troponin-T Degradation Product Research Articles

Myosin heavy chain isoforms expression, calpain system and quality characteristics of different muscles in goats

Myosin heavy chain (MHC) isoforms in goat muscles and their possible relationships with meat quality have not been fully elucidated. This study characterized the MHC isoforms in different caprine muscles using sodium dodecyl sulphate glycerol gel electrophoresis (SDS-GGE). The relationships between MHC isoforms, calpain systems and meat quality characteristics of different muscles in goats were examined. Four muscles, namely infraspinatus (IF), longissimus dorsi (LD), psoas major (PM) and supraspinatus (SS) were obtained from ten Boer crossbred bucks (7–10 months old; 26.5 ± 3.5 kg, BW). The percentages of MHC I, MHC IIa and MHC IIx in SS, IF, PM and LD were 47.2, 38.3, 32.1, 11.9; 28.0, 42.1, 33.0, 36.4; and 24.8, 19.6, 34.9 and 51.7, respectively. IF and SS had higher levels of calpastatin, total collagen and insoluble collagen contents than did PM and LD. PM had longer sarcomere length than did other muscles. LD had higher collagen solubility, troponin-T degradation products and glycogen content than did other muscles. These results infer that variable fiber-type composition could account partially for the differences in the physicochemical properties of goat muscles.

Influence of Beef Production System Technology on Calpain-1 Autolysis and Troponin-T Degradation

ObjectivesBeef production systems utilize implants and β-agonists to improve beef cattle feed efficiency and promote muscle growth. Warner-Bratzler shear force values can be greater in strip loin steaks from cattle treated with implants or β-agonists. Calpain-1 degrades myofibrillar proteins post-mortem, thus altering calpain-1 activation or autolysis which can influence meat tenderness and proteolysis. The objective of this study was to determine the impact beef production system technologies on calpain-1 autolysis and troponin-T degradation as an indicator of tenderness formation and postmortem proteolysis.Materials and MethodsFrom a larger study, beef striploins (n = 16, n = 4/treatment) from cattle finished utilizing four different production systems were collected for analysis: 1) no antibiotics (NA; receiving no technology); 2) non-hormone treated cattle (NHTC; fed 300 mg monensin and 90 mg tylosin during the finishing phase); 3) implant (IMPL; same technologies as NHTC and administered a series of three implants including a low- potency calf implant [36 mg zeranol], a moderate-potency initial feedyard implant [80 mg trenbolone acetate and 16 mg estradiol], and a high potency finishing implant [200 mg of trenbolone acetate and 20 mg estradiol]; and 4) all previous technologies plus fed a β-agonist (IMBA; same technologies as IMPL and fed 200 mg ractopamine hydrochloride per steer per d). Striploins were vacuum packaged, aged for 7 d, and frozen. Western Blots were conducted for calpain-1 autolysis and troponin-T degradation (30 kDa). Abundance of calpain-1 bands and troponin-T degradation product was normalized by a reference on each gel. Treatments were evaluated in PROC MIXED of SAS 9.2 where least squares means and SEM were computed and separated using least significant differences (PDIFF) when tests for fixed effects were significant at P < 0.05 and trending P ≤ 0.10.ResultsCalpain-1 autolysis differed (P < 0.05) in the IMPL group compared to the NHTC group for both active, 78 kDa band, and the fully autolyzed, 76 kDa band. The IMPL group had a greater percentage (P = 0.0048) of active calpain-1 and a lower percentage (P = 0.0048) of fully autolyzed calpain-1 compared to the NHTC group. Also, a trend was detected when comparing both the active, 78 kDa band, and fully autolyzed, 76 kDa band, in the IMBA and IMPL group where the IMPL group had a greater percentage (P = 0.0727) of active calpain-1 and a lower percentage (P = 0.0727) of fully autolyzed calpain-1. Production system did not influence (P > 0.05) 30 kDa troponin-T product abundance.ConclusionThese data indicate level of technology may play a role in the activation and autolysis of calpain-1 from the 80 kDa inactive form to the 78 kDa active product and finally to the 76 kDa autolyzed product. Calpain-1 autolysis was not measured; however, these data suggest calpain-1 autolysis in the IMPL group may be limited compared with NHTC and IMBA groups. Consequently, calpain-1 may remain in the 78 kDa active form in the implanted cattle, actively degrading myofibrillar proteins. However, production system did not influence troponin-T 30 kDa degradation products. Further analysis of the rate of calpain-1 autolysis and troponin-T degradation at different days of postmortem aging could provide further evidence that different beef production technologies impact calpain-1 autolysis and postmortem proteolysis.

