Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activity.
Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N-acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity.
Zimowska
M
M
Department of Cytology, Faculty of Biology, Warsaw University, Poland.
Constantin
B
B
Papy-Garcia
D
D
Raymond
G
G
Cognard
C
C
Caruelle
J-P
JP
Moraczewski
J
J
Martelly
Isabelle
I
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000032:analysis / Q000378:metabolism*; D002454:Cell Differentiation; D002457:Cell Extracts / Q000494:pharmacology; D002459:Cell Fusion; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D006025:Glycosaminoglycans / Q000737:chemistry / Q000494:pharmacology*; D007150:Immunohistochemistry; D008297:Male; D018716:Molecular Mimicry; D018482:Muscle, Skeletal / Q000166:cytology*; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002457:Cell Extracts; D006025:Glycosaminoglycans; D002154:Calpain; D002118:Calcium