Calpastatin Activity Research Articles

Effect of sire on mu- and m-calpain activity and rate of tenderization as indicated by myofibril fragmentation indices of steaks from Brahman cattle.

The objectives of this study were to assess the influence of sire on mu- and m-calpain activities, to evaluate the relationships of activities of these enzymes to other traits related to beef palatability, and to assess the influence of sire on the rate of tenderization (as measured by myofibril fragmentation index [MFI]) in Brahman longissimus muscle. Brahman calves (n = 87), sired by nine bulls, were born, weaned, fed, and slaughtered in central Florida. Traits evaluated were mu- and m-calpain activities and MFI after 1, 7, 14, and 21 d of aging. Other traits were analyzed to determine their associations with mu- and m-calpain activity and MFI, including calpastatin activity, percentage of raw and cooked lipids, Warner-Bratzler shear force (WBSF) values after 7, 14, and 21 d of aging, and sensory panel rating of tenderness, juiciness, and connective tissue amount after 14 d of aging. Data were analyzed using a model with sire, sex, year, and slaughter group (calves of the same sex slaughtered on the same date) as fixed effects, and adjusted to a constant adjusted 12th-rib fat thickness. Sire affected mu-calpain activity (P < 0.04), calpastatin activity (P < 0.01), d-14 MFI (P < 0.02), d-7 WBSF (P < 0.05), d-14 WBSF (P < 0.04), and sensory panel juiciness score (P < 0.01), but not (P < 0.75) m-calpain activity. Measures of tenderness and palatability were generally moderately to strongly correlated (both simple and residual correlations) with calpastatin and m-calpain activity. Myofibril fragmentation index residuals (adjusted for all model components except sire) after all aging periods were fitted using nonlinear regression to the exponential curve (MFI(i) = kappa0 + kappa1 exp[kappa2 t(i)] + epsilon(i), where t(i) represents aging in days, k0 is ultimate MFI after aging, kappa1 is the difference between initial and ultimate MFI, kappa2 is the rate of increase in MFI, and epsilon(i) is the error term associated with the ith observation, assumed to be independent and identically distributed normally). Sires had different estimates and combinations of estimates, which were used to plot MFI change with time. These curves visually differed for sires and suggested that postmortem tenderization extent and rate differ as well. Use of a combination of these estimated parameters in a selection/carcass sorting program represents an alternative consideration for tenderization improvement programs.

Characterization and consumer acceptance of three muscles from Hampshire × Rambouillet cross sheep expressing the callipyge phenotype1

Eight Hampshire x Rambouillet crossbred wethers expressing the callipyge phenotype and eight Hampshire x Rambouillet half-sibling wethers with a normal phenotype were slaughtered when they reached 59 kg. The supraspinatus (SPM), longissimus (LM), and semitendinosus (STM) muscles were analyzed to determine callipyge effects on calpain and calpastatin activities, sarcomere length, percentage of muscle fiber types, and muscle fiber areas. After 14 d of aging, chops were frozen until analyses for trained sensory panel evaluations, Warner-Bratzler shear force values, and consumer perceptions of tenderness, flavor, juiciness, and overall satisfaction of chops were conducted. Calpastatin activity was 57% greater (P < 0.05) and m-calpain activity was 33% greater (P < 0.05) in muscles from carcasses of callipyge than normal sheep. Sarcomeres were shorter (P < 0.001) in the LM than the SPM or STM, regardless of phenotype. Muscle fiber area was 76% larger (P < 0.05) in the LM of callipyge than normal sheep, but muscle fiber area was not affected (P > 0.05) by phenotype in the SPM or STM. Phenotype had no effect (P = 0.12) on the percentage of slow-twitch, oxidative fiber types in any of the three muscles. In STM and LM from callipyge lambs, the percentage of fast-twitch, oxidative/glycolytic fibers was lower (P < 0.05) and that of fast-twitch-glycolytic fibers was higher (P < 0.05) than in their normal counterparts. Phenotype did not affect (P = 0.90) the fiber type percentage in the SPM. Callipyge LM were less tender and normal LM were more tender than other chops (P < 0.05). Callipyge loin chops had higher Warner-Bratzler shear force values than other chops (P < 0.001). Consumers rated fewer (P < 0.05) callipyge loin and shoulder chops acceptable in juiciness, tenderness, and overall acceptability than normal chops, but phenotype did not affect (P > 0.05) consumer acceptability of leg chops. These results indicate that LM from Hampshire x Rambouillet sheep displaying the callipyge phenotype had higher calpastatin activity and were less tender than the LM from normal sheep. In addition, consumer perceptions indicated that only one in 10 leg chops, one in five shoulder chops, and one in four loin chops from callipyge sheep were unacceptable.

