Related Experiment Videos
Rat myoblast fusion requires exteriorized m-calpain activity
J J Brustis1, N Elamrani, D Balcerzak
1Institut des Sciences et Techniques des Aliments de Bordeaux, Laboratoire de Biochimie et Technologie des Aliments, Université Bordeaux I, Talence, France.
Abstract:
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.
Insights
Extracellular calcium-activated protease (m-calpain) promotes muscle cell fusion. Inhibiting m-calpain or using antibodies significantly reduces fusion, indicating its crucial extracellular role in myogenesis.
Area of Science:
- Muscle cell biology
- Enzymology
- Extracellular matrix interactions
Background:
- Previous studies showed fibronectin proteolysis by m-calpain during muscle differentiation.
- Recent findings indicated m-calpain exteriorization and association with extracellular matrix.
- The extracellular role of m-calpain in myogenesis remained unclear.
Purpose of the Study:
- To investigate the physiological function of extracellular m-calpain in myogenesis.
- To analyze the effect of purified m-calpain and inhibitors on myoblast fusion kinetics.
Main Methods:
- Cultured myoblasts were treated with purified m-calpain at varying concentrations.
- Myoblast fusion was assessed following treatment with m-calpain inhibitors (leupeptin, calpastatin) and m-calpain antibodies.
- Extracellular m-calpain was detected using immunofluorescence after antibody incubation.
Main Results:
- Low concentrations of added m-calpain (0.01 µg/ml) induced precocious fusion and increased fusion by 78%.
- High concentrations of m-calpain (10 µg/ml) did not affect viability but inhibited fusion.
- M-calpain inhibitors and antibodies significantly reduced myoblast fusion by 70-76%.
- Immunofluorescence confirmed the presence of exteriorized m-calpain on the myoblast cell surface.
Conclusions:
- Extracellular m-calpain plays a significant role in regulating myoblast fusion during myogenesis.
- The enzyme appears to function extracellularly, influencing cell-cell fusion dynamics.
- These findings highlight a novel extracellular role for m-calpain in muscle development.