Roles of mu-calpain in cultured L8 muscle cells: application of a skeletal muscle-specific gene expression system.
The goal of this work was to characterize the roles of mu-calpain in skeletal muscle protein degradation. Three approaches were developed to alter mu-calpain activity in rat myotubes. These included over-expression of antisense mu-calpain (mu-AS), dominant negative mu-calpain (mu-DN) and the antisense 30-kDa calpain subunit (30-AS). Constructs were expressed in rat L8 myotubes, and their effects on protein degradation and on concentrations of intact and/or degraded fodrin, desmin and tropomyosin were examined. An ecdysone-inducible expression system, in which we replaced a constitutively active CMV promoter with a skeletal muscle-specific alpha-actin promoter, was used to drive expression. Cell lines were evaluated by expression of the gene-of-interest following addition of ponasterone A (PA; ecdysone analog) to culture medium. Changes in calpain activity were assessed by evaluating fodrin degradation. 30-AS, which should alter both mu- and m-calpain activities, increased intact fodrin concentration. mu-DN and mu-AS reduced fodrin degradation products. mu-DN reduced total protein degradation by 7.9% (P<0.01) at 24 h and by 10.6% (P<0.01) at 48 h. mu-AS reduced total protein degradation by 6.4% at 24 h (P<0.05). 30-AS reduced total protein degradation by 13.4% (P<0.05) and 7.3% (P<0.05) following 24 and 48 h of PA administration, respectively. We assessed effects of mu-DN, mu-AS and 30-AS on concentrations of desmin and tropomyosin. Inhibition of calpains stabilized desmin, but had no effect on tropomyosin. These data indicate that fodrin and desmin are mu-calpain substrates and that mu-calpain accounts for a small proportion of total protein degradation in muscle cells. Tropomyosin is not degraded by calpain in muscle cells.
Xiao
Ying-yi
YY
Department of Animal Sciences, Oregon State University, Corvallis, OR 97331-6702, USA.
Wang
Mei-Chuan
MC
Purintrapiban
Juntipa
J
Forsberg
Neil E
NE
D000818:Animals; D002154:Calpain / Q000502:physiology*; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000201:enzymology* / Q000502:physiology*; D051381:Rats
D002154:Calpain; C449002:mu-calpain