Contribution of Protein Degradation and Sarcomere Length to Aged Pork Loin Warner-Bratzler Shear Force

ObjectivesPostmortem aging of fresh pork loins improves tenderness through protein degradation. Sarcomere length (SL) of postmortem muscle can vary between animals, and this could impact access and efficacy of proteinases to degrade proteins within, but not outside of the myofibril. The relationship between SL and protein degradation is not well documented in pork. Therefore, the objective of this experiment was to compare protein degradation of troponin-T with desmin and SL in aged pork loins over 21d.Materials and MethodsPaired sides of fresh pork loins (n = 20) were collected 1 d postmortem. Criteria for inclusion in the study were a pH between 5.85 and 6.10 and a visual color score (NPPC) between 3 and 4. Eight loin chops (2.54 cm) containing only the longissimus muscle were fabricated. Two chops from each pair of loins were aged for 1, 8, 14 or 21 d and immediately evaluated. After aging, chops were cooked to 68°C and Warner-Bratzler shear force (WBSF) was measured. Whole muscle proteins were solubilized from samples at each aging period (10mM sodium phosphate, pH 7.0 and 2% wt/vol sodium dodecyl sulfate). Abundance of degraded troponin-T (30 kDa) and intact desmin (55 kDa) in the whole muscle sample was determined by immunoblotting. Abundance of troponin-T degradation product and intact desmin was normalized by a reference sample on each gel. A helium-neon laser diffraction method was used to determine SL (total of 36 SL per sample were recorded). The distance between primary diffraction bands was used to calculate SL. Correlation coefficients were determined using PROC CORR of SAS 9.4 and significance determined by P < 0.05.ResultsOverall and across all days aging, SL was not strongly correlated to intact desmin (r = –0.198; P = 0.07) or troponin-T degradation (r = 0.236; P = 0.04). Troponin-T degradation was not detected at d1 in any samples but overall and across all days was highly correlated with WBSF (r = –0.671; P < 0.01)). Intact desmin was correlated with WBSF (r = 0.661; P < 0.01). Across all samples, SL was correlated with WBSF (r = –0.445; P < 0.01). Intact desmin and troponin-T degradation were correlated (r = –0.818; P < 0.01).Correlations within day of aging revealed that protein degradation was not significantly correlated with WBSF at d 1. In contrast, troponin-T degradation was correlated (P < 0.01) with WBSF at 8, 14, 21 d postmortem (r = –0.733, –0.641, and –0.772, respectively). Similarly, intact desmin was correlated (P < 0.01) with WBSF at 8, 14, and 21 d postmortem (r = 0.447, 0.553, and 0.824, respectively). SL was correlated (P < 0.01) with WBSF at each d postmortem (r = –0.445, –0.562, –0.714, and –0.512, respectively).ConclusionThe correlation results suggest that SL is consistently correlated with WBSF across aging periods and is more strongly correlated with WBSF early postmortem than protein degradation. After aging, troponin-T degradation and intact desmin demonstrate greater correlations with WBSF than SL. Finally, SL correlation to troponin-T and desmin were generally similar and not strong, suggesting that SL does not affect the efficacy of proteinases to degrade proteins within the myofibril differently than extra-myofibrillar proteins.

