Effect of postmortem storage on activity of mu- and m-calpain in five bovine muscles

An in situ system involving incubation of 60- to 80-g pieces of muscle at 4 degrees C under different conditions was used to determine the effects of time of postmortem storage, of pH, and of temperature on activities of mu- and m-calpain activity in bovine skeletal muscle. Casein zymograms were used to allow measurement of calpain activity with a minimum of sample preparation and to ensure that the calpains were not exposed to ionic strengths of 100 or greater before assay of their activities. In 4 of the 5 muscles (longissimus dorsi, lumbar; longissimus dorsi, thoracic; psoas major; semimembranosus; and triceps brachii) studied, mu-calpain activity decreased nearly to zero within 48 h postmortem. Activity of m-calpain also decreased in the in situ system used but at a much slower rate. Activities of both mu- and m-calpain decreased more slowly in the triceps brachii muscle than in the other 4 muscles during postmortem storage. Although previous studies have indicated that mu-calpain but not m-calpain is proteolytically active at pH 5.8, these studies have used calpains obtained from muscle at death. Both mu- and m-calpain are proteolytically inactive if their activities are measured at pH 5.8 and after incubating the muscle pieces for 24 h at pH 5.8. Western analysis suggested that neither the large 80-kDa subunit nor the small 28-kDa subunit of m-calpain was autolyzed during postmortem storage of the muscle pieces. As has been reported previously, the 80-kDa subunit of mu-calpain was autolyzed to 78- and then to a 76-kDa polypeptide after 7 d postmortem, but the 28-kDa small subunit was not autolyzed; hence, the autolyzed mu-calpain molecule in postmortem muscle is a 76-/28-kDa molecule and not a 76-/18-kDa molecule as previously assumed. Because both subunits were present in the postmortem calpains, loss of mu-calpain activity during postmortem storage is not due to dissociation of the 2 subunits and inactivation. Although previous studies have shown that the 76-/18-kDa mu-calpain molecule is completely active proteolytically, it is possible that the 76-/28-kDa mu-calpain molecule in postmortem muscle is proteolytically inactive and that this accounts for the loss of mu-calpain activity during postmortem storage. Because neither mu- nor m-calpain is proteolytically active at pH 5.8 after being incubated at pH 5.8 for 24 h, other proteolytic systems such as the caspases may contribute to postmortem proteolysis in addition to the calpains.     

The calpain system in three muscles of normal and callipyge sheep

Activities of mu- and m-calpain and of calpastatin were measured at four different times during postmortem storage (0, 1, 3, and 10 d) in three muscles from either callipyge or noncallipyge (normal) sheep. The weights of two muscles, the biceps femoris and the longissimus, are greater in the callipyge phenotype, whereas the weight of the infraspinatus is not affected. The activity of m-calpain was greater (P < 0.05) in the biceps femoris and longissimus from callipyge than in those from normal sheep, but it was the same in the infraspinatus in the two phenotypes. The extractable activity of m-calpain did not change (biceps femoris and infraspinatus) or decreased slightly (longissimus) during postmortem storage. Extractable activity of mu-calpain decreased to zero or nearly zero after 10 d postmortem in all muscles from both groups of sheep. The rate of decrease in mu-calpain activity was the same in muscles from the callipyge and normal sheep. At all time points during postmortem storage, calpastatin activity was greater (P < 0.05) in the biceps femoris and longissimus from the callipyge than from the normal sheep, but it was the same in the infraspinatus from callipyge and normal sheep. Calpastatin activity decreased (P < 0.05) in all three muscles from both phenotypes during postmortem storage; the rate of this decrease in the callipyge biceps femoris and longissimus and in the infraspinatus from both the callipyge and normal sheep was slow, especially after the first 24 h postmortem, whereas calpastatin activity in the biceps femoris and longissimus from the normal sheep decreased rapidly. During postmortem storage, the 125-kDa calpastatin polypeptide was degraded, but the 80-kDa subunit of mu-calpain was cleaved only to 76- and 78-kDa polypeptides even though extractable mu-calpain activity declined nearly to zero. Approximately 50 to 60% of total mu-calpain became associated with the nonextractable pellet after 1 d postmortem. The myofibril fragmentation index for the biceps femoris and longissimus from normal sheep increased significantly during postmortem storage. The fragmentation index for the infraspinatus from the callipyge and normal sheep increased to an intermediate extent, whereas the index for the biceps femoris and longissimus from the callipyge did not change during 10-d postmortem storage. The results suggest that postmortem tenderization is related to the rate of calpastatin degradation in postmortem muscle and that calpastatin inhibition of the calpains in postmortem muscle is modulated in some as yet unknown manner.     

