Postmortem role of calpains in Pekin duck skeletal muscles

BACKGROUD: It is generally agreed that calpains are involved in postmortem proteolysis of skeletal muscle and improve meat tenderness. However, little information regarding the postmortem role of calpains in duck skeletal muscle is known. Therefore, the purpose of this study was to examine the role of calpains in Pekin duck postmortem breast muscles (BM) and leg and thigh muscles (LM) muscles at 5 °C. RESULTS: The postmortem pH was lower (P < 0.05) in BM than in LM. Western blots indicated that postmortem desmin degradation and the 30/32 kDa troponin-T degradation product accumulation were more rapid in BM than in LM. Casein zymograms showed that at-death μ-calpain activity was higher in BM than in LM. As time post mortem increased, μ-calpain was activated and autolyzed more rapidly and extensively in BM than in LM, but μ/m-calpain was activated at a relative slower rate compared with μ-calpain. Correlation results showed that μ-calpain activity, rather than μ/m-calpain activity, in BM samples was highly correlated with the abundance of desmin and the 30/32 kDa troponin-T degradation components across the postmortem period. However, no such correlations were found with LM μ- and μ/m-calpains. CONCLUSION: Therefore, our results suggest that BM μ-calpain with a faster and more extensive activation and autolysis would play a relatively dominant role in dictating degradation of desmin and troponin-T in postmortem duck muscle.     

The effect of temperature on the activity of μ- and m-calpain and calpastatin during post-mortem storage of porcine longissimus muscle

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 μM from 440 μM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain.     

Partial Purification and Characterization of Chinook Salmon (Oncorhynchus tshawytscha) Calpains and an Evaluation of Their Role in Postmortem Proteolysis

The involvement of calpains in the proteolysis of salmon muscle during refrigerated storage was investigated. Salmon m-calpain and μ-calpain were purified and partially purified, respectively. Salmon μ-calpain had similar calcium requirements and specific activity against casein as ovine m-calpain. Salmon μ-calpain had similar calcium requirements as ovine μ-calpain but a lower specific activity against casein than ovine μ-calpain. Autolysis patterns of both calpains differed from those of ovine calpains, and their activities were less than those of mammalian muscles. Calpastatin activity was relatively high and comparable to that of bovine muscles. Little proteolysis and fiber fragmentation resulted during refrigerated storage. However, proteolysis could be reproduced by incubation of myofibrils with m-calpain.     

Identification of calpastatin, μ-calpain and m-calpain in Atlantic salmon (Salmo salar L.) muscle

A soft fish muscle is generally considered as a poor quality trait among consumers and producers. This degradation and softening of post mortem muscle is thought to be partly caused by proteolytic enzymes such as the calpain system. Separation and identification of μ-calpain and m-calpain and their inhibitor – calpastatin, from Atlantic salmon (Salmo salar) muscle were for the first time assessed in this study. A two-step chromatography approach was used, starting with a hydrophobic interaction column and followed by an anion exchange column. Calpastatin was successfully separated from calpain by hydrophobic interaction chromatography, and following the anion exchange chromatography, two forms of calpastatin (I and II) and two forms of calpain (micro (μ) and milli (m)) were revealed. The proteolytic activity of μ-calpain was detectable with column chromatography, but not consistently detected with casein zymography, and m-calpain was detected with both chromatography and casein zymogram. The proteolytic activity of m-calpain per g muscle was 15 times higher than that of μ-calpain. μ-Calpain had a temperature optimum of 15 °C and a maximum calcium requirement at 0.2 mM, while m-calpain had temperature optimum at 25 °C and a maximum calcium requirement of 0.6 mM. The two forms of calpastatin differed in inhibitory activity with calpastatin II having the highest activity. Both calpastatins tolerated heat treatment, as previously seen for mammals, and they kept their activity when stored at −80 °C, but not at −20 °C. The calpain to calpastatin ratio was 1:4.5 as observed for beef muscle. This study provides evidence that two calpain isoforms, likely to be μ- and m-calpain, in addition to two forms of calpastatin exist in Atlantic salmon muscle.     