Influence of Beef Production System Technology on Calpain-1 Autolysis and Troponin-T Degradation

ObjectivesBeef production systems utilize implants and β-agonists to improve beef cattle feed efficiency and promote muscle growth. Warner-Bratzler shear force values can be greater in strip loin steaks from cattle treated with implants or β-agonists. Calpain-1 degrades myofibrillar proteins post-mortem, thus altering calpain-1 activation or autolysis which can influence meat tenderness and proteolysis. The objective of this study was to determine the impact beef production system technologies on calpain-1 autolysis and troponin-T degradation as an indicator of tenderness formation and postmortem proteolysis.Materials and MethodsFrom a larger study, beef striploins (n = 16, n = 4/treatment) from cattle finished utilizing four different production systems were collected for analysis: 1) no antibiotics (NA; receiving no technology); 2) non-hormone treated cattle (NHTC; fed 300 mg monensin and 90 mg tylosin during the finishing phase); 3) implant (IMPL; same technologies as NHTC and administered a series of three implants including a low- potency calf implant [36 mg zeranol], a moderate-potency initial feedyard implant [80 mg trenbolone acetate and 16 mg estradiol], and a high potency finishing implant [200 mg of trenbolone acetate and 20 mg estradiol]; and 4) all previous technologies plus fed a β-agonist (IMBA; same technologies as IMPL and fed 200 mg ractopamine hydrochloride per steer per d). Striploins were vacuum packaged, aged for 7 d, and frozen. Western Blots were conducted for calpain-1 autolysis and troponin-T degradation (30 kDa). Abundance of calpain-1 bands and troponin-T degradation product was normalized by a reference on each gel. Treatments were evaluated in PROC MIXED of SAS 9.2 where least squares means and SEM were computed and separated using least significant differences (PDIFF) when tests for fixed effects were significant at P < 0.05 and trending P ≤ 0.10.ResultsCalpain-1 autolysis differed (P < 0.05) in the IMPL group compared to the NHTC group for both active, 78 kDa band, and the fully autolyzed, 76 kDa band. The IMPL group had a greater percentage (P = 0.0048) of active calpain-1 and a lower percentage (P = 0.0048) of fully autolyzed calpain-1 compared to the NHTC group. Also, a trend was detected when comparing both the active, 78 kDa band, and fully autolyzed, 76 kDa band, in the IMBA and IMPL group where the IMPL group had a greater percentage (P = 0.0727) of active calpain-1 and a lower percentage (P = 0.0727) of fully autolyzed calpain-1. Production system did not influence (P > 0.05) 30 kDa troponin-T product abundance.ConclusionThese data indicate level of technology may play a role in the activation and autolysis of calpain-1 from the 80 kDa inactive form to the 78 kDa active product and finally to the 76 kDa autolyzed product. Calpain-1 autolysis was not measured; however, these data suggest calpain-1 autolysis in the IMPL group may be limited compared with NHTC and IMBA groups. Consequently, calpain-1 may remain in the 78 kDa active form in the implanted cattle, actively degrading myofibrillar proteins. However, production system did not influence troponin-T 30 kDa degradation products. Further analysis of the rate of calpain-1 autolysis and troponin-T degradation at different days of postmortem aging could provide further evidence that different beef production technologies impact calpain-1 autolysis and postmortem proteolysis.

Effects of ultrasonic processing on caspase-3, calpain expression and myofibrillar structure of chicken during post-mortem ageing

In this investigation, ultrasonic treatments at a frequency of 40kHz and power of 1500W made caspase-3 and calpain activities significantly higher in chicken muscle after slaughter during 5d storage (p<0.01). Additionally Western blotting analysis of α-spectrin showed that ultrasonic treatments caused the production of α-spectrin degradation products of 120 and 150kDa more dense than the control (p<0.01). Degradation of calpastatin during chicken meat ageing was induced by ultrasound treatments (p<0.01), which suggested the ability of caspase-3 to cleave calpastatin. SDS–PAGE showed that all treatments enhanced accumulation of the 30kDa troponin-T degradation product (p<0.01), and transmission electron microscopy images of myofibrils displayed that damage of ultrasound treatment for 60min was more extensive than at 30min. The findings proved the low-frequency and high power ultrasonic treatments improved disruption of myofibrillar structures and increased proteolysis of chicken meat faster by triggering an apoptosis cascade.