Glucocorticoids preserve calpastatin and troponin I during cardiopulmonary bypass in immature pigs.

Degradation of troponin I (TnI) by calpain occurs with myocardial stunning in ischemia-reperfusion injury. Glucocorticoids attenuate myocardial ischemia-reperfusion injury, but their effect on TnI degradation is unknown. A piglet model was used to test the hypotheses that cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) are associated with TnI degradation and that TnI alterations could be prevented by glucocorticoid treatment. Piglets were cooled to 18 degrees C, subjected to 2 h of circulatory arrest, rewarmed to 37 degrees C, and allowed to recover for 2 h. Methylprednisolone was administered 6 h before surgery (3 0 mg/kg) and at initiation of CPB (30 mg/kg). The untreated group received saline. Left ventricular tissue was collected after recovery and analyzed by Western blot for TnI, calpain, and calpastatin (the natural inhibitor of calpain). CPB/DHCA animals had 27.4 +/- 0.2% of total detected TnI present in degraded form. Glucocorticoid treatment significantly decreased the percentage of degraded TnI (12.0 +/- 0.1%, p < 0.05). Calpain I and calpain II increased after CPB/DHCA compared with non-CPB/DHCA controls (p < 0.05), with or without glucocorticoid treatment. Calpastatin significantly decreased in untreated CPB/DHCA animals compared with non-CPB/DHCA controls (p < 0.05), but levels were preserved by glucocorticoids. Glucocorticoids were associated with preservation of maximum rate of increase of left ventricular pressure at 95 +/- 10% of baseline, whereas maximum rate of increase of left ventricular pressure decreased to 62 +/- 12% of baseline without steroids. TnI degradation occurs after CPB/DHCA in neonatal pigs. Reduction in reperfusion injury by glucocorticoids may depend partly on preservation of calpastatin activity and intact TnI.

Structural Determinants of the Calpain Inhibitory Activity of Calpastatin Peptide B27-WT

Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible for the pathological role of calpain. An important key to understanding calpain regulation by calpastatin is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. A 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from subdomain 1B of the repetitive domains of calpain, named peptide B27-WT, was previously shown to be a potent inhibitor of mu- and m-calpain. In this report, a combination of beta-alanine scanning mutagenesis and kinetic measurements was used to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of the amino acid side chain and backbone functionalities to the overall calpain-inhibitory activity of B27-WT. The study identified two "hot spots," Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19), in B27-WT within which the residues critical for inhibitory function are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity. Furthermore, it was shown that a restricted conformation of the Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19) backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. The information on the specific contribution of the amide bond and side chain of each key residue to the bioactivity of B27-WT will contribute to a better understanding of the mechanism of calpain inhibition and lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.

Estimated genetic parameters for palatability traits of steaks from Brahman cattle.

Heritabilities and genetic and phenotypic correlations were estimated from carcass and beef palatability data collected from Brahman calves (n = 504) born in central Florida from 1996 to 2000. Traits evaluated included Warner-Bratzler shear force (after 7, 14, and 21 d of aging), panel tenderness score, connective tissue amount, juiciness, flavor intensity, and off flavor (after 14 d of aging), percentages of raw and cooked lipids, and milligrams per gram of muscle calpastatin activity. Parameters were estimated using an animal model and derivative-free restricted maximum likelihood procedures. Estimated heritabilities for d 7, 14, and 21 shear force were 0.14,0.14, and 0.06, respectively, indicating that improvement in these traits by selection would be slow. Estimated heritabilities of sensory panel attributes were 0.11, 0.12, 0.05, 0.04, and 0.01 for tenderness, connective tissue amount, juiciness, flavor intensity, and off flavor, respectively. The estimated heritabilities for percentages of raw and cooked lipids, and calpastatin activity were 0.34, 0.17, and 0.07, respectively. Most of the estimated genetic correlations among palatability traits and for palatability traits with fat thickness, marbling score, and loin muscle area were consistent with other estimates from the literature. Results indicated that improvement in tenderness based on selection for favorable shear force, sensory panel tenderness, or calpastatin activity would be slow; therefore, postslaughter intervention programs should also be considered.

