AUTHOR=Wulkan Fanny , Romagnuolo Rocco , Qiang Beiping , Valdman Sadikov Tamilla , Kim Kyung-Phil , Quesnel Elya , Jiang Wenlei , Andharia Naaz , Weyers Jill J. , Ghugre Nilesh R. , Ozcan Bilgehan , Alibhai Faisal J. , Laflamme Michael A. 

TITLE=Stem cell-derived cardiomyocytes expressing a dominant negative pacemaker HCN4 channel do not reduce the risk of graft-related arrhythmias

JOURNAL=Frontiers in Cardiovascular Medicine

VOLUME=11

YEAR=2024

URL=https://www.frontiersin.org/journals/cardiovascular-medicine/articles/10.3389/fcvm.2024.1374881

DOI=10.3389/fcvm.2024.1374881

ISSN=2297-055X

ABSTRACT=<sec><title>Background</title><p>Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI), but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models, representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore, we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation.</p></sec><sec><title>Methods</title><p>We used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs, and their electrophysiological behavior was evaluated <italic>in vitro</italic> by patch-clamp recordings and optical mapping. Next, we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry.</p></sec><sec><title>Results</title><p><italic>In vitro</italic> dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (I<italic><sub>f</sub></italic>) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs, all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However, in contrast to our hypothesis, both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT).</p></sec><sec><title>Conclusions</title><p>While genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs <italic>in vitro</italic>, this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model, implying more complex mechanism(s) are operational <italic>in vivo</italic>.</p></sec>