Connexin43 transduction of skeletal myoblasts improves their electrical coupling with cardiac myocytes

Journal article

Background: Organ transplantation is presently often the only avai lable option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from auto logous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal mu scle cells in the heart milieu is, howeve r, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursu it of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by ov erexpression of the main gap-junctional protein connexin 43 and to study electric al coupling of connexin 43 overexpr essing 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-EG FP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myob lasts in optimised conditions involving a concomitant use of the retrovirus immobilisi ng protein RetroNectin ® and the polycation transdu ction enhancer Transfectam ® . The EGFP- positive transduced ce lls were then enrich ed by flow cytometry. Results: More than four-fold overexpression of connexin 43 in the transduced skel etal myoblasts, compared with non- transduced cells, was shown by Western blotting. Function ality of the overexpressed co nnexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-j unctional intercellular transf er in connexin 43 transduced myoblasts compared with transfer in no n-transduced myoblasts. Rat cardiac my ocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transd uced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in th e co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in th e 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 activati on rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupl ing due to overexpression of connexin 43 in skeletal myoblasts. This study suggests th at retroviral connexin 43 tran sduction can be employed to augment engineering of the electrocompetent cardia c grafts from patients' own skeletal myoblasts.

Authors: Ma, Y; Tolmachov, O; Themis, M; Coutelle, C; Peters, N

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: BioMed Central
• Journal: BMC Cardiovascular Disorders
• Volume: 6
• Article number: 25
• Publication date: 2006-01-01
• DOI: 10.1186/1471-2261-6-25
• ISSN: 1471-2261