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Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro

BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart mi...

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Autores principales: Tolmachov, Oleg, Ma, Yu-Ling, Themis, Michael, Patel, Pravina, Spohr, Hilmar, MacLeod, Kenneth T, Ullrich, Nina D, Kienast, Yvonne, Coutelle, Charles, Peters, Nicholas S
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1513252/
https://www.ncbi.nlm.nih.gov/pubmed/16756651
http://dx.doi.org/10.1186/1471-2261-6-25
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author Tolmachov, Oleg
Ma, Yu-Ling
Themis, Michael
Patel, Pravina
Spohr, Hilmar
MacLeod, Kenneth T
Ullrich, Nina D
Kienast, Yvonne
Coutelle, Charles
Peters, Nicholas S
author_facet Tolmachov, Oleg
Ma, Yu-Ling
Themis, Michael
Patel, Pravina
Spohr, Hilmar
MacLeod, Kenneth T
Ullrich, Nina D
Kienast, Yvonne
Coutelle, Charles
Peters, Nicholas S
author_sort Tolmachov, Oleg
collection PubMed
description BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin(® )and the polycation transduction enhancer Transfectam(®). The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.
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spelling pubmed-15132522006-07-20 Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro Tolmachov, Oleg Ma, Yu-Ling Themis, Michael Patel, Pravina Spohr, Hilmar MacLeod, Kenneth T Ullrich, Nina D Kienast, Yvonne Coutelle, Charles Peters, Nicholas S BMC Cardiovasc Disord Research Article BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin(® )and the polycation transduction enhancer Transfectam(®). The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts. BioMed Central 2006-06-06 /pmc/articles/PMC1513252/ /pubmed/16756651 http://dx.doi.org/10.1186/1471-2261-6-25 Text en Copyright © 2006 Tolmachov et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Tolmachov, Oleg
Ma, Yu-Ling
Themis, Michael
Patel, Pravina
Spohr, Hilmar
MacLeod, Kenneth T
Ullrich, Nina D
Kienast, Yvonne
Coutelle, Charles
Peters, Nicholas S
Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro
title Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro
title_full Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro
title_fullStr Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro
title_full_unstemmed Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro
title_short Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro
title_sort overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1513252/
https://www.ncbi.nlm.nih.gov/pubmed/16756651
http://dx.doi.org/10.1186/1471-2261-6-25
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