Sarcomere length influences postmortem proteolysis of excised bovine semitendinosus muscle

The interaction between sarcomere length and postmortem proteolysis as related to meat tenderness is not clear. The extent of thick and thin filament overlap alters actomyosin binding and may alter substrate availability during aging-induced tenderization. The objective of this study was to determine the influence of sarcomere length on proteolytic degradation in beef. Strips from bovine semitendinosus were either stretched 40% and restrained or allowed to shorten unrestrained in an ice bath. After rigor completion, 0.6-cm cross sections were fabricated and were randomly assigned to 2, 4, 7, or 10 d of aging treatments. Myofibrils were isolated for sarcomere length determination. Samples were collected and frozen for shear force analysis, and muscle proteins were extracted for SDS-PAGE and Western blotting analyses to determine troponin T (TnT) proteolysis. Sarcomere length was greater (P < 0.01) in stretched muscle samples compared with shortened samples (2.57 vs. 1.43 microm, respectively). Correspondingly, shear force values were greater (P < 0.05) in shortened samples than stretched samples. Western blots revealed the presence of 3 major intact TnT bands that diminished with time postmortem and 4 bands (TnT degradation products) that accumulated during postmortem storage. Quantification of intact TnT showed increased (P < 0.05) proteolysis at 4 and 7 d postmortem in samples with long sarcomeres. By 10 d, only traces of the greatest molecular weight intact TnT band were evident in both shortened and stretched samples, suggesting this TnT band may be more susceptible to proteolysis than other intact TnT bands. Degradation products of TnT appeared earlier postmortem in samples with long sarcomeres. The 30-kDa TnT fragment appeared after 7 d of postmortem storage in samples with long sarcomeres but not until 10 d in muscle containing short sarcomeres. Collectively, these data show that postmortem TnT proteolysis is sarcomere length-dependent and suggest that thick and thin filament overlap may influence the postmortem aging process in beef.     

The extent of proteolysis is independent of sarcomere length in lamb longissimus and psoas major.

The objective of this experiment was to determine the effect of sarcomere length on postmortem proteolysis and meat tenderness. Eighteen Dorset market-weight sheep were slaughtered conventionally. The longissimus thoracis et lumborum and psoas major from each carcass were either left intact on the carcass (control), which was chilled at 0 degrees C, or excised from the carcass and chilled in an ice slurry (0 degrees C). At 24 h, control muscles were excised, and all muscles were cut into sections and assigned to 1 or 10 d of postmortem storage at 2 degrees C. Sarcomere length was shorter (P < .01), as intended, in the shortened relative to the control treatment and in longissimus relative to psoas major (1.36 vs 1.69 microm, raw longissimus; 1.45 vs 3.03 microm, raw psoas major). Sarcomere length was not affected (P > .05) by aging time. Western blot analysis of troponin-T and desmin indicated no effect (P > .05) of the shortened treatment compared to the control on the extent of proteolysis. Regardless of aging time or treatment, troponin-T was more degraded (P < .01) in longissimus than in psoas major (38.1 vs 23.5%) and desmin tended to be more degraded (P = .08) in longissimus than in psoas major (50.4 vs 35.1%). Regardless of muscle or treatment, aging 10 d compared to 1 d increased degradation of troponin-T (46.3 vs 15.3%) and desmin (69.3 vs 16.1%). Warner-Bratzler shear force was greater (P < .01) in the shortened treatment than in control (6.9 vs 3.8 kg), greater (P < .01) in longissimus than in the psoas major (6.5 vs 4.2 kg), and greater (P < .01) with 1 d than with 10 d of aging time (6.1 vs 4.6 kg). A muscle x aging time interaction (P < .05) indicated shear force declined more in longissimus than in psoas major during aging. We conclude that sarcomere length did not affect the extent of proteolysis. However, sarcomere length may have an indirect effect on tenderization during aging due to its effect on initial tenderness.     

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.     

