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

Abstract

BackgroundOrgan 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.MethodsTo 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.ResultsMore 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.ConclusionThe 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.

Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct

“…Indeed, binding sites for this family of transcription factors are also present on the EF1a promoter. 34 The use of these two promoters in combination in lentiviral vectors has been reported previously; 28,35 however, the possibility of promoter interference was not examined in these studies. Therefore, while gene expression was seen from both promoters, as is the case in our study, promoter interference effects may have gone undetected.…”

Designing Lentiviral Gene Vectors

“…The polycations' transduction enhancer activity is highly cell specific. Alternative polycations, such as positively charged lipid dioctadecylamidoglycylspermine (DOGS), can be more effective than Polybrene for specific cell populations (Tolmachov, Ma et al 2006). Transduction efficiency can be further increased by a very unusual and poorly understood method called "spinfection".…”

Cardiac Tissue Engineering

“…There is evidence that increasing myoblast connexin 43 expression can improve functional integration Tolmachov et al, 2006). There are also multiple ongoing clinical trials with different attempts to use skeletal myoblasts to repair damaged heart muscle, with some positive effects reported (Dib et al, 2009;Haider et al, 2008;Siminiak et al, 2004).…”

“…Disadvantages of using MSCs and adipose-derived stem cells include the potential for a patient's disease state to affect their stem cell pool (Heeschen et al, 2004), and conflicting reports as to whether MSCs actually differentiate into cardiomyocytes (van der Bogt et al, 2009), or if any observed functional improvements are due to cell fusion and/or paracrine effects (Burdon et al, 2011). …”