Rate and extent of pH decline affect proteolysis of cytoskeletal proteins and water-holding capacity in pork

The objective of this study was to determine the extent to which early postmortem (PM) pH decline influences proteolysis of the intermediate filament protein desmin, the costameric proteins vinculin and talin and autolysis of μ-calpain in the longissimus muscle (LM) of pigs from two genetic lines. Based on the LM 3 h pH (H = 3 h pH of LM > 6.0; L = 3 h pH of LM pH < 5.7) PM, 10 carcasses per line and pH group were selected. The average 3 h pH within pH group was 6.23 (H) and 5.44 (L). The LM samples were collected 24, 48, 72, and 120 h PM and percent drip loss was measured after 1, 2, and 4 d of storage. Samples collected at 24, 48, 72, and 120 h PM were used to monitor desmin, vinculin, and talin degradation and samples collected at 24 h PM were used to determine the extent of μ-calpain autolysis by immunoblotting. Higher (P < 0.01) pH values at 45 min, 6 h, and 24 h PM and lower (P < 0.01) drip losses after 1, 2, and 4 d of storage were recorded in the H-compared to the L-group. Abundance of the 76 kDa μ-calpain autolysis product was greater (P < 0.01), proteolysis of talin at all measured time points and proteolysis of desmin after 24 and 48 h PM was greater (P ? 0.03) in the H-group than in the L-group. The current findings indicate activation rate of μ-calpain may be associated with proteolysis of desmin and talin and could play a role in the development of drip loss. The rate of early PM pH decline can partly explain the variation of desmin and talin degradation by affecting the activation of μ-calpain.     

Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system

Tenderness has been repeatedly reported as the most important quality aspect of meat. However, a number of studies have shown that a significant portion of retail meat can be considered tough. As a consequence, a significant consumer segment is willing to pay a premium for guaranteed tender meat. However, apart from measuring the shear force, there is no reliable method to predict tenderness. Most of the branded meat programs therefore attempt to ensure eating quality by controlling some of the factors that affect tenderness. Meat tenderness is determined by the amount and solubility of connective tissue, sarcomere shortening during rigor development, and postmortem proteolysis of myofibrillar and myofibrillar-associated proteins. Given the effect of postmortem proteolysis on the muscle ultrastructure, titin and desmin are likely key substrates that determine meat tenderness. A large number of studies have shown that the calpain proteolytic system plays a central role in postmortem proteolysis and tenderization. In skeletal muscle, the calpain system consists of at least three proteases, μ-calpain, m-calpain and calpain 3, and an inhibitor of μ- and m-calpain, calpastatin. When activated by calcium, the calpains not only degrade subtrates, but also autolyze, leading to loss of activity. m-Calpain does not autolyze in postmortem muscle and is therefore not involved in postmortem tenderization. Results from a number of studies, including a study on calpain 3 knockout mice, have shown that calpain 3 is also not involved in postmortem proteolysis. However, a large number of studies, including a study on μ-calpain knockout mice, have shown that μ-calpain is largely, if not solely, responsible for postmortem tenderization. Research efforts in this area should, therefore, focus on elucidation of regulation of μ-calpain activity in postmortem muscle. Discovering the mechanisms of μ-calpain activity regulation and methods to promote μ-calpain activity should have a dramatic effect on the ability of researchers to develop reliable methods to predict meat tenderness and on the meat industry to produce a consistently tender product.     

Changes in calpain and calpastatin activities of osmotically dehydrated bovine muscle during storage after treatment with calcium

Calpain and calpastatin activities were investigated in calcium-treated beef after osmotic dehydration. Dehydrated beef was soaked in 150 mM calcium chloride solution for 3 h, and then stored for 48 h at 3-4 °C. The untreated sample (control) was soaked in deionized water for 3 h instead of calcium chloride solution, after osmotic dehydration. The increase and decrease in the relative activity of crude calpain were observed in the untreated and the calcium-treated meat, respectively, during the storage. When the crude calpains were subjected to DEAE-Sephacel column chromatography, it was found that μ-calpain activity decreased rapidly during the storage in the untreated meat, whereas there was almost no change in the activity of m-calpain during the storage. The decrease of calpastatin activity was moderate compared with the decrease of μ-calpain activity. In the calcium chloride-treated meat, however, no μ-calpain nor calpastatin activities was detectable after 48 h at cold-room temperature, and m-calpain activity after 48 h had decreased to 6.1% of its activity immediately after thawing. It was concluded that 150 mM calcium chloride treatment after osmotic dehydration was sufficient to introduce calcium ions into the meat. In the presence of sufficient calcium, autolysis of calpains and proteolytic degradation of calpastatin, which eventually related to the rate of decrease in calpain and calpastatin activities, clearly seem to be related to a decrease in meat toughness.     

