Changes in calpain and calpastatin mRNA induced by β‐adrenergic stimulation of bovine skeletal muscle

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Abstract
Administration of beta-adrenergic agonists to mammals can produce skeletal muscle hypertrophy in some species and muscle types. The growth-promoting effect appears to be due to suppression of protein breakdown rather than stimulation of synthesis, although evidence from turnover studies is equivocal. In ovine muscle, changes in the activity of the calcium dependent neutral proteinases (calpains I and II) and their specific inhibitor (calpastatin) accompany beta-agonist-induced hypertrophy. These observations suggest that the calpain system is involved in myofibrillar protein turnover in some way. Alternatively, the relationship with hypertrophy may be indirect, since the calpains also interact with hormone and growth-factor receptors, protein kinase C and transcription factors, in addition to a range of membrane, cytoskeletal and nuclear proteins. In the present study, attempts have been made to determine if the beta-agonist-induced effects on the calpain system are associated with corresponding changes in specific mRNA. The activity of both calpain isoforms and calpastatin was measured in bovine longissimus dorsi samples from trials in which test animals were treated with the beta agonist cimaterol. Total RNA was extracted from the muscle samples. A cDNA probe for calpastatin mRNA was generated from bovine RNA by the polymerase chain reaction. This cDNA and a human calpain-II large-subunit cDNA were used to detect specific mRNA by Northern-blot analysis. beta-agonist treatment of Friesian steers caused significant longissimus dorsi hypertrophy. Increases in muscle mass (+37%, P less than 0.005), calpain-II specific activity (+27%, P less than 0.05) and calpastatin-specific activity (+76%, P less than 0.05) were found in treated animals. Total RNA was unchanged, but there was a 96% overall increase in calpastatin mRNA and a 30% increase in calpain-II large-subunit mRNA in muscle from treated animals. The mRNA changes are similar in direction and degree to the activity changes. Both calpain-II large subunit and inhibitor expression may therefore be stimulated by agonist action at the level of transcription or mRNA stabilisation. Multiple calpastatin mRNA species were detected in steers, as reported for other species. Differential changes in these messages, induced by the beta agonist, suggest that expression or stability of alternative mRNA species may be a factor in calpastatin regulation.
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