The Calpain-Calpastatin System and the Calcium Paradox in the Isolated Perfused Pigeon Heart

To examine whether the calpain-calpastatin system is activated during the calcium paradox in the isolated perfused pigeon heart, we separated the protease from its inhibitor calpastatin and studied its kinetic properties. The protease exhibits kinetic properties similar to those of mammalian m-calpains. Ca²⁺ requirements for half and maximum activities are 220 µM and 2 mM, respectively. In the absence of Ca²⁺ the protease is strongly activated by Mn²⁺ or Sr²⁺. In the presence of Ca²⁺, Mn²⁺ and Sr²⁺ exhibit a synergistic effect; Mg²⁺ and Ba²⁺ have no effect, whereas Co²⁺, Ni²⁺ and Cd²⁺ completely inhibit its activation. Furthermore, we measured the activity of calpain and calpastatin under either conditions inducing a calcium paradox, or protecting the heart against this phenomenon. Although the calpain/calpastatin ratio is lowered during Ca²⁺ depletion, during Ca²⁺ repletion it is markedly inverted. Calpain activation during reperfusion is inhibited by the presence of 200 µM Mn²⁺ or Ba²⁺, in the Ca²⁺-free medium. Gel filtration of calpastatin, isolated from either untreated hearts or during Ca²⁺ depletion, produces two main peaks of ñ150 and 40 kDa of molecular mass, respectively, whereas calpastatin isolated during the 2^nd min of reperfusion appears to be shifted to the 150 kDa form. All the above data suggest that this system may be involved in the induction of the calcium paradox in pigeon heart.

Dietary-induced changes of muscle growth rate in pigs: effects on in vivo and postmortem muscle proteolysis and meat quality.

The effects of various growth rates in pigs induced by four different feeding strategies on the activity of the calpain system and on postmortem (PM) muscle proteolysis and tenderness development were studied. An increased growth rate may be caused by an increased protein turnover, which results in up-regulated levels of proteolytic enzymes in vivo that, in turn, possibly will affect PM tenderness development. It can be hypothesized that increased proteolytic activity pre-slaughter will increase the PM tenderization rate. From postnatal d 28 to d 90 (phase 1) the pigs were divided into two groups, given either ad libitum (A) or restricted (R, 60% of ad libitum) access to feed. The two groups were then divided into two subgroups, given either restricted or ad libitum access to feed from d 91 to slaughter at d 165 (phase 2). Measurements of the activity of mu-calpain, m-calpain, and calpastatin; concentrations of total collagen and the percent of soluble collagen; and RNA, DNA, and elongation factor-2 where made at slaugther. Myofibrillar fragmentation index (MFI) was determined at slaughter and 24 h PM. Warner-Braztler shear force was determined 1 d and 4 d PM. Pigs fed restricted diets in phase 1 and fed ad libitum in phase 2 (RA pigs) had increased growth rates in the last phase compared to pigs fed ad libitum during both phase 1 and phase 2 (AA pigs). The increased growth rate (compensatory growth) was followed by an increased proteolytic potential (mu-calpain:calpastatin ratio), increased MFI values, and higher tenderization rates. There was a positive correlation between the activities of m-calpain and growth rates (r = 0.35, P = 0.03), and between RNA levels and growth rates (r = 0.43, P = 0.006). The proposed hypothesis is largely supported by the results. The activities of both mu- and m-calpain at slaughter were highest in fast-growing pigs. The calpain activity was highest in RA pigs, which in turn also had the fastest growth rates prior tslaughter among the four groups. This implies that the synthesis of these enzymes was up-regulated during the second feeding period to a larger extent in RA pigs. The proteolytic potential and the MFI values indicate that the up-regulated in vivo calpain activity had an effect on PM protein degradation, which also is supported by the higher tenderization rate in RA pigs.

