Calpain proteinase mRNA and beta-agonist induced muscle growth
The mechanism by which β-agonists induce skeletal muscle hypertrophy is believed largely to be through a reduction in protein degradation. These growth promoters are also known to effect the activity of the calcium dependent proteinases (calpains) and their specific endogenous inhibitor calpastatin. The changes in activity appear to be toward a decrease in the calpain system's proteolytic potential. In this study attempts were made to determine whether the altered activity of the enzymes and inhibitor were brought about by induced changes in gene expression as reflected by altered levels of specific mRNAs. Various strategies were employed to generate oligonucleotide and cDNA probes to calpain I and II and calpastatin which would detect their respective mRNAs in L. dorsi total RNA samples originating from a bovine growth trial using the ß-agonist cimaterol. Semi-quantiative measurements of specific mRNAs using Northern blot analysis were related to enzyme and inhibitor activities. In addition ß-agonist-mediated effects on muscle RNA and expression of actin and myosin light chain 2 mRNAs were determined. Using a human calpain cDNA specific hybridization was detected for bovine calpain II mRNA which increased by 34% in the L. dorsi of cimaterol treated animals, similar to the increase in the enzyme activity, 28%. A novel bovine-specific calpastatin cDNA was generated by the polymerase chain reaction and sequence analysis allowed comparison to those already published for other species. Using this PCR cDNA as a probe multiple calpastatin mRNAs were detected in cattle L.dorsi, as had been observed in rabbit. The predominant mRNA increased by 160% in cimaterol treated steers compared to a 76% change in inhibitor activity. There changes were in contrast to the essentially unchanged response of muscle total RNA and actin and myosin light chain 2 specific mRNAs in treated animals. The implications for the calpain system in ß-agonist induced hypertrophy are discussed.