Properties of myofibril-bound calpain activity in longissimus muscle of callipyge and normal sheep

Properties of the calpain bound to myofibrils in longissimus muscle from callipyge or noncallipyge sheep were examined after 0, 1, 3, and 10 d of postmortem storage at 4 degrees C. Western analysis has shown that most of this calpain is mu-calpain, although the sensitivity of the antibodies used in the earlier studies could not eliminate the possibility that up to 10% of the calpain was m-calpain. The calpain is bound tightly, and very little is removed by washing with the detergent Triton X-100; hence, it is not bound to phospholipids in the myofibril. Over 25% of total mu-calpain was bound to myofibrils from at-death muscle, and this increased to approximately 40% after 1 d postmortem. The amount of myofibril-bound mu-calpain increased only slightly between 1 and 10 d of postmortem storage. The percentage of autolyzed mu-calpain increases with time postmortem until after 10 d postmortem, when all myofibril-bound mu-calpain is autolyzed. The specific activity of the myofibril-bound calpain is very low and is only 6 to 13% as high as the specific activity of extractable mu-calpain from the same muscle. It is unclear whether this low specific activity is the result of unavailability of the active site of the myofibril-bound calpain to exogenous substrate. The myofibril-bound calpain degrades desmin, nebulin, titin, and troponin T in the myofibrils, and also releases undegraded alpha-actinin and undergoes additional autolysis when incubated with Ca2+; all these activities occurred slowly considering the amount of myofibril-bound calpain. Activity of the myofibril-bound calpain was partly (58 to 67%) inhibited by the calpain inhibitors, E-64 and iodoacetate; was more effectively inhibited by a broader-based protease inhibitor, leupeptin (84 to 89%); and was poorly inhibited (43 to 45%) by calpastatin. Release of undegraded alpha-actinin and autolysis are properties specific to the calpains, and it is unclear whether some of the myofibril-bound proteolytic activity originates from proteases other than the calpains or whether the active site of myofibril-bound calpain is shielded from the inhibitors. Activities and properties of the myofibril-bound calpain were identical in longissimus muscle from callipyge and normal sheep, although previous studies had indicated that the "normal" longissimus was much more tender than the callipyge longissimus. Hence, it seems unlikely that the myofibril-bound calpain has a significant role in postmortem tenderization of ovine longissimus.     

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.     

Oxidative environments decrease tenderization of beef steaks through inactivation of mu-calpain

This study was designed to test the hypothesis that oxidative conditions in postmortem (PM) tissue decrease calpain activity and proteolysis, subsequently minimizing the extent of tenderization. To achieve different levels of oxidation, the diets of beef cattle were supplemented with vitamin E for the last 126 d on feed, and beef steaks were irradiated early PM. Ten steers were fed a finishing diet with the inclusion of vitamin E at 1,000 IU per steer daily (VITE). Another 10 beef steers were fed the same finishing diet without added vitamin E (CON). At 22 to 24 h PM, strip loins from each carcass were cut into 2.54-cm-thick steaks and individually vacuum packaged. Within 26 h PM, steaks were irradiated at 0 or 6.4 kGy and then aged at 4 degrees C for 0, 1, 3, 7, and 14 d postirradiation. Steaks from each time point were used to determine Warner-Bratzler shear force (WBSF) and calpain activity, and for western blotting of sarcoplasmic proteins and myofibrillar proteins. Calpastatin activity was determined at 0, 3, and 14 d postirradiation. At 1, 3, 7, and 14 d postirradiation, WBSF values of irradiated steaks were higher (P < 0.03) than for nonirradiated steaks. Western blots of troponin-T and desmin showed decreased proteolysis in irradiated samples compared with nonirradiated samples. At 2 d PM, troponin-T degradation products were more evident (P < 0.03) in nonirradiated steaks supplemented with VITE than nonirradiated steaks from the CON diet. Similarly, VITE treatment resulted in steaks with lower (P < 0.05) calpastatin activity at 1 d PM than in steaks from steers fed the CON diet. Irradiation diminished the rate of calpastatin inactivation. Irradiated samples, regardless of diet, had no detectable levels of intact titin or nebulin. Irradiation decreased mu-calpain activity and autolysis, whereas mu-calpain activity was not affected by diet or irradiation. Inactivation of mu-calpain by oxidation during early times PM decreased the amount of myofibrillar proteolysis, thereby decreasing the extent of tenderization of beef steaks.     

