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

• 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
• 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

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.

项目摘要 心律不齐引起的猝死（SCD）在美国和世界上仍然很普遍。许多 旨在纠正或预防心律不齐的靶离子通道的药物，包括电压门控钠 通道（VGSC）。 Perinexus是直接与间隙相邻的插入式椎间盘的专门纳米域 连接。我们的组已表明，VGSC亚基SCN1B/β1对于周氧粘合剂至关重要。 粘附的丧失和延伸性会导致传导速度缓慢和发病率增加 心律不齐。到目前为止，尚未在预防心律不齐时探索靶向β1的药物。我的初步 数据表明，> 24小时用βADP1（模仿细胞外域的模拟物）治疗可能会导致 VGSCβ1亚基的上调，以及表达β1的细胞间粘附水平增加 1610细胞，通过电细胞基底阻抗感应（ECI）测量。拟议的研究旨在 检验总体假设，即用βadp1靶向VGSCβ1亚基的粘附功能 导致质膜中β1的丰度增加，β1介导的粘合剂增加，A 在24-48小时的治疗中，狭窄的Perinexus。此外，我将测试机械假设 βadp1上调β1亚基的膜内蛋白水解（RIP），最近据报道会改变 包括VGSC亚基在内的许多重要的电源蛋白的基因转录。在特定的目标1中 建立的细胞系稳定地表达VGSCβ1亚基（中国仓鼠肺成纤维细胞1610细胞）和 孤立的新生大鼠心肌细胞将用于在48小时内在体外测定βadp1处理的效果 在存在和不存在RIP抑制剂的情况下，时间表。评估将包括ECIS评估 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， 定量IF，RNA-SEQ和QPCR。在特定的目标2中，βadp1治疗在48小时内的影响将是 在几内亚猪中测试，包括使用光学图，会周围超微结构的心脏传导研究 使用透射电子显微镜以及使用相似的蛋白质和基因表达反应监测 目标1的方法。我的研究的目的是进一步了解βadp1行动方式及其ITS 对心脏结构和电生理功能的影响，并将这些知识用作通往的途径 开发预防致命性心律失常的理论。除了完成研究目的之外，目的 这项奖学金是使我能够接受新技术和研究领域的培训，以进行 专业发展，还发展了心理和交流科学方面的技能。研究将 在Rob Gourdie博士的指导和专业知识下进行。电气光学映射的培训 史蒂夫·帕尔辛（Steve Poelzing）博士将进行激活，RNA-Seq分析的培训将在博士的专业知识下进行。 Yassine Sassi和ECIS的培训将与Charles Keese博士进行。