Calpain-calpastatin and toughness in M. longissimus from electrically stimulated lamb and beef carcasses

Shear strength, pH, temperature, μ-calpain, m-calpain and calpastatin levels were measured over a two-week post-slaughter period in Longissimus lumborum et thoracis (LD) from six lamb and six beef carcasses. All carcasses were subjected to high voltage electrical stimulation. The toughness of the beef LD determined by a MIRINZ tenderometer at 24 h post-slaughter showed a strong correlation (r=0.91) with pH of the LD at 3 h. Beef LD toughness at 14 days was correlated (r=0.84) with initial m-calpain levels. In both lamb and beef, LD toughness at 4 and 14 days respectively was also correlated with initial levels of calpastatin (r=0.85, 0.83, respectively). The strong correlation between calpastatin and the rate of tenderisation indicates that the calpain system is closely linked to the proteolytic breakdown of myofibrillar proteins. There is also evidence of an interaction between pH and μ-calpain activity. The μ-calpain, m-calpain, calpastatin, pH and temperature kinetic changes which occurred during the post-mortem ageing of beef and lamb LD were applied to a computer program which predicted rate of meat tenderisation by calculating in situ calpain activity. The closeness of fit between the predicted rate of meat tenderisation and the observed tenderness values of beef and lamb LD indicates that the post-mortem activity of μ-calpain is the major determinant of variations in tenderness. However, application of the meat tenderisation predictive program to LD from individual animals revealed that the program was not sufficiently robust for this use.     

Contribution of postmortem changes of integrin, desmin and μ-calpain to variation in water holding capacity of pork

The purpose of this study was to examine the relationship between integrin, desmin, μ-calpain and water holding capacity in fresh pork. High levels of intact integrin at one day postmortem were negatively correlated with day 1 (P<0.05) and days 1-5 (cumulative) drip loss (P<0.05). High levels of intact integrin at five days postmortem were negatively correlated with days 1-7 (cumulative) purge loss (P<0.05). Intensity of intact desmin at one day postmortem was positively correlated with days 1-7 purge loss (P<0.01). There were positive correlations between intensity of intact desmin at day 7 and day 1 (P<0.01), days 1-5 drip loss (P<0.01) and days 1-7 purge loss (P<0.05). Autolysis of μ-calpain was associated with the degradation of desmin and drip or purge loss postmortem. Our results indicate that low levels of degradation of integrin and high levels of desmin degradation were associated with low drip loss values in fresh pork.     

Small heat shock proteins and toughness in intermediate pHu beef

Bull M. longissimus dorsi (n = 94) categorised into high (n = 28), intermediate (n = 14) and low (n = 52) ultimate pH (pH_u) were aged at − 1.5 °C for 28 days. Shear force was higher and more variable (p < 0.05) in intermediate pH_u samples during ageing. Titin, filamin and desmin degradation was also less extensive in intermediate pH_u samples compared to the other two pH categories. The extent of the decline of HSP20, HSP27 and αβ-crystallin concentrations during post mortem ageing was pH_u related such that high pH_u meat maintained the highest concentration of small heat shock proteins followed by intermediate and low pH_u meat. μ-Calpain autolysis was slowest in intermediate pH_u and cathepsin B activities remained consistently low during ageing in this group (p < 0.05). Meat toughness in the intermediate pH_u group may be attributed to the combination of a larger pool of sHSP with a sub-optimal cathepsin B activity and intermediary μ-calpain activities.     