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.     

Variation in palatability and biochemical traits within and among eleven beef muscles

The objective of this study was to determine the extent of variation in, and relationships among, biochemical and palatability traits within and among 11 major beef muscles. Longissimus thoracis et lumborum (LD), psoas major (PM), gluteus medius (GM), semimembranosus (SM), adductor (AD), biceps femoris (BF), semitendinosus (ST), rectus femoris (RF), triceps brachii (TB), infraspinatus (IS), and supraspinatus (SS) from one side of 31 Charolais x MARC III steer carcasses were vacuum-packaged, stored at 2 degrees C until 14 d postmortem, and then frozen at -30 degrees C. The 2.54-cm-thick steaks were obtained from two or three locations within muscles in order to assess biochemical traits and Warner-Bratzler shear force, and from near the center for sensory trait evaluation. The PM was most tender and was followed by IS in both shear force and tenderness rating (P < 0.05). The other muscles were not ranked the same by shear force and tenderness rating. The BF had the lowest (P < 0.05) tenderness rating. The PM, GM, and LD had lower (P < 0.05) collagen concentration (2.7 to 4.5 mg/g muscle) than muscles from the chuck and round (5.9 to 9.0 mg/g), except for the AD (4.9 mg/g). Desmin proteolysis was highest (P < 0.05) for BF and LD (60.7 and 60.1% degraded), and was lowest (P < 0.05) for PM (20.2%). The PM, TB, IS, RF, and ST had relatively long sarcomere lengths (> 2.1 microm), whereas the GM had the shortest (P < 0.05) sarcomere length (1.7 microm). Cooking loss was lowest (P < 0.05) for BF (18.7%) and was followed by LD and IS (20.7%); it was highest (P < 0.05) for ST (27.4%). Across all muscles, tenderness rating was highly correlated (r > 0.60) with shear force, connective tissue rating, sarcomere length, and collagen content. Within a muscle, correlations among all traits were generally highest in LD and lowest in AD. Within muscle, location effects were detected (P < 0.05) for shear force (PM, ST, BF, SM, and RF), sarcomere length (PM, ST, BF, LD, SS, IS, SM, and RF), collagen concentration (PM, BF, SS, IS, SM. AD, TB, and RF), desmin degradation (PM, GM, BF, SM, AD, and, RF), and cooking loss (all muscles except SS and AD). There is a large amount of variation within and among muscles for tenderness traits and tenderness-related biochemical traits. These results increase our understanding of the sources of variation in tenderness in different muscles and provide a basis for the development of muscle-specific strategies for improving the quality and value of 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.     

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.     

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.     

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.     

Postmortem titin proteolysis is influenced by sarcomere length in bovine muscle

The calpain protease system, in particular, μ-calpain is involved in the disassembly of specific myofibrillar proteins, resulting in tenderization of meat postmortem. Given the size, complexity, and integral nature of titin to the structure of the sarcomere, it is plausible that the length of a sarcomere may alter the susceptibility of various domains of titin to cleavage by the calpains. Therefore, we hypothesized titin degradation differs in a sarcomere-length-dependent manner in beef. After slaughter, beef carcasses were split and sides were either suspended by the Achilles tendon (normal suspension, NS) or by the aitchbone (hip suspension, HS). Immediately after suspension, samples were dissected from the LM, psoas major (PM), and semitendinosus (STN) muscles to serve as 0-d controls. After 24 h, 4 steaks were removed from each muscle and randomly assigned to 1-, 4-, 7-, or 10-d aging treatments. After the assigned aging period, myofibrils were purified for determination of sarcomere length. Warner-Bratzler shear force analysis was also performed to evaluate differences in tenderness. Muscle proteins were solubilized and subjected to SDS-VAGE (vertical agarose gel electrophoresis) to evaluate titin degradation. Sarcomere lengths differed (P < 0.0001) between contralateral muscles of NS and HS carcasses. Quantification of SDS-VAGE gels revealed less (P < 0.05) intact titin in the PM muscle of NS carcasses at each aging period compared with the PM of HS carcasses. No significant differences (P > 0.05) were detected in the disappearance of intact titin among suspension methods in the LM or STN. These data demonstrate that suspension method alters proteolysis of titin and suggest an increase in sarcomere length may contribute to the susceptibility of titin to postmortem proteolysis in beef.     

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.     

