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

项目摘要 由心律失常引起的心脏性猝死(SCD)在美国和世界范围内仍然很普遍。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 频道(VGSC)。周围神经丛是直接与间隙相邻的间盘的一个特殊的纳米结构域。 交汇点。本课题组的研究表明，VGSC的SCN1B/β1亚基对附属膜粘连起关键作用。 周围神经丛的粘连丧失和增宽导致传导速度减慢和发生率增加 心律不齐的症状。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。我的初选 数据表明，用βadp1(一种β1胞外区的模拟物)24小时治疗可能会导致 VGSCβ1亚单位上调以及β1表达的细胞间黏附水平增加 1610个电池，通过电池-底物阻抗传感(ECIS)测量。这项拟议的研究旨在 测试主要假设，靶向VGSCβ1亚单位的黏附功能与βadp1将 结果增加了质膜中β-1的丰度，增加了β-1介导的黏附，并 治疗24-48小时后神经周围神经变窄。此外，我将检验机械假设，即 βadp1上调β1亚单位的膜内蛋白分解，最近报道该亚单位改变 许多重要的电生蛋白的基因转录，包括VGSC亚基。在具体目标1中，一个 稳定表达VGSCβ1亚单位的细胞系(中国仓鼠肺成纤维细胞1610细胞)和 分离的新生大鼠心肌细胞将被用来在体外测试βadp1处理超过48小时的效果。 RIP抑制剂存在和不存在时的时间进程。评估将包括ECIS分析 细胞间黏附，并用Western blotting监测蛋白质和基因表达的反应， 定量IF、RNA-Seq和qPCR。在特定目标2中，βadp1在体内治疗超过48小时的效果将是 在豚鼠身上进行测试，包括使用光学标测、附属器周围超微结构研究心脏传导 使用透射电子显微镜和监测蛋白质和基因表达的反应 通向目标的方法1.我研究的目的是进一步了解βadp1的行为模式及其 对心脏结构和电生理功能的影响，以及利用这些知识作为一种途径 开发预防致命性心律失常的疗法。除了完成研究目标外，目的是 这笔奖学金的目的是使我能够获得新技术和研究领域的培训，从事 专业发展，并培养指导和交流科学的技能。这项研究将 在罗布·古尔迪博士的指导和专业知识下进行。电学光学测绘培训 激活将与Steve Poelze博士一起完成，RNA-Seq分析方面的培训将在Dr. Yassine Sassi和ECIS的培训将与Charles Keese博士一起完成。

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.