Saturday, April 20, 2024
4/20/2024
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/ß1 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 ß1 has yet been explored in preventing arrhythmias. My preliminary data indicates that >24 hour treatment with ßadp1, a mimetic of the ß1 extracellular domain, may result in upregulation of the VGSC ß1 subunit, as well as increased levels of intercellular adhesion in ß1-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 ß1 subunit with ßadp1 will result in increased abundance of ß1 in the plasma membrane, increased ß1-mediated adhesion, and a narrower perinexus over 24-48 hours of treatment. Furthermore, I will test the mechanistic hypothesis that ßadp1 upregulates intramembrane proteolysis (RIP) of the ß1 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 ß1 subunit (Chinese hamster lung fibroblast 1610 cells) and isolated neonatal rat cardiomyocytes will be used to assay effects of ßadp1 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 ßadp1 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 ßadp1 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.
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