Effects of available dietary carbohydrate and preslaughter treatment on glycolytic potential, protein degradation, and quality traits of pig muscles

The current study was conducted to determine the interactive effects of a glycogen-reducing diet fed to finishing pigs and length of preslaughter transportion on muscle metabolic traits, proteolysis of intermediate filament and costameric proteins, and meat quality traits. Large White gilts and barrows (n = 48) were selected at 88 kg of BW and individually fed for 21 d a diet (2.6 kg/d) either high (HC) or low (LC) in available carbohydrates. Six gilts and 6 barrows fed the HC and LC diets were subjected to 0 or 3 h of transportation on the day of slaughter. Muscle temperature and pH were measured at 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, and 24 h postmortem in the LM and 24 h postmortem in the dark (STD) and light (STL) portion of the semitendinosus. At 24 h postmortem, glycolytic potential (GP) was determined in the LM, STD, and STL, as well as proteolysis of titin, nebulin, desmin, vinculin, and talin in the LM and STD. The GP was lower (P < 0.05) in muscles from LC-pigs than in muscles from HC-pigs. The LC diet also resulted in lower (P < 0.05) pH, and a darker (P = 0.03), less (P < 0.01) yellow color in the STL. The LC diet decreased (P = 0.04) cooking losses in the STL and STD. The 3-h journey further decreased (P = 0.05) the GP in the STD, regardless of the diet, but transport had no effect (P > or = 0.67) on the GP of the LM and STL. Ultimate pH of the LM was lower (P = 0.02), and both portions of the semitendinosus were darker (P = 0.01) and less yellow (P < 0.01), in pigs transported 3 vs. 0 h. In pigs transported for 3 h, intact vinculin tended to be more (P = 0.08) degraded in the LM, which coincided with lower (P = 0.04) drip losses in the LM of pigs transported for 3 compared with 0 h. Increased (P < 0.01) proteolysis of titin paralleled lower (P = 0.02) shear force values in the STD of pigs transported 3 vs. 0 h. Although the present results demonstrated the potential of a glycogen-reducing diet to alter the GP of different porcine muscles, the effect of these changes on meat quality traits was limited to higher ultimate pH and darker color in the STL. The positive effects of length of transportation on water-holding capacity (LM and STD) and meat color (STD and STL) were only partially related to the resting muscle glycogen concentration because the 3-h transport lowered the GP only in the muscle with the lowest basal glycogen concentration.     

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 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.     

Influence of packaging method and storage time on shear value and mechanical strength of intramuscular connective tissue of chevon

The objectives of this study were to determine the effects of storage time (ST) and packaging method (PM) on tenderness and changes in intramuscular connective tissue (IMCT) strength of chevon. Spanish does (8 mo of age, average BW 25 kg) were harvested (n = 12), chilled at 4 degrees C for 24 h, and then fabricated into 2.5-cm-thick leg, shoulder/arm, and loin/rib cuts. The cuts from six carcasses were vacuum-packed and aged at 2 degrees C for 0, 4, 8, or 12 d. To assess the influence of a packaging method that favors oxidation on postmortem tenderization, the cuts from the remaining six carcasses were placed on styrofoam trays, overwrapped with polyvinyl-chloride film, and stored at 2 degrees C for similar periods. At each ST, longissimus (LM), semimembranosus (SM), and triceps brachii (TB) muscles were assessed for Warner-Bratzler shear (WBS) values. The WBS of uncooked meat, myofibrillar fragmentation index (MFI), and collagen solubility were assessed on LM. The IMCT samples were prepared to assess changes in mechanical strengths and for scanning electron microscopy (SEM). Intact honeycomb structures of endomysium, with no muscle fiber elements, were observable under SEM. The PM or ST did not influence the mechanical strength of IMCT preparations, as measured by a texture analyzer. Collagen solubility of LM muscles also did not change during aging. For both PM, cooked meat WBS values were higher (P < 0.01) in SM and TB than in LM. In the SM samples, the average WBS values were higher (P < 0.01) at d 0 than at other ST. Although MFI of LM increased with increasing aging time (P < 0.05), changes in WBS over ST were minimal in TB and LM samples. The WBS of uncooked LM decreased sharply up to 8 d postmortem in both PM (P < 0.05). However, there was no PM x ST interaction to indicate any adverse influence of packaging on tenderization of chevon. The results suggest that aging chevon cuts for more than 4 d may not result in significant additional improvement in tenderness.     