Evidence of decreased muscle protein turnover in gilts selected for low residual feed intake1

The objective of this study was to evaluate the contribution of muscle protein turnover (synthesis and degradation) to the biological basis for genetic differences in finisher pigs selected for residual feed intake (RFI). Residual feed intake is defined as the difference between expected feed intake (based on the achieved rate of BW gain and backfat depth of individual pigs) and the observed feed intake of the individual pig. We hypothesized that protein turnover would be reduced in pigs selected for low RFI. Twelve gilts from a line selected for 7 generations for low RFI and 12 from a contemporary line selected for 2 generations for high RFI were paired by age and BW and fed a standard corn-soybean diet for 6 wk. Pigs were euthanized, muscle and liver samples were collected, and insulin signaling, protein synthesis, and protein degradation proteins were analyzed for expression and activities. Muscle from low RFI pigs tended to have less μ- and m-calpain activities (P = 0.10 and 0.09, respectively) and had significantly greater calpastatin activity and a decreased μ-calpain:calpastatin activity ratio (P < 0.05). Muscle from low RFI pigs had less 20S proteasome activity compared with their high RFI counterparts (P < 0.05). No differences in insulin signaling intermediates and translation initiation signaling proteins [mammalian target of rapamycin (mTOR) pathway] were observed (P > 0.05). Postmortem proteolysis was determined in the LM from the eighth generation of the low RFI pigs versus their high RFI counterparts (n = 9 per line). Autolysis of μ-calpain was decreased in the low RFI pigs and less troponin-T degradation product was observed at 3 d postmortem (P < 0.05), indicating slowed postmortem proteolysis during aging in the low RFI pigs. These data provide significant evidence that less protein degradation occurs in pigs selected for reduced RFI, and this may account for a significant portion of the increased efficiency observed in these animals.

Postmortem role of calpains in Pekin duck skeletal muscles

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. 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. 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.

Effects of camptothecin, etoposide and Ca 2+ on caspase-3 activity and myofibrillar disruption of chicken during postmortem ageing

Recently, a novel consideration has focused on the potential relationship of apoptosis and the protease caspases and the underlying mechanism for meat postmortem tenderization. In this study, apoptosis inducers, camptothecin and etoposide as well as Ca 2+ were used to treat chicken muscle immediately after slaughter and follow the changes in caspase-3 activities and changes in the myofibrillar structures during 7 days of ageing. All three treatments resulted in significantly higher caspase-3 activities during storage (p < 0.05), with the natural substrates, whereas Western blotting analysis of the α-spectrin cleavage product, 120 kDa peptide (SBDP 120), showed that Ca 2+ was more effective than either camptothecin or etopside, and all were most active up to day 3 (p < 0.01). According to SDS-PAGE, each treatment enhanced the accumulation of the 30 kD Troponin-T degradation product, especially during the first 3 days (p < 0.05), and this was supported by the degradation of myofibrils observed by electron microscopy (TEM). TEM images showed the treatments resulted in enlargement of the I-bands and shrinkage of A-bands; however Z-lines were only slightly affected, even at day 7. The findings revealed that the three apoptosis inducers could increase myofibrillar dissociation and proteolysis during the first 3 days of chicken meat ageing. Because of the high activity of caspase-3 during the early postmortem period, it is possible that caspase-3 contributes to the conversion of muscle into meat.

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.