Isolation and characterization of mu-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps

Evidence has indicated that mu-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and mu-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of mu-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25 degrees C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca(2+) requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive mu-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active mu-calpain even though very little mu-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this mu-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive mu-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active mu-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of mu-calpain activity remains unknown.     

Effect of postmortem storage on mu-calpain and m-calpain in ovine skeletal muscle.

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.     

Effect of calpastatin on degradation of myofibrillar proteins by mu-calpain under postmortem conditions.

To improve our understanding of the regulation of mu-calpain activity in situ during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system closely mimicking postmortem conditions was studied. Increasing the amount of calpastatin in the incubations limited both the rate and extent of proteolysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpastatin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolysis completely. Western blot analysis revealed that proteolysis of myofibrillar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of incubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-calpain activity in postmortem muscle is discussed.     

Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles

The objective of this study was to determine whether differences in pork tenderness and water-holding capacity could be explained by factors influencing calpain activity and proteolysis. Halothane-negative (HAL-1843 normal) Duroc pigs (n = 16) were slaughtered, and temperature and pH of the longissimus dorsi (LD), semimembranosus (SM), and psoas major (PM) were measured at 30 and 45 min and 1, 6, 12, and 24 h postmortem. Calpastatin activity; mu-calpain activity; and autolysis and proteolysis of titin, nebulin, desmin, and troponin-T were determined on muscle samples from the LD, SM, and PM at early times postmortem. Myofibrils from each muscle were purified to assess myofibril-bound (mu-calpain. Percentage drip loss was determined, and Warner-Bratzler shear (WBS) force was analyzed. Myosin heavy-chain (MHC) isoforms were examined using SDS-PAGE. The pH of PM was lower (P < 0.01) than the pH of LD and SM at 30 and 45 min and 1 h postmortem. The PM had a higher (P < 0.01) percentage of the MHC type IIa/IIx isoforms than the LD. The-LD had the greatest proportion of (P < 0.01) MHC IIb isoforms of any of the muscles. The PM had the lowest (P < 0.01) percentage of MHC IIb isoforms and a greater (P < 0.05) percentage of type I MHC isoforms than the LD and SM. The PM had less (P < 0.01) drip loss after 96 h of storage than the SM and LD. The PM had more desmin degradation (P < 0.01) than the LD and SM at 45 min and 6 h postmortem. Degradation of titin occurred earlier in the PM than the LD and SM. At 45 min postmortem, the PM consistently had some autolysis of mu-calpain, whereas the LD and SM did not. At 6 h postmortem, some autolysis of mu-calpain (80-kDa subunit) was observed in all three muscles. The rapid pH decline and increased rate of autolysis in the PM paralleled an earlier appearance of myofibril-bound mu-calpain. The SM had higher calpastatin activity (P < 0.05) at 45 min, 6 h, and 24 h and had higher WBS values at 48 h (P < 0.01) and 120 h (P < 0.05) postmortem than the LD. At 48 and 120 h postmortem, more degradation of desmin, titin, and nebulin were observed in the LD than in the SM. These results show that mu-calpain activity, mu-calpain autolysis, and protein degradation are associated with differences in pork tenderness and water-holding capacity observed in different muscles.     

Postmortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb biceps femoris during extended postmortem storage.