Effect of proteolytic enzyme activity and heating on the mechanical properties of bovine single muscle fibres

The effect of proteolysis and heating on the mechanical properties of single muscle fibres isolated from bovine M. semitendinosus was investigated. Short incubations with μ-calpain (pH 7.5) and cathepsin B (pH 5.6) at room temperature reduced (P<0.01) the raw fibre strength by 50%. After subsequent heating, μ-calpain-incubated fibres were 20% weaker and cathepsin B-incubated fibres 50% weaker than heated controls. Fibres were incubated for 8 days at 2?°C under post-mortem like pH conditions (pH 5.6) or pH conditions optimal for the calpains (pH 7.5). At pH 5.6 no significant weakening of the fibres was observed when incubations were performed in 0.1, 1 or 10 mM Ca(2+). In contrast, incubation in 1 or 10 mM Ca(2+) at pH 7.5 decreased the strength of the fibres. Addition of the protease inhibitors E-64 or PD150,606 prevented this weakening, suggesting that the weakening is caused by proteolyses and not directly by Ca(2+).     

The protection of bovine skeletal myofibrils from proteolytic damage post mortem by small heat shock proteins

This study aimed to determine how small heat shock proteins (sHSPs) protect myofibrillar proteins from μ-calpain degradation during ageing. Immunoprecipitation experiments with M. longissimus dorsi (LD) from Angus heifers (n = 14) examined the interaction between αβ-crystallin, desmin, titin, HSP20, HSP27 and μ-calpain. Results showed that αβ-crystallin associated with desmin, titin, HSP20, HSP27 and μ-calpain. Exogenous αβ-crystallin reduced desmin and titin degradations in myofibrillar extracts and attenuated μ-calpain activity. In a second experiment, bull LD (n = 94) were aged at − 1.5 °C for up to 28 days post mortem. μ-Calpain autolysed faster in high ultimate pH (pH_u) meat (pH_u ≥ 6.2) and this was concomitant with the more rapid degradation of titin and filamin in this pH_u group. Desmin stability in intermediate pH_u meat (pH_u 5.8 to 6.19) may be due to the protection of myofibril-bound sHSPs combined with the competitive inhibition of μ-calpain by sHSPs.     

钙蛋白酶系统与肌肉嫩度的关系

钙蛋白酶系统主要由钙蛋白酶(μ-calpain,m-calpain)及钙蛋白酶抑制蛋白(calpastatin)组成,calpain是存在于细胞质中的依赖于Ca2+的中性蛋白酶,calpastatin是钙蛋白酶的内源抑制蛋白。本文综述了钙蛋白酶系统各种酶的结构、作用、活性调节机能及其与肉质嫩度的关系。     

Studies of Desmin and α-Actinin Degradation in Bovine Semitendinosus Muscle

The degradation of desmin and α-actinin was studied in post-mortem bovine semitendinosus muscle. Using a desmin-specific monoclonal antibody, SDS-PAGE, and immunoblotting we show that desmin is easily degraded at 4°C during the aging process. Within 96 hr, fragments of degraded desmin are detected with our antibody probe. Further storage at 4°C results in an increase of proteolytic fragments and concomitant loss of intact desmin. By 3 wk post-mortem, little un-degraded desmin remains in the muscle. In contrast, α-actinin was degraded slowly at 4°C. Proteolytic fragments of α-actinin were not detectable with anti-α-actinin polyclonal antisera until the second week of incubation at 4°C. However, the degradation of α-actinin was accelerated when meat was incubated at 25°C or at 37°C.     

Identification of myofibrillar substrates for μ-calpain

To identify myofibrillar substrates of μ-calpain under post-mortem conditions, a combination of SDS–PAGE, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used. Purified myofibrils were incubated with μ-calpain under post-mortem-simulated conditions for two or four days at 4 °C. The resulting protein changes were analyzed by SDS–PAGE and 2DE. The μ-calpain-mediated protein changes were identified by peptide-mass mapping using MALDI-TOF MS and revealed that desmin, actin, myosin heavy chain, myosin light chain I, troponin T, tropomyosin 1, tropomyosin 4, thioredoxin and CapZ are all degraded in vitro by μ-calpain. The findings that actin and myosin heavy chain are substrates of μ-calpain were rather surprising, as it has previously been reported that these proteins are resistant to μ-calpain degradation. However, both actin and myosin heavy chain are poor substrates compared with desmin.     

