Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1.

Anna M D Végh,Anke M Smits,Lucía Cócera Ortega,Ruby Nobel,Dirk Geerts,Anke J Tijsen,Jianan Wang,Hanno L Tan,Gerard J J Boink,Mischa Klerk,Arie O Verkerk,Marie José T H Goumans,Kirsten Lodder,Vincent M Christoffels,Max Medina-Ramírez,Ronald Wilders,Harsha D Devalla

doi:10.3389/fphys.2020.588679

Abstract

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a “funny current” and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

Highlights

At present, electronic pacemaker therapy is the standard of care for patients with heart block and/or sinus node dysfunction
We investigated cellular gene delivery of Hcn2 and SkM1 in an effort toward engineering longterm biological pacemaker activity
Both nucleofection and lentiviral transduction were effective gene transfer tools to manipulate cardiomyocyte progenitor cells (CPCs), lentiviral transduction was superior with regard to survival of gene-modified cells in the context of in vivo transplantation

Summary

Introduction

Electronic pacemaker therapy is the standard of care for patients with heart block and/or sinus node dysfunction. Shortterm proof-of-concept studies have demonstrated that this is feasible either via direct gene transfer or via transplantation of stem cells With the latter approach, cells are either coaxed into a lineage of pacemaker cells, or are merely used as a vehicle for delivery of ion channel function, following ex vivo gene transfer. To explore potential alternative cell sources that can provide function over a much longer follow-up period we have previously explored the use of human cardiomyocyte progenitor cells (CPCs) These CPCs have previously been shown to reside in the mouse heart for at least 12 weeks after transplantation and are able to functionally couple to mouse cardiomyocytes, without manifesting unwanted proliferation or differentiation beyond the cardiac lineage (Smits et al, 2009a; Végh et al, 2019). In vitro studies have demonstrated that CPCs can function as an effective vehicle for HCN-based biological pacing (Végh et al, 2019)

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