Glutathione depletion impairs myogenic differentiation of murine skeletal muscle C2C12 cells through sustained NF-kappaB activation.
Skeletal muscle differentation is a complex process regulated at multiple levels. This study addressed the effect of glutathione (GSH) depletion on the transition of murine skeletal muscle C2C12 myoblasts into myocytes induced by growth factor inactivation. Cellular GSH levels increased within 24 hours on myogenic stimulation of myoblasts due to enhanced GSH synthetic rate accounted for by stimulated glutamate-L-cysteine ligase (also known as gamma-glutamylcysteine synthetase) activity. In contrast, the synthesis rate of GSH using gamma-glutamylcysteine and glutamate as precursors, which reflects the activity of the GSH synthetase, did not change during differentiation. The stimulation of GSH stores preceded the myogenic differentiation of C2C12 myoblasts monitored by expression of muscle-specific genes, creatine kinase (CK), myosin heavy chain (MyHC), and MyoD. The pattern of DNA binding activity of NF-kappaB and AP-1 in differentiating cells was similar both displaying an activation peak at 24 hours after myogenic stimulation. Depletion of cellular GSH levels 24 hours after stimulation of differentiation abrogated myogenesis as reflected by lower CK activity, MyHC levels, MyoD expression, and myotubes formation, effects that were reversible on GSH replenishment by GSH ethyl ester (GHSEE). Moreover, GSH depletion led to sustained activation of NF-kappaB, while GSHEE prevented it. Furthermore, inhibition of NF-kappaB activation restored myogenesis despite GSH depletion. Thus, GSH contributes to the formation of myotubes from satellite myoblasts by ensuring inactivation of NF-kappaB, and hence maintaining optimal GSH levels may be beneficial in restoring muscle mass in chronic inflammatory disorders.
Servei de Pneumologia, Instituto Clinic de Pneumologia y Cirugía Torácica, Barcelona, Spain.
Esther
E
Ardite
Joan Albert
JA
Barbera
Josep
J
Roca
Jose C
JC
Fernández-Checa
D000818:Animals; D016923:Cell Death; D002454:Cell Differentiation; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000172:deficiency*; D051379:Mice; D032342:Muscle Cells / Q000166:cytology*; D024510:Muscle Development / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology*; D017570:MyoD Protein / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology*; D018995:Myosin Heavy Chains / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D018808:Transcription Factor AP-1
D017570:MyoD Protein; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D018808:Transcription Factor AP-1; D004247:DNA; D003402:Creatine Kinase; D018995:Myosin Heavy Chains; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione