Effect of dietary protein on the calpain/calpastatin proteolytic system in canine skeletal muscle
The cysteine proteinases [Mu-] and m-calpain along with their inhibitor, calpastatin, and possibly skeletal muscle specific p94, have been hypothesized to play a role in skeletal muscle protein degradation. Previous studies have indicated a nutritional influence on calpastatin. Our working hypothesis is that protein nutrition can influence regulation of the calpain system in muscle. Our objectives were to determine the effects of dietary protein on the expression of calpastatin and p94 in canine skeletal muscle. A biopsy was taken from the semitendinosis of 56 dogs prior to and after 12 weeks on their respective diets. This experimental design allowed us to examine change within individual dogs. Our study consisted of 8 diets with 7 dogs per diet. Diets 1-4 were 12% total protein and contained ratios of chicken to corn gluten protein of 100:0, 67:33, 33:67, and 0:100%, respectively. Diets 5-8 were 28% total protein with identical protein ratios to diets 1-4. We examined these differences qualitatively using SDS-PAGE and immunoblotting, and quantitatively with densitometric analyses. Western blots were completed using an anti-calpastatin antibody (MA3945, Affinity Bioreagents). p94 blots were examined with an anti-p94 antibody (NCL-CALP-12A2, Novocastra Labs). The majority of our calpastatin blots showed an expression of three distinct calpastatin bands, the uppermost appearing at approximately 110 kDa. Diet 5 resulted in an increase in the expression of the 110 kDa calpastatin band. A significant difference (P < 0.05) was obtained from comparison of the ratio of relative intensity in all three bands when comparing diet 5 (100:0) to diet 8 (0:100). Our results showed no treatment differences in detection of a 94 kDa protein examined using an anti-p94 antibody. Our calpastatin data suggest that dogs fed a diet containing a higher percentage of chicken protein may have a greater potential to regulate calpain-mediated degradation of muscle protein.