Effects of a soy protein diet on exercise-induced muscle protein catabolism in rats

OBJECTIVE: We examined effects of dietary soy protein isolate on muscle calpain activity and myosin heavy chain (MHC) degradation in rats performing an acute running exercise. METHODS: In rats fed a 20% casein diet, the treadmill running exercise, fixed at 80 kg/m, transiently increased calpain activity in gastrocnemius muscles in parallel with the release of creatine kinase into plasma. The fixed running also caused an accumulation of immunoreactive degradation fragments of MHC in the muscle. Feeding a 20% soy protein isolate diet as opposed to the control casein diet to rats significantly suppressed the running-induced activation of μ- and m-calpains, fragmentation of MHC, and release of creatine kinase into plasma ( P < 0.05). RESULTS: Rats fed the soy protein isolate diet had significantly higher calpastatin activity in gastrocnemius muscle than did rats fed the casein diet ( P < 0.05), indicating that this increase ihibits the exercise-induced autoactivation of calpain. Activities of proteasome, cathepsin B + L, and antioxidant enzymes and the levels of glutathione and thiobarbituric acid-reactive substances in the muscle did not differ between the diet groups at the end of the exercise. CONCLUSIONS: Our results suggest that diets containing soy protein prevent exercise-induced protein degradation in skeletal muscle, possibly through inhibiting the calpain-mediated proteolysis.

Effect of growth rate on tenderness development and final tenderness of meat from Friesian calves

AbstractThe present study was conducted to determine the effect of growth rate of calves on the activity of the calpain system post mortem, post mortem desmin degradation, myofibrillar fragmentation index and meat tenderness of m. longissimus lumborum (LL) and m. supraspinatus (SS). Twenty-four Friesian heifer calves were allocated to two treatment groups: MM and HH. The MM calves were given food to achieve a moderate growth rate (678 g/day from 5 days of age to 90 kg body weight (BW) (period I) and 770 g/day from 90 kg BW to slaughter at 250 kg BW (period II)) and the HH calves were given food to achieve a high growth rate (period I: 895 g/day and period II: 1204 g/day). The myofibril fragmentation index (MFI), desmin degradation and the activity of µ-calpain, m-calpain and calpastatin were measured in LL and SS at slaughter, and 1 and 7 days post mortem. Shear force was measured in LL and SS after 1 and 7 days of ageing and a sensory panel evaluated the eating quality of the loin aged 7 days. MFI (P &lt; 0·01) and tenderness (P &lt; 0·01) were higher and shear force was lower (P &lt; 0·001) in meat from HH calves compared with meat from MM calves. However, growth rate did not affect the activity of µ-calpain, m-calpain or calpastatin or the degradation pattern of desmin at any time post mortem. Besides growth rate and MFI at day 7 post mortem, which both correlated positively with meat tenderness (P &lt; 0·001 and P &lt; 0·01, respectively), fibre type traits and DNA or RNA concentrations were not generally related to final tenderness. Thus, it was concluded that increased growth rate of calves before slaughter increased the ease of fragmentation (MFI) of the meat and improved the tenderness but these findings could not be related to any changes in the activity of the calpain system.

Influence of growth rate and muscle type on muscle fibre type characteristics, protein synthesis capacity and activity of the calpain system in Friesian calves

AbstractThe objective of this study was to determine the effect of growth rate on muscle fibre characteristics, concentration of nucleic acids (RNA and DNA) as indicators of muscle protein synthesis capacity and activity of the calpain system at time of slaughter in m. longissimus lumborum (LL) and m. supraspinatus (SS) from calves, in order to elucidate the effect of growth rate on muscle protein turn-over at time of slaughter. Twenty-four Friesian heifer calves were allocated to two different feeding regimens that allowed for a moderate/moderate (MM) or high/high (HH) growth rate from 5 days of age to 90 kg body weight (BW) (period I) and from 90 kg BW to slaughter at 250 kg BW (period II), respectively. The growth rates in the two periods and the weight of LL and SS at slaughter were recorded. Within 30 min after exsanguination, samples were removed from LL and SS, snap-frozen, and later analysed for muscle fibre type frequency and cross-sectional area, DNA and RNA concentration and the activity of the calpain system. High growth rate (i.e. 895 g/day and 1204 g/day in periods I and II, respectively), compared with moderate growth rate (678 g/day and 770 g/day in periods I and II, respectively) had a marked effect on muscle weight and muscle characteristics. High compared with moderate growth rate resulted in hypertrophic growth of type I, IIA and IIB fibres in LL and of type IIA and type IIB fibres in SS, but had no effect on the muscle fibre type frequency in either of the muscles. High growth rate increased total DNA and RNA content and the RNA: DNA ratio in LL, indicating a greater potential for protein synthesis in this muscle, whereas the effect of growth rate was smaller in SS. The activity of µ-calpain, m-calpain and calpastatin was higher in the red SS muscle compared with the whiter LL muscle. However, these enzyme activities were not affected by growth rate, and thus, did not indicate a higher myofibrillar proteolysis in vivo in calves exhibiting high growth rate compared with moderate growth rate. Overall the results showed that different types of muscles react differently to high versus moderate growth rate. High growth rate induced muscle hypertrophy and increased protein synthesis capacity especially in LL and less in SS, but the activities of the enzymes in the calpain system did not show any concomitant increase in muscle protein degradation that would be in favour of improved meat tenderness.