Postmortem proteolysis in longissimus muscle from beef, lamb and pork carcasses

Postmortem proteolysis in skeletal muscle and factors affecting this process were examined in pork, lamb and beef longissimus muscles (LM) to determine the cause of differences in meat tenderness among these species. Fat thickness differed among species in the following order: pork greater than beef greater than lamb. The following patterns were observed for rate of temperature and pH decline: lamb greater than pork greater than beef and pork greater than beef greater than lamb, respectively. At 1 d postmortem, pork was the most tender, followed by beef and lamb, respectively. Between 1 and 14 d of postmortem storage, lamb LM was the most improved in tenderness, followed by beef and pork, respectively. Species did not differ (P greater than .05) in LM collagen solubility. Pork LM from fed pigs had the highest (P less than .05) level of cathepsins B + L and cystatin(s) activities, whereas no differences (P greater than .05) were observed among the species for cathepsin B activity. The lowest (P less than .01) Ca2(+)-dependent protease (CDP)-II and CDP inhibitor activities were observed in pork LM. Beef LM had the highest CDP inhibitor activity (P less than .05) but was intermediate in CDP-II activity. No differences were observed among species for CDP-I activity. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of myofibrils isolated at 0, 1 and 14 d postmortem indicated that by d 1, desmin hydrolysis was most extensive in pork muscle, followed by lamb and beef.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in proteolysis, sarcomere length, collagen content, and tenderness among major pork muscles

The objectives of this experiment were to determine the extent of variation in proteolysis, sarcomere length, and collagen content among pork muscles and the association of those factors with tenderness variation among muscles at 1 d postmortem. Twenty-three white composite barrows were slaughtered and carcasses (66 kg) were chilled at 0 degrees C for 24 h. At 1 d postmortem, the longissimus lumborum, biceps femoris, semimembranosus, semitendinosus, and triceps brachii, long head were dissected from one side of each carcass and frozen. Trained sensory panelists evaluated tenderness, amount of connective tissue, juiciness, and pork flavor intensity of grilled (70 degrees C) chops on 8-point scales. Raw chops were used for total collagen content, sarcomere length, and the extent of desmin proteolysis. Tenderness ratings were highest (P < .05) for semitendinosus (7.2) and triceps brachii (7.1), followed by longissimus lumborum (6.4) and semimembranosus (5.7) and were lowest (P < .05) for biceps femorus (4.0). The simple correlations between longissimus lumborum tenderness and the tenderness of other muscles were .54 (semimembranosus), .34 (semitendinosus), .36 (triceps branchii), and .17 (biceps femorus). Total collagen was highest (P < .05) for biceps femorus (7.1 mg/g muscle), followed by triceps branchii (6.0 mg/g) and semitendinosus (5.3 mg/g), and lowest for semimembranosus (4.5 mg/g) and longissimus lumborum (4.1 mg/g). Sarcomere length was longest (P < .05) for semitendinosus (2.5 microm) and triceps branchii (2.4 microm), followed by semimembranosus (1.8 microm), longissimus lumborum (1.8 microm), and biceps femorus (1.7 microm). Proteolysis of desmin was greatest (P < .05) in longissimus lumborum (39.3%), followed by semimembranosus (21.0%) and biceps femoris (18.5%), then semitendinosus (.2%) and triceps brachii (.2%). Multiple linear regression using total collagen, sarcomere length, and proteolysis accounted for 57% of the variation in tenderness rating among all samples. Piecewise linear regression was used to account for the interaction of sarcomere length with proteolysis and collagen. This analysis accounted for 72% of the variation in tenderness rating. Variation in collagen, proteolysis, and sarcomere length and the degree of their interaction with one another determine the tenderness of individual muscles.     

Micro-calpain is essential for postmortem proteolysis of muscle proteins

The objective of this investigation was to test the hypothesis that -calpain is largely responsible for postmortem proteolysis of muscle proteins. To accomplish this objective, we compared proteolysis of known muscle proteins in muscles of wild type and micro-calpain knockout mice during postmortem storage. Knockout mice (n = 6) were killed along with control mice (n = 6). Hind limbs were removed and stored at 4 degrees C. Muscles were dissected at 0, 1, and 3d postmortem and subsequently analyzed for degradation of nebulin, dystrophin, metavinculin, vinculin, desmin, and troponin T. In a separate experiment, hind limb muscles from knockout (n = 4) and control mice (n = 4) were analyzed at 0, 1, and 3 d postmortem using casein zymography to confirm that mu-calpain activity was knocked out in muscle and to determine whether or not m-calpain is activated in murine postmortem muscle. Cumulatively, the results of the first experiment indicated that postmortem proteolysis was largely inhibited in micro-calpain knockout mice. The results of the second experiment established the absence of micro-calpain in the muscle tissue of knockout mice and confirmed the results of an earlier study that m-calpain is active in postmortem murine muscle. The results of the current study show that even in a species in which m-calpain is activated to some extent postmortem, micro-calpain is largely responsible for postmortem proteolysis. This observation excludes a major role for any of the other members of the calpain family or any other proteolytic system in postmortem proteolysis of muscle proteins. Therefore, understanding the regulation of micro-calpain in postmortem muscle should be the focus of further research on postmortem proteolysis and tenderization of meat.     