Unique expression patterns of myosin heavy chain genes in the ductus arteriosus and uterus of rabbits.

In smooth muscle tissue, two smooth muscle myosin heavy chain (MHC) isoforms (SM1, SM2) and two non-muscle MHC isoforms (NMA, NMB) have been identified. The purpose of our study was to clarify whether smooth muscle MHC mRNA expression reflects the physiological and functional state of the muscle. We studied the expression pattern of MHC mRNAs, using the S1-nuclease mapping procedure, in functionally and morphologically changeable organs; the ductus arteriosus (DA) during development (25 and 29 days of gestation, and from 3-day-old neonates) and uteri from virgin, day-10 pregnant (P10) and day-29 pregnant (P29) rabbits. The results demonstrated that SM2 expression was greater in the fetal DA than in the fetal aortic and pulmonary arteries, but that it decreased significantly following closure of DA. In the gravid uterus, SM1 expression was significantly (P<0.05) strong compared to other MHC mRNAs from virgin to P10 rabbits. During pregnancy, NMB expression showed a tendency to increase until P10, and after P10, SM2 expression increased dramatically and NMB expression decreased to give almost a mirror image of the SM2 expression. Smooth muscle type (SM1, SM2) was significantly (P<0.05) strong compared to non-muscle type expression (NMA, NMB) at P29. These data suggest that smooth muscle MHC mRNA, especially SM2 expression reflects the physiological and functional state of the smooth muscle.     

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.     

Chilling and cooking rate effects on some myofibrillar determinants of tenderness of beef

Our objectives were to examine the effects of prerigor excision and rapid chilling vs. conventional carcass chilling of two muscles on proteolysis and tenderness during the postmortem storage, as well as the effects of fast and slow rates of cooking on myofibrillar characteristics and tenderness. The longissimus thoracis (LT) and triceps brachii (TB), long head muscles were removed 45 min after exsanguination from the left side of 12 carcasses and chilled in an ice bath to induce cold shortening (excised, rapidly chilled). At 24 h postmortem, the corresponding muscles were removed from the right side (conventionally chilled). All muscles were cut into 2.54-cm-thick steaks and assigned to one of two postmortem times (1 or 14 d), and to raw and cooking treatments. Steaks were cooked at 260 degrees C (FAST) or 93 degrees C (SLOW) in a forced-air convection oven to an internal temperature of 70 degrees C. Cooking loss, cooking time, and Warner-Bratzler shear force (WBSF) were measured on cooked steaks. Sarcomere length (SL) and the extent of proteolysis of desmin were measured on raw and cooked steaks. As expected, the excised, rapidly chilled muscles had a much more rapid (P < 0.05) temperature decline than those that were conventionally chilled. The excised, rapidly chilled treatment resulted in shorter (P < 0.05) SL, and SL was shorter (P < 0.05) in LT than in TB steaks. Raw steaks had longer (P < 0.05) SL than cooked steaks, regardless of chilling treatment. The FAST cooking resulted in shorter (P < 0.05) SL than SLOW cooking in conventionally chilled steaks, but cooking rate had no effect (P > 0.05) on SL of rapidly chilled steaks. Generally, TB steaks required longer (P < 0.05) cooking times and had higher (P < 0.05) cooking losses than LT steaks, and FAST-cooked steaks had greater (P < 0.05) cooking losses than SLOW-cooked steaks. Rapidly chilled steaks had less (P < 0.05) degradation of desmin than conventionally chilled steaks (31 vs. 41%). Aging for 14 d increased (P < 0.05) desmin degradation. Rapid chilling of muscles resulted in much higher (P < 0.05) WBSF values, whereas aging resulted in lower (P < 0.05) WBSF values. The SLOW-cooked TB steaks were more tender (P < 0.05) than FAST-cooked TB steaks and LT steaks cooked at either rate. Excised, rapidly chilled muscles underwent proteolysis, but it occurred at a slower rate during the first 24 h postmortem than it did in conventionally chilled muscles. Cooking rate did not affect tenderness of LT steaks, but SLOW cooking resulted in more tender TB steaks.     

