Roles of μ-calpain in cultured L8 muscle cells: application of a skeletal muscle-specific gene expression system

Abstract

The goal of this work was to characterize the roles of μ-calpain in skeletal muscle protein degradation. Three approaches were developed to alter μ-calpain activity in rat myotubes. These included over-expression of antisense μ-calpain (μ-AS), dominant negative μ-calpain (μ-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 α-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 μ- and m-calpain activities, increased intact fodrin concentration. μ-DN and μ-AS reduced fodrin degradation products. μ-DN reduced total protein degradation by 7.9% ( P<0.01) at 24 h and by 10.6% ( P<0.01) at 48 h. μ-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 μ-DN, μ-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 μ-calpain substrates and that μ-calpain accounts for a small proportion of total protein degradation in muscle cells. Tropomyosin is not degraded by calpain in muscle cells.