Use of 25-hydroxyvitamin D3 and dietary calcium to improve tenderness of beef from the round of beef cows1,2

The objective of this trial was to determine how 25-hydroxyvitamin D(3) (25-OH D(3)) supplementation, altering supplemental dietary calcium, or their combination influence postmortem biochemical and tenderness changes in muscles from the round of mature cows. Twenty-seven Angus cows (3 to 7 yr old) were allotted randomly to 9 pens with 3 cows per pen. Treatments were arranged in a 3 x 3 factorial design with 3 dosages of 25-OH D(3) (0, 250, or 500 mg of 25-OH D(3) administered as a 1-time oral bolus 7 d before slaughter) and 3 percentages of supplemental limestone (0.5, 0.75, and 1.0%) replenished in the diet for 3 d before slaughter and after a 2-wk limestone withdrawal. Plasma samples were obtained during the feeding period. Upon slaughter, adductor, gracilus, pectineus, sartorius, semimembranosus, vastus intermedius, and vastus lateralis muscles were obtained and aged for 1, 3, or 7 d. Calcium concentrations were increased in plasma when 250 or 500 mg of 25-OH D(3) were administered (P </= 0.05). Calcium concentrations in muscle increased (P </= 0.001) when 500 mg of 25-OH D(3) were administered. Concentrations of 25-OH D(3) in meat and in plasma and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2) D(3)] in plasma were increased when 25-OH D(3) was administered (P </= 0.05). The percentage of limestone replenished in the diet had no effect on 25-OH D(3) or 1,25-(OH)(2) D(3) in meat or in plasma. Calpastatin activity was affected by treatments only in the gracilus and vastus intermedius muscles (P </= 0.05). Among all muscles and aging periods, calpastatin activity and intensity of troponin-T degradation product were related inversely. Results indicate that supplemental 25-OH D(3) has some influence on muscle characteristics known to improve tenderness, but improved tenderness was not observed.

EFFECT OF HYDRODYNAMIC PRESSURE PROCESSING AND AGING ON THE TENDERNESS AND MYOFIBRILLAR PROTEINS OF BEEF STRIP LOINS*

ABSTRACT This study evaluated the effects of hydrodynamic pressure (HDP) processing and aging on the tenderness and myofibrillar proteins of beef strip loins. Loins (n = 12) were halved at 48 h postmortem and assigned to HDP or control treatments. Following treatment, each half was divided into three portions for aging (0, 5 or 8 days). Samples were removed for Warner–Bratzler shear force (WBSF) determination and myofibrillar protein isolation. HDP decreased (P &lt; 0.0001) WBSF values 23% at 0, 5 and 8 days of aging. Myofibrillar fragmentation and myofibrillar protein solubility increased (P &lt; 0.01) with HDP and aging. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Western blotting analysis of myofibrillar proteins showed that HDP and aging decreased the intensity of the troponin T (TnT) band and enhanced the accumulation of the 30 kD TnT degradation product. These data suggest that HDP is more effective than aging tenderization, and that HDP tenderization is caused by both protein degradation and physical disruption of the myofibril apparatus.PRACTICAL APPLICATIONSHydrodynamic pressure (HDP) processing was shown to instantaneously tenderize tough cuts of beef to tenderness levels attained following 8 days of postmortem aging. For the meat industry, these results demonstrate the potential benefits of alternative tenderization techniques, such as high‐energy shock waves. Observed changes in protein characteristics due to HDP and aging allow researchers to understand better potential mechanisms of meat tenderization.

Oxidative environments decrease tenderization of beef steaks through inactivation of μ-calpain1

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.

Effects of biological type and dietary fat treatment on factors associated with tenderness: I. Measurements on beef longissimus muscle.