The present experiment was conducted to determine whether calpastatin inhibits only the rate, or both the rate and extent, of calpain-induced postmortem proteolysis. Biceps femoris from normal (n = 6) and callipyge (n = 6) lamb was stored for 56 d at 4 degrees C. Calpastatin activity was higher (P < .05) in the callipyge muscle at 0 and 14 d postmortem, but not at 56 d postmortem. The activity of mu-calpain did not differ between normal and callipyge biceps femoris at 0 and 56 d postmortem (P > .05), but was higher at 14 d postmortem in the callipyge muscle (P < 0.05). The activity of m-calpain was higher in the callipyge muscle (P < 0.05). Western blot analyses of titin, nebulin, dystrophin, myosin heavy chain, vinculin, alpha-actinin, desmin, and troponin-T indicated that postmortem proteolysis was less extensive in callipyge than in normal biceps femoris at all postmortem times. The results of this experiment indicate that calpastatin inhibits both the rate and extent of postmortem proteolysis.     

Ionic strength-induced inactivation of mu-calpain in postmortem muscle

The present study was conducted to study the stability of autolyzed mu-calpain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmortem muscle. Ionic strength and pH affected the stability of partially autolyzed mu-calpain. Complete loss of activity was observed as a result of binding of autolyzed mu-calpain to DEAE-Sephacel when the large subunit of mu-calpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpain by standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle. The activity of autolyzed mu-calpain was stabilized by inclusion of glycerol in the buffers, and this permitted us to investigate whether the apparent loss of mu-calpain activity in postmortem muscle is an artifact of the methodology. Despite the inclusion of glycerol in the buffers, a decrease in mu-calpain activity was observed during postmortem storage of muscle, even though the autolyzed enzyme was readily detectable by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for the decline in mu-calpain activity in postmortem muscle.     

Indicators of tenderization are detectable by 12 h postmortem in ovine longissimus

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.     

Effect of pH, temperature, and inhibitors on autolysis and catalytic activity of bovine skeletal muscle mu-calpain.

To improve our understanding of the regulation of calpain activity in situ during postmortem storage, the effects of pH, temperature, and inhibitors on the autolysis and subsequent proteolytic activity of mu-calpain were studied. Calpains (mu- and m-calpain) and calpastatin were purified from bovine skeletal muscle. All autolysis experiments were conducted in the absence of substrate at different pH (7.0, 6.2, and 5.8) and temperatures (25 and 5 degrees C). Autolysis of mu-calpain generated polypeptides with estimated masses of 61, 55, 40, 27, 23, and 18 kDa. The rate of autolysis was significantly increased with decreasing pH. The rate of degradation of the 80-kDa subunit was significantly decreased with decreasing temperature. However, degradation of the 30-kDa subunit was not affected by decreasing temperature. By conducting autolysis experiments at 5 degrees C and immunoblotting of autolytic fragments with anti-80 kDa, it was demonstrated that with the exception of 18 kDa, which originates from 30 kDa, all other fragments probably originate from degradation of the 80-kDa subunit. Calpastatin, leupeptin, and E-64 did not inhibit the initial step of autolysis, but they did inhibit further breakdown of these fragments. However, zinc, which also inhibits the proteolytic activity of calpain, only reduced the rate of autolysis, but did not inhibit it. The possible significance of these results in terms of the regulation of calpain in postmortem muscle is discussed.     

Combined effect of epinephrine and exercise on calpain/calpastatin and cathepsin B and L activity in porcine longissimus muscle.

The objective of the study was to improve the understanding of the relationship between the effect of epinephrine plus exercise and meat tenderness. The calpain, calpastatin, and cathepsin B + L activities and postmortem proteolysis in porcine longissimus muscle were studied. The muscle glycogen stores were depleted in five pigs by s.c. injection of epinephrine (.3 mg/kg) at 15 h antemortem and exercise on a treadmill (5 min, 3.8 km/h) immediately before slaughter. Antemortem injection of epinephrine and treadmill exercise resulted in higher ultimate pH (6.32 vs 5.66 in control) and decreased (P < .05) thaw loss, cooking loss, and shear force values. The muscle energy depletion treatment increased (P < .05) the muscle mu-calpain activity measured 42 min postmortem, and at 24 h mu-calpain activity was still approximately 50% greater in the high ultimate pH group. Also, as the ratio of mu-calpain to calpastatin increased (P < .01), the overall proteolytic potential of the calpain system were greater. These observations suggest that the muscle energy level may influence the activity of the calpain system in the living animal. The high ultimate pH group showed lower (P < .001) cathepsin B + L activity in the myofibrillar and the soluble fractions after 8 d of storage, suggesting that the increased ultimate pH increased the stability of the lysosomal membrane and thereby reduced the release of cathepsins from the lysosomes during storage. The SDS-PAGE showed increased (P < .001) degradation of a 39-kDa band in the epinephrine and exercise-treated samples. Degradation products at 30, 31, and 32 kDa were labeled by troponin-T antibody in western blot. An appearing 24-kDa band was identified as a troponin-I degradation product in western blot. The proteolytic degradation pattern of myofibrillar proteins during storage differed in control and treated samples, supporting the hypothesis that calpain-mediated proteolysis was affected after treatment, resulting in meat with high ultimate pH.     

