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Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1

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 “fu...

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Autores principales: Végh, Anna M. D., Verkerk, Arie O., Cócera Ortega, Lucía, Wang, Jianan, Geerts, Dirk, Klerk, Mischa, Lodder, Kirsten, Nobel, Ruby, Tijsen, Anke J., Devalla, Harsha D., Christoffels, Vincent M., Medina-Ramírez, Max, Smits, Anke M., Tan, Hanno L., Wilders, Ronald, Goumans, Marie José T. H., Boink, Gerard J. J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815531/
https://www.ncbi.nlm.nih.gov/pubmed/33488393
http://dx.doi.org/10.3389/fphys.2020.588679
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author Végh, Anna M. D.
Verkerk, Arie O.
Cócera Ortega, Lucía
Wang, Jianan
Geerts, Dirk
Klerk, Mischa
Lodder, Kirsten
Nobel, Ruby
Tijsen, Anke J.
Devalla, Harsha D.
Christoffels, Vincent M.
Medina-Ramírez, Max
Smits, Anke M.
Tan, Hanno L.
Wilders, Ronald
Goumans, Marie José T. H.
Boink, Gerard J. J.
author_facet Végh, Anna M. D.
Verkerk, Arie O.
Cócera Ortega, Lucía
Wang, Jianan
Geerts, Dirk
Klerk, Mischa
Lodder, Kirsten
Nobel, Ruby
Tijsen, Anke J.
Devalla, Harsha D.
Christoffels, Vincent M.
Medina-Ramírez, Max
Smits, Anke M.
Tan, Hanno L.
Wilders, Ronald
Goumans, Marie José T. H.
Boink, Gerard J. J.
author_sort Végh, Anna M. D.
collection PubMed
description 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.
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spelling pubmed-78155312021-01-21 Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1 Végh, Anna M. D. Verkerk, Arie O. Cócera Ortega, Lucía Wang, Jianan Geerts, Dirk Klerk, Mischa Lodder, Kirsten Nobel, Ruby Tijsen, Anke J. Devalla, Harsha D. Christoffels, Vincent M. Medina-Ramírez, Max Smits, Anke M. Tan, Hanno L. Wilders, Ronald Goumans, Marie José T. H. Boink, Gerard J. J. Front Physiol Physiology 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. Frontiers Media S.A. 2021-01-06 /pmc/articles/PMC7815531/ /pubmed/33488393 http://dx.doi.org/10.3389/fphys.2020.588679 Text en Copyright © 2021 Végh, Verkerk, Cócera Ortega, Wang, Geerts, Klerk, Lodder, Nobel, Tijsen, Devalla, Christoffels, Medina-Ramírez, Smits, Tan, Wilders, Goumans and Boink. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Physiology
Végh, Anna M. D.
Verkerk, Arie O.
Cócera Ortega, Lucía
Wang, Jianan
Geerts, Dirk
Klerk, Mischa
Lodder, Kirsten
Nobel, Ruby
Tijsen, Anke J.
Devalla, Harsha D.
Christoffels, Vincent M.
Medina-Ramírez, Max
Smits, Anke M.
Tan, Hanno L.
Wilders, Ronald
Goumans, Marie José T. H.
Boink, Gerard J. J.
Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1
title Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1
title_full Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1
title_fullStr Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1
title_full_unstemmed Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1
title_short Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1
title_sort toward biological pacing by cellular delivery of hcn2/skm1
topic Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815531/
https://www.ncbi.nlm.nih.gov/pubmed/33488393
http://dx.doi.org/10.3389/fphys.2020.588679
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