Overexpression of connexin 43 in skeletal myoblasts: Relevance to cell transplantation to the heart.

OBJECTIVE Skeletal myoblast transplantation is a promising strategy for treating end-stage heart failure. One potential problem in the development of functional, synchronously contracting grafts is the degree of intercellular communication between grafted myoblasts and host cardiomyocytes. Thus it is expected that enhancement of intercellular gap junction formation would result in improved efficiency of skeletal myoblast transplantation. In this study we investigated whether myoblasts overexpressing connexin 43, a major cardiac gap junction protein, would enhance this intercellular communication. METHODS AND RESULTS L6 rat skeletal myoblast cell lines overexpressing connexin 43 were generated by means of gene transfection and clonal selection. Connexin 43 overexpression of these myoblasts, which continued both in undifferentiated and differentiated states (up to 17-fold greater protein level in comparison with control-transfected myoblasts, as measured with Western blotting), was observed on cell surfaces where gap junctions should exist. Both dye microinjection and scrape loading with fluorescent dyes showed enhancement in intercellular dye transfer between connexin 43-transfected myoblasts compared with that found in control-transfected cells. Morphologically, these myoblasts fused and differentiated into multinucleated myotubes more rapidly, demonstrating a higher level of cellular creatine kinase activity as a marker of myogenic differentiation throughout the culture period compared with that of control-transfected myoblasts. CONCLUSIONS We have generated connexin 43-overexpressing skeletal myoblast cell lines that resulted in improved formation of functional intercellular gap junctions, which could be relevant to synchronous contraction of grafted myoblasts in the heart. In addition, these cells demonstrated more rapid differentiation, which would also be advantageous in a graft for transplantation to the heart.

[1]  M. Yacoub,et al.  Development of a Novel Method for Cell Transplantation Through the Coronary Artery , 2000, Circulation.

[2]  C. Murry,et al.  Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair. , 2000 .

[3]  S. Hunt.  Current status of cardiac transplantation. , 1998, JAMA.

[4]  Doris A Taylor,et al.  Regenerating functional myocardium: Improved performance after skeletal myoblast transplantation , 1998, Nature Medicine.

[5]  H. Jongsma,et al.  Characterization of gap junction channels in adult rabbit atrial and ventricular myocardium. , 1997, Circulation research.

[6]  P. Farnham,et al.  Expression patterns of the E2F family of transcription factors during murine epithelial development. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[7]  R. Kao,et al.  Cellular Cardiomyoplasty: Myocardial Repair with Cell Implantation , 1997 .

[8]  T. Tsukamoto,et al.  Gap junction genes Cx26 and Cx43 individually suppress the cancer phenotype of human mammary carcinoma cells and restore differentiation potential. , 1996, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[9]  C. Naus,et al.  Role of connexin genes in growth control. , 1996, Carcinogenesis.

[10]  S. Okazaki,et al.  Effects of calcitonin gene‐related peptide and interleukin 6 on myoblast differentiation , 1996, Cell proliferation.

[11]  D. Paul,et al.  Connexins, connexons, and intercellular communication. , 1996, Annual review of biochemistry.

[12]  T. Steinberg,et al.  Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43 , 1995, The Journal of cell biology.

[13]  A. Lau,et al.  A connexin 43 antisense vector reduces the ability of normal cells to inhibit the foci formation of transformed cells , 1994, Molecular carcinogenesis.

[14]  M. Nicolet,et al.  Is intercellular communication via gap junctions required for myoblast fusion? , 1994, Cell adhesion and communication.

[15]  C. Naus,et al.  Expression of gap junctions in cultured rat L6 cells during myogenesis. , 1993, Developmental biology.

[16]  C. Naus,et al.  Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[17]  E. Bernt,et al.  Creatine kinase in serum: 1. Determination of optimum reaction conditions. , 1976, Clinical chemistry.

[18]  D. Yaffe,et al.  The in vitro cultivation and differentiation capacities of myogenic cell lines. , 1970, Developmental biology.