Effects of Dietary Treatment, Gender, and Implantation on Calpain/Calpastatin Activity and Meat Tenderness in Skeletal Muscle of Korean Native Cattle

The objectives of this study were to examine calpain activity and meat tenderness by three different feeding patterns in Korean native cattle (KNC). Total forty-five animals were assigned each fifteen in long term restriction feeding (LTFR), long-term restriction feeding and hormone treatment (LTFR-tH), and short term non-restriction feeding (STFNR), respectively. Concentrate was restricted based on body weight in exp 1 and 2. However, it was fed ad libitum in exp. 3. Hormonal implantation was made with M- PO TM for bulls and with F-TO TM for heifers at 18, 20, 22 months of age in exp. 2. Animals were purchased (3-5 month old) from local cattle market and managed in two local farms and university research unit at three different years. Animals were slaughtered at 24 months for long-term trial and at 18 month for short term-trial. Loin and tender loin muscle was used for calpain activity and meat quality. Calpain proteolytic system was not changed by treatment. However, calpastatin activity was low in short-term trial. The calpain and calpastatin activity is reciprocal relationship, therefore, the high calpain activity may effect on quality grade. The shear force value was decreased as the processing of aging after postmortem. On the other hand, the cooking loss was significantly higher in short-term than in long-term trial, and then gradually decreased by the aging. Hormone implants to increase meat yield influenced to calpastatin activity more powerfully than calpain activity to meat tenderness. In meat color-a*, there was not significant difference in loin. Meat color-b* was decreased as postmortem aging time increased in tenderloin. Western blots were done to learn whether these proteins are degraded during postmortem storage and whether this degradation temporally parallels the decrease of shear force value. Vinculin was detected at 0 day and 1 day and degraded after 3 day. In conclusion, Calpain activity was affected slightly on meat tenderness. But meat tenderness was influenced by calpastatin, more effectively. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 11 : 1653-1658)

Calpain, calpastatin activities and ratios during myocardial ischemia-reperfusion.

The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a 'calpain burst' impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or 60 min of ischemia followed by 30 min of reperfusion with and without pre-administration (s.c.) of a cysteine protease inhibitor (E-64c). Myocardial function, calpain activities (casein release assay), calpastatin inhibitory activity and release of CK, LDH, cTnI and cTnT were determined (n = 8 for all groups). No detectable changes in calpain activities were observed following I/R with and without E-64c (p > 0.05). Both I/R conditions reduced calpastatin activity (p < 0.05) while E-64c pre-treatment was without effect, implicating a non-proteolytic event underlying the calpastatin changes. A similar result was noted for calpain-calpastatin ratios and the release of all marker proteins (p < 0.05). In regard to cardiac function, E-64c resulted in transient improvements (15 min) for left ventricular developed pressure (LVDP) and rate of pressure development (p < 0.05). E-64c had no effect on end diastolic pressure (LVEDP) or coronary pressure (CP) during I/R. These findings demonstrate that restricting the putative early burst in calpain activity, suggested for I/R, by pre-treatment of rats with E-64c does not prevent downregulation of calpastatin inhibitory activity and/or protein release despite a transient improvement in cardiac function. It is concluded that increases in calpain isoform activities are not a primary feature of l/R changes, although the role of calpastatin downregulation remains to be elucidated.