The objective was to evaluate chemical, mechanical, and sensory attributes associated with tenderness in divergent cattle breeds--Wagyu (W; n = 12), Limousin (L; n = 12) and F1-cross (WxL; n = 12)--fed two dietary treatments (0 or 6% sunflower oil (DM basis)). A randomized complete block repeated measures design in a 3 x 2 factorial arrangement of treatments was used, and effects of breed, diet, block, and associated interactions were tested. Cattle were fed barley-based diets for an average of 259 d. Twenty-four hours postmortem (PM), steaks from the longissimus muscle (LM) were sliced, vacuum-packaged, aged (1, 3, 7, 14, 28, and 56 d PM) at 2 degrees C, and frozen (-40 degrees C) until analyzed. Wagyu steaks had lower (P < 0.05) Warner-Bratzler shear force (WBSF) values than L steaks across all aging times. At 1 d PM, W steaks required slightly more (P > 0.05) force to shear than WxL or L (0.30 and 0.11 kg, respectively); however, by d 14 PM, W steaks required 0.77 kg less (P < 0.05) force to shear than L. Wagyu steaks received higher (P < 0.05) sensory panel sustained tenderness scores at d 14 PM than L. The pH decline was slower (P < 0.05), and temperature decline more (P < 0.05) rapid, in W carcasses than L or WxL carcasses. Breed and diet did not affect (P > 0.10) free calcium levels (FCL) over time (0, 1, 3, 7, and 14 d PM), 0-h calpastatin activity (CA), d-1 percent collagen (OH-PRO), or d-1 collagen cross-linking (HP). Western blot analysis for the presence of the troponin-T (TNT) 30-kDa fragment, conducted only on samples from steers fed the 0% sunflower oil diet, demonstrated more proteolysis by d 3 PM in L than W or WxL. Overall, breed differences in mechanical and sensory measures of tenderness were not explained by FCL, CA, OH-Pro, and HP. Even though the initial appearance of the TNT 30-kDa fragment was greater in L, linear slopes for appearance of TNT degradation product across aging time were greater for W and WxL (P < 0.01 and P = 0.056, respectively) than for L, suggesting that tenderness differences due to breed may have been facilitated by more-rapid proteolytic degradation over time.

Solubilization, two-dimensional separation and detection of the cardiac myofilament protein troponin T.

Proteomics strongly relies on the separation of complex protein mixtures, with two-dimensional electrophoresis (2-DE) being a commonly used technique. However efficient separation requires adequate solubilization of the original sample which will determine whether all proteins are accurately represented. Cardiac muscle has presented a particular challenge to solubilization. Here we have optimized the solubilization, separation and detection of the myofilament protein troponin T (TnT). Human left ventricular tissue from a rejected donor transplant heart was homogenized under 19 different conditions and subjected to 2-DE and Western blot analysis for TnT. The optimal conditions for isoelectric focusing of intact TnT requires homogenization in 6 M urea, 2.5 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, with the addition of NaCl (2.5 M final concentration). TnT degradation products present in this severely damaged heart however, were differentially extracted from both each other and the intact molecule under the various conditions used. Despite adequate focusing of TnT it was found that a nonglutaraldehyde silver staining protocol, that is compatible with subsequent mass spectrometry, has greatly reduced sensitivity for TnT compared to Coomassie blue. Thus, care is required to avoid misrepresentation of troponin T in proteomic analysis in cardiac tissue.

Measuring the effects of phenotype and mechanical restraint on proteolytic degradation and rigor shortening in callipyge lamb longissimus dorsi muscle during extended aging

The purpose of this study was to determine if tenderness of callipyge (CLPG) longissimus dorsi muscle (LM) could be improved by: (1) extending the aging period to 48 days postmortem or (2) preventing rigor shortening by clamping. In CLPG and normal (NML) chops respectively, initial Warner-Bratzler shear values (WBS) were lower ( P<0.05) in clamped (CL) (5.5 and 3.6 kg) compared to unclamped (UCL) (7.4 and 4.9 kg) LM. In CLPG, an acceptable WBS (3.6 kg) was reached at 48 days PM, whereas, NML lambs reached an acceptable level (3.8 kg) by 3 days PM. Sarcomere lengths (SL) of CL (1.68 μm) were longer ( P <0.05) than for UCL (1.44 μm) and were negatively correlated with WBS ( r=−0.55; P<0.1). The appearance of Troponin-T (TNT) degradation product coincided with tender WBS values; 3 days postmortem in NML UCL and 48 days postmortem in CLPG. In conclusion, clamping reduced WBS possibly by reducing rigor shortening. Extended aging resulted in CLPG LM with acceptable WBS values, concurrent with the appearance of TNT degradation products.