Technical note: Sampling methodology for relating sarcomere length,collagen concentration,and the extent of postmortem proteolysis to beef and pork longissimus tenderness

The objective of this study was to determine the effect of sampling methodology on the relationship between longissimus tenderness and measures of biochemical meat traits. Sampling methodology included measurements of sarcomere length, collagen concentration, and postmortem desmin proteolysis on raw samples and measurements of these same traits on the same cooked meat used for shear force measurement. Twenty crossbred steers and 20 crossbred barrows were used for these studies. The beef longissimus thoracis were vacuum-packaged, stored at 2 degrees C until 14 d postmortem, then frozen and stored at -30 degrees C. The pork longissimus thoracis et lumborum were vacuum-packaged, stored at 2 degrees C until 7 d postmortem, then frozen and stored at -30 degrees C. Trained sensory panel tenderness rating ranged from 3.1 to 7.6 for beef and 4.1 to 7.4 for pork. The coefficient of variation was lower for sarcomere length than for all other traits. Simple correlation coefficients between measurements on raw and cooked samples were 0.58 (beef) and 0.11 (pork) for sarcomere length, 0.66 (beef) and 0.59 (pork) for collagen, and 0.74 (beef) and 0.76 (pork) for desmin degradation. Simple correlation coefficients between biochemical traits and measures of tenderness (Warner-Bratzler shear force and trained sensory tenderness rating) were higher or not different for cooked compared to raw samples. Correlation coefficients between biochemical traits and tenderness rating were 0.38 (raw) and 0.22 (cooked) for sarcomere length, -0.12 (raw) and -0.45 (cooked) for collagen, and 0.48 (raw) and 0.80 (cooked) for desmin degradation in beef longissimus and 0.14 (raw) and 0.15 (cooked) for sarcomere length, -0.38 (raw) and -0.33 (cooked) for collagen, and 0.53 (raw) and 0.67 (cooked) for desmin degradation in pork longissimus. The coefficients of determination for explaining variation in tenderness rating using sarcomere length, collagen concentration, and desmin degradation for raw and cooked samples were 0.43 and 0.73 (beef) and 0.48 and 0.57 (pork), respectively. This study indicates that measurements of biochemical traits on the same cooked meat as used for shear force determination account for more of the variation in measures of tenderness than biochemical measurements made on a separate raw sample.     

Myofibrillar proteolysis in chick muscle cell cultures during heat stress

The effect of heat stress on protein oxidation and myofibrillar proteolysis in chick myotubes was investigated. Myotubes were incubated at 37 or 41°C for 6 and 24 h. Protein carbonyl content, as an index of protein oxidation, increased more at 41°C than at 37°C for 6 and 24 h incubations. N^τ‐methylhistidine release as an index of myofibrillar proteolysis also increased more at 41°C than at 37°C for 6 and 24 h incubations. Proteasome activity also increased more under those same conditions. Calpain and cathepsin D but not B + L activities showed a greater increase at 41°C than at 37°C for 24 but not the 6 h incubation. These results indicate that heat stress increases protein oxidation and proteasome activity, resulting in an increase in myofibrillar proteolysis for short‐term incubation and, for long‐term incubation, it increases calpain, proteasome and cathepsin D activities, finally accelerating myofibrillar proteolysis in chick myotubes.     

Estrogen receptor in bovine skeletal muscle

In connection with investigations of the anabolic action of estrogens, we examined skeletal muscle of veal calves for estradiol receptors. The high speed supernatant of muscle homogenate was incubated with .5 nM 3H-estradiol and for the determination of nonspecific binding with .5 nM 3H-estradiol plus 13 nM estradiol at 0 C overnight. After treatment with charcoal two times, the supernatant was analyzed by agar gel electrophoresis. Specific binding was found in the typical position of cytosolic estradiol receptor. Ninety percent of 3H-estradiol binding was suppressed by estradiol-17 beta, zeranol, estrone or diethylstilbestrol, but was not affected by testosterone, dihydrotestosterone, trenbolone or progesterone. The specific binding activity varied between .3 and 2.0 fmol/mg protein and the dissociation constant of the receptor was Kd = 60 pM. After an enrichment up to 42 fmol/mg cytosolic protein using heparin sepharose, the receptor remained unchanged as determined by agar gel electrophoresis. Although uterine tissue generally contains 1,000 times more estradiol receptors, these results clearly demonstrate that skeletal muscle also contains estradiol receptors with identical properties. This indicates that one possible component of the anabolic action of estrogens may be the direct stimulation of the muscle via the estradiol receptor.     