Altered calpain levels in longissimus muscle from normal pigs and heterozygotes with the ryanodine receptor mutation

The calpain proteolytic system was examined in the longissimus muscle (LD) of heterozygote pigs carrying a single copy of a mutation in the skeletal muscle ryanodine receptor gene (RyR1) that is associated with porcine stress syndrome and reduced meat quality. Conventional British White-type pigs (n = 30) were selected from a commercial line on the basis of slaughter weight, backfat depth, and pH at 45 min postmortem > 6.0; based on DNA analysis, 11 were heterozygous RyR1 mutants (Nn), and 19 were normal genotype (NN). The LD samples were taken from carcasses at 2, 4, and 24 h postmortem for calpain analysis with enzyme assay and immunoblotting, using specific antisera raised against recombinant polypeptides derived from calpain large subunits and calpastatin. Shear force (SF) was measured after conditioning for 8 d at 2 degrees C and did not differ between Nn and NN groups. The extractable activity of mu-calpain decreased over 24 h postmortem (P < .001), with no significant difference in activity between NN and Nn animals at any time. The activity of m-calpain also decreased with time (P < .001), but it was lower at all times in Nn than in normal genotypes (P < .001). After Western blotting, the immunoreactivity of mu- and m-calpain large subunit bands declined over 24 h postmortem (P < .001); values for mu-calpain were higher (P < .05) and for m-calpain were lower (P < .001) in heterozygotes than in normal animals at each sampling time. The calpastatin antibody detected a major band of 135 kDa that declined with time postmortem but did not differ between Nn and NN genotypes at any sampling time. These data indicate that the levels of extractable mu- and m-calpain, but not calpastatin, may be different in pigs that carry the RyR1 mutation.     

Alterations in postmortem degradation of myofibrillar proteins in muscle of lambs fed a beta-adrenergic agonist

Dietary administration of 4 ppm of the beta-agonist L-644,969 (Merck Sharpe and Dohme Research Laboratories) to finishing lambs induced a decrease (10 to 14%, P less than .05) in extractable calpain I activity in the longissimus muscle (LD) at death (d 0). At 4 d postmortem (d 4), extractable calpain I levels in the LD of both control and treated lambs were reduced (P less than .001) from those present at d 0, but the extractable activity in the LD was reduced to a greater extent in control than in treated lambs. Calpain II activity was increased 42% (P less than .005) in LD of treated lambs; however, no significant differences were observed between d 0 and d 4 calpain II activity within treated or control LD samples (P greater than .1). Calpastatin activity was higher in the LD of treated lambs (74% on d 0, P less than .001 and 430% on d 4, P less than .001) than in the LD of control lambs. Measurable cathepsin B activity was decreased (29% on d 0, P less than .05) and measurable cathepsin H activity was increased (10% on d 0, P less than .05 and 10% on d 4, P less than .05) in the LD of treated lambs compared with controls. On d 2, 4 and 6 postmortem, degradation in myofibrils isolated from the LD was lower for treated than for control lambs. Warner-Bratzler shear values for loin chops from treated lambs were higher on both d 3 (111%) and 6 (108%) postmortem than for chops from control lambs (P less than .001). L-644,969-induced decreases in muscle proteolytic capacity may limit postmortem myofibril degradation and contribute to the reduced tenderness observed. This decreased proteolytic capacity may contribute to increased muscularity of L-644,969-treated lambs.     

