RIP)，最近有报道称其会改变许多重要的生电蛋白的基因转录，包括 VGSC 亚基。在具体目标 1 中，建立稳定表达VGSC β1亚基的细胞系（中国仓鼠肺成纤维细胞1610细胞）分离的新生大鼠心肌细胞将用于检测 βadp1 体外治疗 48 小时的效果存在和不存在 RIP 抑制剂的时间进程评估将包括 ECIS 分析。细胞间粘附，并通过蛋白质印迹监测蛋白质和基因表达反应， IF、RNA-Seq 和 qPCR 在具体目标 2 中，将在体内定量 βadp1 处理超过 48 小时的效果。在豚鼠身上进行测试，包括使用光学测绘、周周超微结构研究心脏传导使用透射电子显微镜并使用类似的方法监测蛋白质和基因表达反应实现目标的方法 1. 我的研究目标是进一步了解 βadp1 的作用模式及其作用对心脏结构和电生理功能的影响，以及利用这些知识作为途径开发预防致命性心律失常的疗法除了完成研究目标外。这项研究金的目的是使我能够获得新技术和研究领域的培训，以便进行专业发展，并培养指导和传播科学的技能。在 Rob Gourdie 博士的指导和专业知识下进行电气光学测绘培训。激活将由 Steve Poelzing 博士完成，RNA-Seq 分析培训将在 Steve Poelzing 博士的专业知识下进行。 Yassine Sassi 和 ECIS 培训将由 Charles Keese 博士完成。