Effects of fasting and transportation on pork quality development and extent of postmortem metabolism

One hundred seventy-seven pigs were used to determine the interaction effects of fasting and length of transport prior to harvest on pork muscle quality. The study design was a 2 x 2 x 3 factorial, which involved two genetic sources, fasting (F) or no fasting (N) of pigs 48-h prior to harvest, and three transport times (0.5, 2.5, or 8.0 h) on a semitrailer to the packing plant. Genetic source was a significant source of variation (P < 0.05) for most composition and muscle quality variables. Fasting reduced hot carcass weight 3.6% (P < 0.05), but length of transport did not affect hot carcass weight (P > 0.05). There were no differences (P > 0.05) in percent lean among fasting and transport treatments. Fasted pigs had higher longissimus dorsi (LD) ultimate pH (pHu), darker lean color, higher marbling score and lower 7-d purge loss, 24-h drip loss, and cooking loss (P < 0.05) than nonfasted pigs. Meat from pigs that were transported 8.0 h had lower glycolytic potential (GP), higher LD and semimembranosus (SM) pHu, darker lean color, and lower L*, 7-d purge loss, 24-h drip loss, cooking loss, and shear force values than meat from pigs transported 0.5 h (P < 0.05). Meat from pigs transported 2.5 h had higher LD and SM pHu and lower L*, 7-d purge loss, 24-h drip loss, and cooking loss than meat from pigs transported 0.5 h (P < 0.05). Meat from pigs transported 8.0 h had higher LD pHu and color scores and lower L* and cooking loss than meat from pigs transported 2.5 h (P < 0.05). The fasting x transport interaction was significant for SM pHu, L*, color score, and drip loss. Fasting improved SM pHu, L*, color score, and drip loss for pigs that were transported 0.5 h (P < 0.05), but when pigs were transported for 2.5 h or 8.0 h, fasting had little or no effect on these muscle quality traits. Fasting lowered GP and increased LD pHu for pigs from the genetic source with the higher initial pork quality (P < 0.05), while fasting had no effect on pork quality for pigs from the genetic source with the lower initial pork quality (P > 0.05). Longer transport times resulted in lower GP and higher LD pHu regardless of genetic source. Fasting and length of transport each had positive effects on pork quality, but length of transport effects was greater in magnitude. When pigs were transported for 0.5 h, fasting for 48 h prior to harvest improved pork quality, but when pigs were transported 2.5 or 8.0 h, fasting had little effect on pork quality.     

The use of vitamin D3 and its metabolites to improve beef tenderness

Three experiments were conducted to determine whether feeding 25-hydroxyvitamin D3 (25-OH D3) or 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) improves the tenderness of longissimus dorsi (LD), semimembranosus (SM), and infraspinatus (IF) muscles similar to supplemental vitamin D3 without leaving residual vitamin D3 and its metabolites in muscle. In the first two experiments, 24 crossbred steers were used to determine the effects of different oral amounts of 1,25-(OH)2 D3 (Exp. 1; n = 12) and 25-OH D3 (Exp. 2; n = 12) on plasma Ca2+ concentrations. In the third experiment, crossbred steers were allotted randomly to one of four treatments: 1) control placebo (n = 7); 2) 5 x 10(6) IU of vitamin D3/d (n = 9) for 9 d and harvested 2 d after last treatment; 3) single, 125-mg dose of 25-OH D3 (n = 8) 4 d before harvest; or 4) single, 500-microg dose of 1,25-(OH)2 D3 (n = 9) 3 d before harvest. The LD and SM steaks from each animal were aged for 8, 14, or 21 d, whereas steaks from the IF were aged for 14 or 21 d. All steaks were analyzed for tenderness by Warner-Bratzler shear force and for troponin-T degradation by Western blot analysis. Supplementing steers with vitamin D3 increased (P < 0.01) the concentration of vitamin D3 and 25-OH D3 in all muscles sampled. Feeding steers 25-OH D3 increased (P < 0.05) the concentration of 25-OH D3 in meat, but to an amount less than half that of cattle treated with vitamin D3. Supplemental 1,25-(OH)2 D3 did not affect (P < 0.10) shear force values; however, there was a trend (P < 0.10) for supplemental vitamin D3 and 25-OH D3 to produce LD steaks with lower shear values after 8 and 14 d of aging, and lower (P < 0.10) shear force values for the SM aged for 21 d. Analysis of Western blots indicated that LD steaks from cattle supplemented with vitamin D3 and 25-OH D3 had greater (P < 0.05) troponin-T degradation. Antemortem supplementation of 25-OH D3 seems to increase postmortem proteolysis and tenderness in the LD and SM without depositing large concentrations of residual vitamin D3 and its metabolite 25-OH D3.