Postmortem proteolysis is reduced in transgenic mice overexpressing calpastatin

Using both in vitro and in vivo approaches, numerous studies have provided evidence that mu-calpain is responsible for postmortem proteolysis. This paper reports the effect of overexpression of calpastatin on postmortem proteolysis in transgenic mice. Transgenic mice (n = 8) with a human calpastatin gene, whose expression was driven by the human skeletal muscle actin promoter, were killed along with control nontransgenic littermates (n = 5). Hind limbs were removed and stored at 4 degrees C, and muscle samples were dissected at 0, 1, 3, and 7 d postmortem and analyzed individually. At time 0, active human calpastatin was expressed in transgenic murine skeletal muscle at a level 370-fold greater (P < 0.001) than calpastatin in control mice. Although the native isoform of this protein was degraded with storage, at 7 d postmortem, approximately 78% of at-death activity remained, indicating that degraded calpastatin retains activity. Calpain (mu- and m-) expression was unaffected (P > 0.05) by the transgene as assessed by immunoreactivity at d 0. Over 7 d, 33% of at-death 80-kDa isoform immunoreactivity of mu-calpain was lost in transgenics compared to an 87% loss in controls, indicating that autolysis of mu-calpain was slowed in transgenic mice. Desmin degradation was also inhibited (P < 0.05) in transgenics when compared to controls. Control mice lost 6, 78, and 91% of at-death native desmin at 1, 3, and 7 d postmortem, respectively; conversely, transgenic mice lost only 1, 3, and 17% at the same times. A similar trend was observed when examining the degradation of troponin-T. Interestingly, m-calpain seemed to undergo autolysis in control mice, which in postmortem tissue is indicative of proteolysis. Further investigation revealed that both mu- and m-calpain are active postmortem in normal murine skeletal muscle. In conclusion, a high level of expression of active calpastatin was achieved, which, by virtue of its inhibitory specificity, was determined to be directly responsible for a decrease in postmortem proteolysis.     

Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle.

A negative correlation exists between calpastatin activity and meat tenderness. Therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. To accomplish this, purified calpastatin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees C, and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.     

Intermuscular variation in tenderness: association with the ubiquitous and muscle-specific calpains

The biochemistry of intermuscular variation in tenderness is not fully understood. To investigate the role of the calpains in this process we performed two experiments using bovine and ovine species. In the bovine experiment, two distinct muscles, longissimus thoracis et lumborum (LT) and psoas major (PM), were used. In the ovine experiment, four muscles, LT, PM, semimembranosus (SM), and semitendinosus (ST), were used. Muscles were sampled at death for the determination of the steady-state mRNA level of calpains and calpastatin and the activities of calpain 1, 2, and calpastatin. Muscles were also sampled to determine the temporal changes in pH, tenderness, and the activity of the ubiquitous calpain system during postmortem aging. The results of the relative rate of tenderization in both species was found to be related to muscle type; LT had the highest value in both species. Within species, the mRNA steady-state levels of calpain 1 and calpastatin were similar in various bovine and ovine muscles. Bovine calpain 2 mRNA level was significantly lower in the LT than in the PM. Ovine calpain 2 mRNA level was lower, but not significantly different, in the LT compared to the other muscles. The mRNA level of bovine calpain 3 was significantly higher in the LT muscle than in the PM. In the ovine, the mRNA level of calpain 3 was highest in the LT, followed by SM, PM, and ST. Results on the activity of the ubiquitous calpain system in various muscles at death were dependent on muscle type and species. Temporal changes in the activity of calpains and calpastatin during the first 24 h of postmortem aging were similar in the muscles studied: calpain 1 and calpastatin declined significantly and calpain 2 remained relatively unchanged. The temporal changes in muscle pH in both experiments indicated that the extent and rate of pH decline during aging was related to muscle type. Correlation analysis between the relative rate of tenderization and mRNA expression of calpains revealed a strong relationship with calpain 3 in both species.     