Relationship between calpastatin activity and the slow and fast myosin heavy chain content of ovine skeletal muscles

Calpastatin, the specific endogenous inhibitor of the calpain system, is considered to be a principle contributor to variations in meat tenderisation (Parr et al., 1999). Previous studies have suggested that the differences in calpastatin activity in different ovine skeletal muscles could be influenced by muscle metabolic and contractile characteristics according to myofibrillar ATPase activity (Ouali and Talmant, 1990). The type of myofibrillar ATPase activity is largely determined by the content of slow or fast myosin heavy chains (Rivero et al., 1999). The present study was designed to investigate the relationship between calpastatin inhibitory activity and slow myosin heavy chain (MHC-s) and fast myosin heavy chain (MHC-f) expression.

Comparative Studies on Metabolic Rate and Calpain/Calpastatin Activity between Hanwoo and Holstein Beef

The objectives of this study were to examine the effect of early short-term temperature conditioning on metabolic rate and calpain/calpastatin system and to compare variations in metabolic rate and calpain/calpastatin system between Hanwoo and Holstein beef. Longissimus thoracis et lumborum of the right carcass from 3 Hanwoo and 3 Holstein bulls were removed within 30 min of exsanguinations, cut into three pieces, and then temperature conditioned until 3 h postmortem (PM) at 2, 16, and 30°C, respectively. Rigor values (R-values; R248, R250, and R258), pH, muscle temperature, glycogen content, µ- and m-calpain activities, and calpastatin acitivity were measured at 1, 3, 9, and 24 h PM, respectively. Hanwoo beef had higher muscle temperature, faster metabolic rate at early PM stage in R-values, and lower µ-calpain activity than Holstein beef (p 0.60; p<0.001). Among R-values, R258 had higher correlations with other metabolic traits than those of R248 and R250. These data suggest that early PM metabolic rate, µ-calpain activity, and calpastatin activity may be closely related to each other. Variations in metabolic rate and µ-calpain activity at early PM stage between Hanwoo and Holstein beef may imply variations in meat quality between both breeds. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 12 : 1747-1753)

Age-dependent Degradation of Calpastatin in Kidney of Hypertensive Rats

Hypertensive rats from the Milan strain show a significant decrease in calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of calpastatin activity and mRNA are consistent with an increase of calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of calpastatin protein as compared with control rats. Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca(2+)-activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca(2+) influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa calpastatin fragment. These data allow the conclusion that calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.

POSTMORTEM PROTEOLYSIS AND TENDERIZATION OF TOP LOIN STEAKS FROM BRANGUS CATTLE1

ABSTRACTVariations in calpastatin activity (CA) and postmortem (PM) degradation of myofibrillar protein are hypothesized to be related to variations in PM tenderization of beef. The objective of this study was to define the relationships between CA, calpastatin specific activity (CSPA), PM degradation of myofibrillar protein, and Warner‐Bratzler shear (WBS) force values in steaks aged 2, 7, and 14 days PM. Brangus bulls (n=43) and steers (n=51) were utilized to define these relationships. Steaks from bulls had higher WBS, and CA, in samples aged 2 and 7 days PM, but not after aging 14 days PM, as compared to steaks from steers. CSPA was correlated with WBS after 7 (r=0.570) and 14 (r=0.424) days PM. Degradation of troponin‐T after aging 2 days PM was a consistent indicator of proteolysis and tenderization. WBS, CA, CSPA, and PM degradation of myofibrillar protein after aging 2 days PM may be reliable predictors of rate of tenderization during the aging process.

