Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures.
Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.
Gorbe
Aniko
A
Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary.
Krenacs
Tibor
T
Cook
Jeremy E
JE
Becker
David L
DL
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015726:Giant Cells / Q000378:metabolism / Q000502:physiology; D007365:Intercellular Junctions / Q000502:physiology; D024510:Muscle Development; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D032446:Myoblasts / Q000378:metabolism / Q000502:physiology*; D032448:Myoblasts, Skeletal / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D018031:Connexin 43