Submitted to: FASEB Letters
Publication Type: Abstract Only
Publication Acceptance Date: July 11, 2010
Publication Date: N/A
Technical Abstract: Protein turnover ultimately requires proteolytic enzymes to degrade skeletal muscle proteins. The calpain system has been identified as a potential candidate due to its role in a variety of cellular processes such as cytoskeletal remodeling, myogenesis and signal transduction; and involvement in muscular dystrophies including Duchenne disease and muscle wasting associated with muscle disuse. The objective of this study was to evaluate proteolytic enzymes during growth and development of skeletal muscle in mu-calpain knockout mice (KO) compared to wild type C57/BL6 mice (WT). At 3, 5, 10, 20 and 30 weeks of age, WT and KO mice (n=6) were sacrificed and extensor digitorum longus (EDL), tibulus anterior (TA), quadriceps, gastrocnemius and soleus muscles were quantitatively dissected. Muscle weights and total RNA, DNA and protein content of muscles were unaffected by genotype. However, at 30 weeks of age muscle from KO mice had higher RNA:DNA (P = 0.016) and also higher protein:DNA (P = 0.0349), which provides an indirect measure of ribosomal abundance and myonuclear domain size respectively. This nucleic acid data indicates KO mice have an increased potential for protein synthesis and skeletal muscle hypertrophy at 30 weeks, although these differences were not reflected in muscle weights. Genotype significantly affected m-calpain expression (P < 0.0001), measured by zymography, with an age*genotype interaction at 3, 5, and 10 weeks (P = 0.012, P < 0.0001, P = 0.0078, respectively). No differences in m-calpain expression were observed between WT and KO mice at either 20 or 30 weeks. Western blotting analysis for m-calpain revealed a similar pattern of expression to that of the zymographs, with a significant increase in m-calpain expression in KO mice in comparison to WT (P = 0.0033). Protein expression of the calpain specific inhibitor, calpastatin, was significantly higher in KO mice compared to WT mice (P = 0.0039). An age*genotype interaction was observed at 5 weeks (P = 0.0466), which coincides with the largest difference in m-calpain expression between genotypes. This could indicate that more calpastatin is required to control the elevated levels of m-calpain in KO mice. The activity of another proteolytic enzyme family, caspase 3/7, was significantly higher in KO mice, compared to WT mice (P = 0.0492). Differences in caspase 3/7 activity between KO and WT mice were not observed at either 3 or 30 weeks of age. The increased expression and activity of other proteases, including m-calpain and caspase 3/7, during the growth and development stages of the KO mice suggest that they are up-regulated to compensate for the lack of mu-calpain expression. This provides an explanation for the lack of differences in muscle growth between genotypes.