Carcass traits, muscle characteristics, and palatability attributes of lambs expressing the callipyge phenotype

Dorset×Suffolk crossbred wethers expressing a normal phenotype (normal, n=10) and callipyge phenotype (CLPG, n=9) were fed individually and slaughtered upon reaching maximum growth potential. Carcass weight did not differ ( P>0.05), but fat thickness was lower (0.30 vs. 0.56 cm), ribeye area was greater (24.4 vs. 16.6 cm 2), and USDA yield grade was improved (1.6 vs. 2.6) for CLPG carcasses ( P < 0.05). Carcasses from CLPG demonstrated higher cutability and a more desirable muscle to bone ratio, with less fat and more lean as a percentage of total side weight ( P<0.05). Biceps femoris (BF), semitendinosus (ST), semimembranosus (SM), longissimus thoracis et lumborum (LTL), and triceps brachii (TB) were used to compare muscle characteristics of both phenotypes. Muscle from normal lambs had lower calpastatin activity, higher fat percentages and lower moisture percentages ( P<0.05). Total amount and solubility of collagen were not affected by phenotype nor was sarcomere length. Shear force and trained sensory attributes were evaluated for BF and LTL only. Shear force values did not differ between normal and CLPG BF while CLPG LTL had higher ( P< 0.05) shear force values than normal LTL. Biceps femoris did not differ between phenotypes in trained sensory panel ratings. However, CLPG LTL received the lowest sensory ratings for connective tissue amount and myofibrillar and overall tenderness (6.11, 4.97, and 4.95, respectively) and the normal LTL received the highest ratings ( P < 0.05; 7.10, 7.00, and 6.90, respectively). The CLPG phenotype had a positive influence on carcass characteristics but had a negative influence on tenderness of some muscles, possibly through the myofibrillar component of the muscle.

Activation of Ca(2+)-dependent proteolysis in skeletal muscle and heart in cancer cachexia.

Cachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFα are involved in its triggering (Costelli and Baccino, 2000), yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca2+-dependent proteolysis, which may provide a rapidly activated system in response to the extracellular signals. In the present work we have evaluated the status of the Ca2+-dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca2+-ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca2+-ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca2+-ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca2+-ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca2+-dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia. © 2001 Cancer Research Campaignhttp://www.bjcancer.com

Effect of low voltage electrical stimulation and temperature conditioning on postmortem changes in glycolysis and calpains activities of Korean native cattle (Hanwoo)

The combined effects of low voltage electrical stimulation (LVES) and temperature conditioning during early postmortem (PM) ageing on glycolytic rates and calpains activities of Korean native cattle (Hanwoo) were determined. M. longissimus was taken after splitting course, divided into three pieces and temperature conditioned at 2, 16, and 30°C for 3 h PM. The PM glycolytic rates, calpains and calpastatin activities were measured at 1, 3, 9, and 24 h PM. Although both LVES and the 30°C treatment accelerated glycolytic rates and resulted in improved enzyme activities, LVES was more effective for the improvement of enzyme activities than the 30°C treatment. Among tested Rigor-values (R-value; R248, R250, and R258), R258 showed the highest correlation with pH (r=0.814, P<0.01), glycogen content (r=0.784, P<0.01) and μ-calpain (r=0.838, P<0.01) and selected as a suitable parameter to predict glycolytic rate. The high correlation coefficients between μ-calpain activity and metabolic rate parameters suggest that the change in the enzyme activity is closely related to glycolytic rates. The LVES in combination with the 30°C treatment until 3 h PM was the best treatment to accelerate the glycolytic rate and to improve the calpains activities in Hanwoo tissue.

The interaction between pH and temperature decline early postmortem on the calpain system and objective tenderness in electrically stimulated beef longissimus dorsi muscle

The study investigated the effects of pH and temperature decline early postmortem (pm) on calpains and calpastatin activities and objective tenderness in M. longissimus thoracis et lumborum. A total of 40 sides were used to generate a range of pH and temperature declines, by different stimulation treatments (i.e. low voltage stimulation applied for either 10 or 40 s 3 min pm), and chilling treatments (fast or slow). Both rapid glycolysis, or a slow chilling regime significantly reduced μ-calpain level at 4 h pm ( P< 0.001 and P< 0.05, respectively). There were significant interactions between pH and temperature at 1.5 h pm on μ-calpain and calpastatin activities at 24 h pm ( P< 0.05). Rapid glycolysis resulted in lower shear force and compression measurements at day 1, although because of low ageing rates, an intermediate pH decline (i.e. pH 5.9–6.2 at 1.5 h pm) resulted in the most tender meat after 14 days ageing. Higher shear forces in the slow glycolysing sides appeared to be associated with the later activation of tenderising process, as well as physical shortening. The optimum pH decline early pm varied with days ageing, with an intermediate pH decline resulting in the lower shear forces for meat aged for 14 days.