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 心律失常引起的心源性猝死（SCD）在美国和全世界仍然普遍存在。许多 旨在纠正或预防心律失常的药物靶向离子通道，包括电压门控钠离子通道。 通道（VGSC）。perinexus是直接邻近间隙的闰盘的专门纳米结构域 交叉点我们的研究小组已经表明，VGSC亚基SCN 1B/β1对会阴粘连至关重要。 粘连丧失和perinexus增宽导致传导速度减慢和发病率增加 心律不齐到目前为止，还没有针对β1的药物被探索用于预防心律失常。我的初步 数据表明，用β ADP 1（β1胞外结构域的模拟物）处理>24小时可能导致 VGSC β1亚单位的上调，以及β1表达的细胞间粘附水平的增加， 1610个细胞，如通过电细胞基质阻抗感测（ECIS）测量的。拟议的研究旨在 检验以下总体假设：用β adp 1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， 在24-48小时的治疗中，此外，我将检验机械假说， β adp 1上调β1亚基的膜内蛋白水解（RIP），这是最近报道的改变 许多重要的产电蛋白的基因转录，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）， 将使用分离的新生大鼠心肌细胞来测定β ADP 1在体外处理48小时以上的效果 在RIP抑制剂的存在和不存在下的时间过程。评估将包括ECIS检测， 细胞间粘附，并通过蛋白质印迹法监测蛋白质和基因表达反应， 定量IF、RNA-Seq和qPCR。在具体目标2中，将在体内观察β adp 1处理48小时以上的效果。 在豚鼠中进行测试，包括使用光学标测、perinxal超微结构 使用透射电子显微镜和使用类似的方法监测蛋白质和基因表达反应 接近目标1。本研究的目的是进一步了解β adp 1的作用模式及其在 对心脏结构和电生理功能的影响，以及使用这些知识作为一种途径， 开发出预防致命性心律失常的疗法。除了完成研究目标外， 这项奖学金的目的是使我能够获得新技术和研究领域的培训， 专业发展，并在指导和传播科学发展的技能。这项研究将 在Rob Gourdie博士的指导和专业知识下进行。电子光学测绘培训 激活将由Steve Poelzing博士完成，RNA-Seq分析的培训将由Dr. Yassine Sassi和ECIS的培训将与Charles Keese博士一起完成。