Brianna Ferguson
Research Mentor(s): Adam Helms
Mentor Department: Internal Medicine / Cardiovascular Medicine
Authors: Brianna Ferguson, Karen Jin, Sabrina Friedline, Yao-Chang Tsan, Eric Smith, Adam Helms
Session: Session 1 (9:00am – 9:50am)
Presentation Type: Poster 11
Abstract
Background: Desmoplakin (DSP) cardiomyopathy is a heart disease characterized by arrhythmias and excessive cardiac fibrosis and is caused by truncating variants in desmoplakin (DSPtv) which result in decreased DSP protein at myocardial cell junctions. Cardiomyocyte muscle bundles harboring DSPtv show increased susceptibility to injury with mechanical stress. We hypothesized that transcriptional repression of DSP can recapitulate stress induced injury seen in DSPtv muscle bundles. Methods: Induced pluripotent stem cells with green fluorescent protein tagged DSP were differentiated into cardiomyocytes (iPSC-CMs), assembled into 2D microbundles and exposed to contractile stress using endothelin-1 (ET-1). Transcriptional repression was performed using lentiviral expression of deactivated Cas9 fused with KRAB and a guide RNA targeting the promoter of DSP (CRISPRi). Relative expression levels were quantified using immunofluorescence. Contractility and intercellular fractures were measured from live cell time lapse images of 2D microbundles. Results: CRISPRi of DSP caused down-regulation of DSP protein colocalized to n-cadherin at cell borders (XXX, p=XXX). Contractility of 2D microbundles was similar between DSP +/+ and DSP CRISPRi cardiomyocytes at baseline (contraction velocity, XXXvsXXX, p=ns) and showed a similar contractile response to ET-1 (XX% vs XX% increase in contractile velocity). DSP CRISPRi microbundles demonstrated a high degree of intercellular tears and microbundle pattern loss compared to control cells (XX% vs XX%, p=XX). Conclusion: We show that CRISPR-KRAB based transcriptional repression can reduce the amount of DSP expression in IPSC cardiomyocytes. This results in mechanical injury when cardiac muscle bundles are exposed to contractile stress. These results suggest an important role of DSP in support of cardiomyocyte structural integrity.