Postmortem magnesium concentration in bovine vitreous humor: comparison with antemortem serum magnesium concentration

The range for postmortem vitreous humor Mg2+ concentration in 97 healthy cattle was 1.8 mg/dl to 2.72 mg/dl at 23 C for a 48-hour postmortem interval. The postmortem vitreous Mg2+ concentration closely paralleled the antemortem serum Mg2+ concentration at 23 C. Low environmental temperature (4 C) had no effect on postmortem vitreous concentration. However, high environmental temperatures (30 C) significantly (P less than 0.05) reduced postmortem vitreous Mg2+ concentration at the 36-hour postmortem interval. It was concluded that postmortem vitreous humor Mg2+ determination could be a useful diagnostic aid in cattle for detecting Mg2+ imbalances for at least 48 hours after death, provided the postmortem environmental temperature did not exceed 23 C after 24 hours.     

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.     

牛肌肉中的蛋白质组学研究进展

蛋白质组学的研究是生命科学进入后基因时代的特征,它已在医学、畜牧学、食品科学以及农业等领域得到广泛的研究与应用。牛肉因其蛋白质含量高、脂肪含量少、血红素铁丰富等,深受人们的喜爱。但目前对我国而言,牛肉仍需要大量进口,因此对于如何提高牛肉品质一直是我国畜牧和食品行业科研工作者不断努力研究的一大课题。随着基因组学的深入研究发展,从蛋白质水平研究牛肌肉及其形成机制,对提高牛肉品质有着重要的意义。肌肉主要是由水、蛋白质等成分构成,其性状及品质主要是由蛋白质来表达的。因此,研究蛋白质组学在牛肌肉中的认知对于调控牛肉的品质具有重要意义。本文主要对蛋白质组学的研究技术及其在牛肌肉中的研究做简要介绍。     

Susceptibility of Pacific herring to viral hemorrhagic septicemia is influenced by diet

Groups of specific-pathogen-free Pacific herring Clupea pallasii were highly susceptible to infection by viral hemorrhagic septicemia virus (VHSV); however, the level of mortality was influenced by diet during the 40-71 d before, during, and after the first exposure to the virus. Cumulative mortality was highest among the herring maintained on an experimental soy-based pellet, intermediate among those maintained on a commercially available fish-meal-based pellet, and lowest among those maintained on a second commercially available fish-meal-based pellet containing beta-glucans. Additionally, the herring maintained on the experimental soy-based feed demonstrated less growth than those on the commercially available feeds. The results indicate the importance of standardizing diet during empirical determinations of disease susceptibility and provide insights into the risk factors affecting VHS susceptibility in wild populations.     

Abundance of some skeletal muscle mitochondrial proteins is associated with increased blood serum insulin in bovine fetuses

The aim of this study was to investigate the evolution of the abundance of cytochrome oxidase c subunit IV (NCOIV) and beta subunit of ATP synthase (β-ATP) during the last third of gestation in bovine skeletal muscles. Semitendinosus, longissimus thoracis and rectus abdominis muscles were chosen for the immunoblotting of the respective protein levels. Muscle and blood samples from bovine fetuses of randomly selected breeds were collected at 180, 210, and 260 days post-conception (dpc). The muscle tissue expressions of NCOIV, β-ATP were compared to blood glucose and insulin. At 260 dpc, protein levels of NCOIV raised in skeletal muscles. Additionally, β-ATP in semitendinosus and longissimus thoracis were elevated and paralleled by higher concentrations of blood serum insulin. It corroborates our previous observations indicating that accelerated metabolic differentiation of bovine skeletal muscles is associated with elevated blood insulin and occurs during the last trimester of gestation. Our observations point to the connection between insulin-sensitivity and the molecular mechanisms of mitochondrial contribution to ontogenesis of skeletal muscles.