Relationship between skeletal muscle-specific calpain and tenderness of conditioned porcine longissimus muscle

Tenderization of skeletal muscle in meat animals has been closely linked to the postmortem activity of the calpain proteolytic enzyme system, which includes the specific inhibitor calpastatin. Increased understanding of the skeletal muscle-specific calpain isoform p94 has prompted suggestions as to whether it too could have a role in the tenderization process. In this study, two groups of pigs were identified in which shear force measurements after 8 d of conditioning indicated a large variation in the tenderness of longissimus muscle. The quantity of p94 in the muscle was monitored by immunoblotting, using a porcine-specific polyclonal antibody raised against a recombinant peptide fragment generated as a fusion protein. The antiserum recognized a 94-kDa protein associated with myofibrils in skeletal but not cardiac muscle, as expected for this calpain isoform, although it could not be tested with the native protein because of the extreme instability of p94. In the first experiment, the mean shear force for the tough group was 6.71 +/- .28 kg (n = 12, SEM) and that of the tender group was 3.87 +/- .12 kg (n = 12), but there was no difference in the normalized absorbance of the immunopositive 94 kDa band on Western blots from samples collected at approximately 40 min postmortem. In the second experiment, the stability of p94 in chilled carcasses was investigated over 24 h, using a further two groups of 10 tough and 10 tender pigs of mean shear force values 5.36 +/- .14 kg and 2.81 +/- .15 kg, respectively. In tough and tender animals, there was a decline (P < .05) in the 94-kDa immunostaining material of mean half-lives of 13.8 and 12.9 h, respectively, although there was considerable variability. Despite this variability in half lives and shear force values, no correlation was seen between these factors. Thus, in porcine longissimus muscle, the variability in tenderness after 8 d of conditioning cannot be attributed to an underlying variability in p94.     

Effect of oxidation, pH, and ionic strength on calpastatin inhibition of mu- and m-calpain

The objective of this study was to evaluate the effect of oxidation on mu- and m-calpain activity at varying pH and ionic strength conditions in the presence of calpastatin. In 2 separate experiments, purified porcine skeletal muscle mu- or m-calpain (0.45 units of caseinolytic activity) was incubated in the presence of calpastatin (0, 0.15, or 0.30 units) at pH 7.5, 6.5, or 6.0 with either 165 or 295 mM NaCl. The reactions were initiated with the addition of CaCl2 (100 microM for mu-calpain; 1 mM for m-calpain). In Experiment 1, mu- or m-calpain was incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC (170 microM). Either 0 or 16 mu microM H2O2 was added to each assay. Activity was measured at 60 min. In Experiment 2, calpain was incubated with highly purified porcine myofibrils (4 mg/mL) under conditions described. Either 0 or 100 microM H2O2 was added immediately prior to the addition of calpain. Degradation of desmin was determined on samples collected at 2, 15, 60, and 120 min. Results from Experiment 1 indicated that oxidation decreased (P < 0.01) activity of mu-calpain. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5, and m-calpain had the greatest (P < 0.01) activity at pH 7.5 at 60 min. m-Calpain activity was not detected at pH 6.0. Mu- and m-calpain activity were lower (P < 0.01) at 295 mM NaCl than at 165 mM NaCl at all pH conditions. Oxidation lowered (P < 0.01) calpastatin inhibition of mu-and m-calpain at all pH and ionic strength combinations. In Experiment 2, oxidation decreased proteolytic activity of mu-calpain against desmin at pH 6.0 (P < 0.05 at 15, 60, and 120 min) and decreased m-calpain at all pH conditions. However, desmin degradation by mu-calpain was not as efficiently inhibited by calpastatin at pH 7.5 and as at pH 6.5 (P = 0.03 at 60 min) when oxidizing conditions were created. This is consistent with the results from Experiment 1, which indicated that oxidation decreased the ability of calpastatin to inhibit mu-calpain. These studies provide evidence that oxidation influences calpain activity and inhibition of calpains by calpastatin differently under varying environmental conditions. The results suggest that, at the higher pH conditions used, calpastatin may limit the possibility of oxidation-induced inactivation of mu-calpain.     