Effect of stress-induced high post-mortem pH on protease activity and tenderness of beef

Forty-four Swiss Brown young bulls were stressed by regrouping unfamiliar animals before slaughter. M. longissimus thoracis (6-9th ribs) of carcasses were analysed for post-mortem pH, protease activities (m- and α-calpain, calpastatin and cathepsin B + L), Warner-Bratzler shear force and sensory tenderness and juiciness. Muscles were classified into three groups, according to ultimate pH values: > 6.3, 6.3-5.8 and < 5.8. The most significant difference related to high pH was a higher activity of m-calpain at 7th day post mortem. It was also found that meat showing the highest pH was significantly more tender and juicy. Sensory tenderness was highly correlated with activity of m-calpain at 7th day post mortem (r = 0.776) and with ultimate pH (r = 0.708). It is concluded that high ultimate pH induced by stress significantly increases m-calpain activity, and this results in a greatly enhanced tenderisation of beef meat.     

Hind-limb protein metabolism and calpain system activity influence post-mortem change in meat quality in lamb

This study is concerned with the rate of protein turnover in the hind limb muscle bed of intact lambs, the activity of calpain proteolytic system in the M. biceps femoris, and subsequent rates of myofibre breakdown and tenderisation in the M. longissimus dorsi. Feed restriction increased protein degradation in hind-limb muscle of lambs (p<0.1), with a concominant decrease in the extractable activity of calpastatin (p<0.01), the endogenous inhibitor of calpain. IGF-1 analog treatment decreased both protein degradation and assayed μ-calpain activity (p<0.05) with no effect on the activity of calpastatin. β-Agonist treatment increased hind-limb protein synthesis (p<0.01), calpastatin activity (p<0.1) and decreased (p<0.01) μ-calpain activity, but did not effect protein degradation. Significant correlations were observed between Myofibril Fragmentation Index (MFI) values during post-mortem storage and initial post-slaughter calpastatin activity at days 3 (r=-0.34, p<0.1), 5 (r=-0.58, p<0.01) and 9 (r=-0.58, p<0.1), and μ-calpain activity at days 5 (r=0.35, p<0.1) and 9 (r=0.41, p<0.05). However, stronger correlations were observed between the ratio of μ-calpain to calpastatin, an estimate of potential μ-calpain proteolytic activity, and the rate of myofibril fragmentation (r=0.75, p<0.001) and tenderisation (r=-0.64, p<0.01) during aging. These results are consistent with the calpain system being the major proteolytic system involved in myofibril fragmentation and hence aging-related tenderisation of meat.     

Post-mortem kinetics of meat tenderness and the components of the calpain system in bull skeletal muscle

Eight strip loins (M. longissimus dorsi) from pasture fed Friesian bulls were aged at 15 °C for a range of times from 1 to 120 h. pH declined from 6.29 (SE 0.119) one hour post slaughter to an ultimate pH of 5.48 (SE 0.013). The activities of the components of the calpain system (μ-calpain, m-calpain and calpastatin) were determined after separation on a DEAE-sephacel column. There was a dramatic decline in μ-calpain activity post slaughter with a complete disappearance within 48 h. The rates of decline in m-calpain and calpastatin activity were slower with 30% and 50% remaining 120 h post slaughter, respectively. The rapid decline in μ-calpain activity relative to the calpastatin activity is likely to reduce the degree of tenderisation and ultimate tenderness of the meat.     

Effect of added μ-calpain and post-mortem storage on the mechanical properties of bovine single muscle fibres extended to fracture

The effects of μ-calpain and post-mortem storage on the strength of single muscle fibres were investigated. During the 10 min of incubation at pH 7.5, μ-calpain became evenly distributed throughout the fibre. μ-Calpain-incubation resulted in thinner (P <0.001) Z-lines and reduced (P <0.001) the strength of the fibres compared to controls. These results demonstrate that μ-calpain is capable of mechanically weakening the muscle fibres. Post-mortem storage of meat for 10 days at 2?°C weakened (P <0.001) the muscle fibres compared to 24-h fibres. The presence or absence of Ca(2+) affected fibre stiffness. Fibres incubated at pH 7.5 in 100 μM Ca(2+) were less stiff than fibres incubated in 200 μM EGTA. Breaking stress and strain were not affected by Ca(2+). We hypothesise that Ca(2+) causes conformational changes in some of the load-bearing proteins, which alters their initial resistance to extension, but does not affect the breaking strength of the fibres.     

Factors regulating lamb longissimus tenderness are affected by age at slaughter

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