Our objectives for this manuscript are to review the mechanisms of muscle growth, the biological basis of meat tenderness, and the relationship between these two processes. Muscle growth is determined by hyperplasia and hypertrophy. Muscle cell size is determined by the balance between the amount of muscle protein synthesized and the amount of muscle protein degraded. Current evidence suggests that the calpain proteolytic system is a major regulator of muscle protein degradation. Sarcomere length, connective tissue content, and proteolysis of myofibrils and associated proteins account for most, if not all, of the explainable variation in tenderness of meat after postmortem storage. The relative contribution of each of the above components is muscle dependent. The calpain proteolytic system is a key regulator of postmortem proteolysis. While changes in muscle protein degradation affect meat tenderization/tenderness, changes in muscle protein synthesis are not expected to affect meat tenderization/tenderness. Published by Elsevier Science Ltd.

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Casein zymography was used to determine the effect of postmortem storage on the proteolytic activity of mu-calpain and m-calpain in lamb longissimus. Casein zymography assays were conducted on crude muscle extracts (only one centrifugation). Six market weight crossbred lambs were slaughtered and a portion of the longissimus lumborum was removed at death (within 15 min of exsanguination) and after 3, 6, 9, 12, 24, 72, and 360 h postmortem. Muscle samples were snap-frozen in liquid nitrogen and stored at -70 degrees C. Soluble muscle proteins were extracted from muscle samples and analyzed by in-gel casein assay to measure calpain proteolytic activity. There was a gradual decline in mu-calpain activity (P < 0.05) such that after 24 and 72 h postmortem, mu-calpain had lost 42 and 95% of its activity, respectively. After 360 h postmortem, no mu-calpain activity could be detected (under the conditions used in this study). Autolysis of mu-calpain could be detected as early as 3 h postmortem. It was demonstrated that the detectable level of mu-calpain activity is a function of the amount of muscle protein electrophoresed. Hence, the activity data reported are in relative terms, rather than absolute values. Furthermore, it was demonstrated that the activity data also are a function of the assay methods used. Different methods have different lower detection limits. Of the three assays examined, 14C-labeled casein was the most sensitive, then the in-gel casein assay, and the least-sensitive method was the standard casein assay. Unlike mu-calpain, postmortem storage had no effect on m-calpain (P > 0.05). When the calcium concentration of a muscle extract was increased to the level that induces m-calpain autolysis, m-calpain was autolyzed and its autolysis was readily detected by the in-gel casein assay. Collectively, these results demonstrate that calcium concentration in postmortem muscle is only high enough to activate mu-calpain. These results support the widely believed conclusion that mu-calpain-mediated proteolysis of key myofibrillar and cytoskeletal proteins is responsible for postmortem tenderization. Hence, understanding the regulation of mu-calpain in postmortem muscle should be the focus of future studies.

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The objective of this experiment was to determine age-related changes in collagen concentration, sarcomere length, calpain (μ- and m-) and calpastatin activities, postmortem proteolysis and Warner-Bratzler shear force (WBSF) in ovine longissimus thoracis et lumborum. Rambouillet lambs were slaughtered at 2, 4, 6, 8 and 10 months of age and samples of longissimus were collected at 0, 2 and 10 days postmortem. Collagen concentration and sarcomere lengths were determined from the cores used for WBSF measurements and reflected changes in the background toughness. Longissimus collagen concentration did not change (P>0.05) due to lamb age. Sarcomere lengths also showed age-related changes, increasing (P<0.05) from 1.35 μm at 6 months to 1.48 and 1.55 μm at 8 and 10 months, respectively. The extent of calpain mediated proteolysis determines the improvement in meat tenderness with postmortem storage. The most notable change in the calpain proteolytic system was the decline (P<0.05) in calpastatin activity from 4.18 to 1.91 U/g muscle between 2 and 10 months. The activity of μ-calpain showed a 16% increase (P<0.05) from 4 to 6 months, before it dropped again at 8 and 10 months. There was a gradual decline (P<0.05) in m-calpain activity with age, and by 10 months m-calpain activity had reduced to 80% of 2 months levels. The ratio of μ-calpain to calpastatin activities increased (P<0.05) from 2 to 6 months (from 0.31 to 0.56) with no further changes (P>0.05) at 8 or 10 months. There were no age-related changes (P>0.05) in desmin degradation at day 2, however, examination of day 10 samples showed increased (P<0.05) degradation from 2 to 6 months. Thus, the changes observed in the ratio of μ-calpain to calpastatin activities are reflected in the extent of postmortem proteolysis. Meat tenderness was measured using WBSF at 2 and 10 days postmortem. Because little proteolysis had taken place at 2 days postmortem, the decline in day 2 WBSF from 6 to 8 months could be explained by changes in sarcomere length. However, at 10 days postmortem, where WBSF was shown to decrease from 2 to 8 months, the improvement in tenderness could be explained by the amount of postmortem proteolysis. The data presented in this paper show evidence that sarcomere length is the main determinant of background toughness in ovine longissimus, and that postmortem proteolysis, resulting from μ-calpain activity regulated by calpastatin, is the main determinant of ovine longissimus tenderization during aging. Thus, lamb longissimus tenderness after refrigerated storage is determined by postmortem proteolysis and its interaction with sarcomere length.

Postmortem changes in osmotic pressure; ionic strength; pH; temperature; mu- and m-calpain; calpastatin; desmin degradation; and myofibril fragmentation index (MFI) were determined in ovine longissimus muscle. Our objectives were to characterize changes in these variables and to identify postmortem time points at which significant proteolysis and tenderization (as measured by change in MFI) could be detected. Seven crossbred (Dorset x Romanov) lambs were slaughtered, and samples of the longissimus muscle were removed at 0, 3, 6, 9, 12, 24, 72, and 360 h postmortem. Osmotic pressure increased (P < 0.05) from 379 to 528 mOsm during the postmortem storage period, with two-thirds of the increase occurring within the first 24 h. By measuring conductivity, we showed that ionic strength increased (P < 0.05) from 8.13 to 9.78 mS/cm during the storage period, which is equivalent to 79 and 97 mM NaCl solutions, respectively. In accordance with pH and temperature, conductivity reached ultimate levels at 24 h postmortem. Within 9 h postmortem, mu-calpain activity had decreased (P < 0.05) from at-death values and continued to decrease until 72 h, at which time it was undetectable. It was still possible to detect the 76-kDa isoforms (a product of the autolysis of the 80-kDa subunit of mu-calpain) immunologically, which implies that the loss of activity was not caused by extensive autolysis. In contrast, m-calpain activity remained constant throughout the aging period, whereas calpastatin activity was stable until 24 h postmortem, after which it gradually decreased. Autolysis products of mu-calpain were detected at 3 h postmortem, indicating that mu-calpain was activated some time between 0 and 3 h postmortem. Moreover, the effect of mu-calpain activity on myofibrillar substrates was first observed at 9 h postmortem, when a 23% loss of native desmin was detected. This degradation translated into an increase in MFI at 12 h. Collectively, these results imply that mu-calpain is active in postmortem muscle in the presence of calpastatin, and that effects of mu-calpain activity as determined by increased MFI are detectable during the first 12 h postmortem.

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