title: Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Section of Molecular and Cellular Medicine, Division of Biomedical Sciences, Faculty of Life Sciences, Imperial College London, London, UK. o.tolmachov@imperial.ac.uk
authors.forename: Oleg
authors.initials: O
authors.lastname: Tolmachov
authors.forename: Yu-Ling
authors.initials: YL
authors.lastname: Ma
authors.forename: Michael
authors.initials: M
authors.lastname: Themis
authors.forename: Pravina
authors.initials: P
authors.lastname: Patel
authors.forename: Hilmar
authors.initials: H
authors.lastname: Spohr
authors.forename: Kenneth T
authors.initials: KT
authors.lastname: Macleod
authors.forename: Nina D
authors.initials: ND
authors.lastname: Ullrich
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Kienast
authors.forename: Charles
authors.initials: C
authors.lastname: Coutelle
authors.forename: Nicholas S
authors.initials: NS
authors.lastname: Peters
mesh_terms: D000818:Animals; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D004576:Electromyography; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D008297:Male; D032448:Myoblasts, Skeletal / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012190:Retroviridae / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D018031:Connexin 43