Rat myoblast fusion requires exteriorized m-calpain activity.
Our previous studies demonstrated that fibronectin could be proteolyzed by m-calpain during muscle cell differentiation. Recent results indicated also that m-calpain could be exteriorized and more particularly associated to extracellular matrix components. To clarify one of the possible physiological functions of this proteinase during myogenesis, we have analyzed the incidence of added purified m-calpain and calpain inhibitors on the fusion kinetics of cultured myoblasts. Our results provided evidence that at low concentration (0.01 microgram/ml), added m-calpain induces precocious fusion and increases myoblast fusion by 78%. At high concentrations (10 micrograms/ml), the viability of the cells was not affected but the myoblasts were unable to fuse. Leupeptin and calpastatin--potent m-calpain inhibitors--added to the culture medium reduced myoblast fusion by 70%. On the other hand, the addition of monospecific m-calpain polyclonal antibodies to the culture medium induced a 76% decrease of myoblast fusion. In order to trap exteriorized m-calpain, myoblasts were incubated for 24 h with m-calpain antibodies. Following this treatment, nonpermeabilized myoblasts exposed to labeled secondary antibodies showed fluorescent spots scattered at the cell surface. These results strongly support that m-calpain which was involved in myoblast fusion was exteriorized and suggest therefore that this enzyme may play an important role extracellularly.
Brustis
J J
JJ
Institut des Sciences et Techniques des Aliments de Bordeaux, Laboratoire de Biochimie et Technologie des Aliments, Université Bordeaux I, Talence, France.
Elamrani
N
N
Balcerzak
D
D
Safwate
A
A
Soriano
M
M
Poussard
S
S
Cottin
P
P
Ducastaing
A
A
D000818:Animals; D001681:Biological Assay; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D002459:Cell Fusion / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D007074:Immunoglobulin G; D007150:Immunohistochemistry; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar
D002135:Calcium-Binding Proteins; D003470:Culture Media; D007074:Immunoglobulin G; D007976:Leupeptins; C033668:calpastatin; D002154:Calpain; C032854:leupeptin