Effect of pH and ionic strength on mu- and m-calpain inhibition by calpastatin

The objectives of this study were to determine the extent to which pH and ionic strength influence mu- and m-calpain activity and the inhibition of calpains by calpastatin. Calpastatin, mu-calpain, and m-calpain were purified from at-death porcine semimembranosus. Mu-calpain or m-calpain (0.45 U) were incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin in the presence of calpastatin (0, 0.15, or 0.30 U of calpain inhibitory activity) under the following pH and ionic strength conditions: pH 7.5 and 165 mM NaCl or 295 mM NaCl; pH 6.5 and 165 mM NaCl or 295 mM NaCl; and pH 6.0 and 165 mM NaCl or 295 mM NaCl. The reactions were initiated with addition of 100 microM (mu-calpain) or 1 mM CaCl2 (m-calpain), and calpain activity was recorded at 30 and 60 min. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased mu-calpain activity (P < 0.01) at all pH conditions. Inhibition percent of mu-calpain by calpastatin was not affected by pH; however, it was influenced by ionic strength. Inhibition of mu-calpain by calpastatin was higher (P < 0.01) at 295 mM NaCl than at 165 mM NaCl when 0.3 units of calpastatin were included in the assay. Activity of m-calpain was greater (P < 0.01) at pH 7.5 than at pH 6.5. m-Calpain activity was not detected at pH 6.0. Inhibition of m-calpain was greater (P < 0.01) when 0.15 and 0.3 U calpastatin were added at pH 6.5 than 7.5 at 165 mM NaCl, whereas percentage inhibition of m-calpain was greater (P < 0.01) at 295 mM than 165 mM NaCl at pH 7.5 and 6.5. These observations provide new evidence that defines further the influence of pH decline and increased ionic strength on mu-calpain, m-calpain, and calpastatin activity, thereby helping to more accurately define a role for these enzymes in the process of postmortem tenderization.     

Effect of the callipyge gene on muscle growth, calpastatin activity, and tenderness of three muscles across the growth curve

Changes in muscle growth, calpastatin activity, and tenderness of three muscles were assessed in 20 callipyge and 20 normal wether lambs slaughtered at live weights (LW) of 7, 20, 36, 52, and 69 kg. At 24 h postmortem, the longissimus (LM), semimembranosus (SM), and supraspinatus (SS) muscles were removed and weighed and samples were obtained for calpastatin activity (CA; 24 h) and Warner-Bratzler shear force (WBS; aged 6 d). For muscle weights and calpastatin activity, the weight group x muscle x phenotype interaction was significant (P < 0.05). Muscle weights were similar (P > 0.05) between phenotypes for all three muscles at 7 kg LW. At 20 kg LW, the LM and SM muscles from the callipyge lambs were heavier (P < 0.05) than those from normal lambs; however, the SS did not differ (P > 0.05) between phenotypes at 7, 20, or 52 kg. From 20 to 69 kg LW, the LM and SM weights were 42 and 49% heavier (P < 0.05) for callipyge than for normal lambs. Calpastatin activity of the callipyge LM was greater (P < 0.05) than that of normal LM at 36, 52, and 69 kg. In the callipyge LM, CA was similar (P > 0.05) at 20, 36, and 52 kg LW and did not differ (P > 0.05) from 7-kg or 69-kg values. Calpastatin activity declined (P < 0.05) across the growth curve for the SM and SS, but values were higher (P < 0.05) in the SM in callipyge than in normal lambs. Shear force values of the LM were lower (P < 0.05) for normal lambs at 36, 52, and 69 kg LW than for callipyge lambs. In the SM and SS, WBS values decreased (P < 0.05) across the growth curve, but values were higher (P < 0.05) for callipyge lambs in the SM only. These data indicate that the selective muscular hypertrophy of the callipyge phenotype develops during the postnatal growth period between 7 and 20 kg LW (19 and 100 d of age). Longissimus and semimembranosus muscles in the callipyge lambs were over 40% heavier from 20 to 69 kg LW; however, they also had higher levels of calpastatin activity and Warner-Bratzler shear force during this time period, indicating the need for postmortem tenderization treatments to improve palatability.