N-terminal amino acid sequences of troponin T fragments, including 30 kDa one, produced during postmortem aging of bovine longissimus muscle

We have determined the amino (N)-terminal amino acid (AA) sequences of five troponin T (TnT) fragments produced during postmortem aging of bovine longissimus muscle. Western blot analysis showed that 32.1, 28.8, 27, and 25.8 kDa anti-fast-type TnT (fTnT)-positive fragments and a 31 kDa anti-slow-type TnT (sTnT)-positive fragment were present at 14 d postmortem. The N-terminal AA sequences of the 32.1, 28.8 (conventional 30 kDa), 27, and 25.8 kDa fragments were APPPPAEV, EVHEPEEK, EKPRPRLT, and APKIPEGE, respectively, and they were mapped to the N-terminal region of bovine fTnT isoforms. The N-terminal sequences of the 31 kDa fragment, EAPEEPEP, were mapped to the sTnT isoforms. These findings indicate that the two isoform types of fTnT predominantly expressed in the longissimus muscle are cleaved specifically at Glu(21)-Ala(22) and Glu(15)-Ala(16), His(37)-Glu(38) and His(31)-Glu(32), Glu(43)-Glu(44) and Glu(37)-Glu(38), and/or Thr(51)-Ala(52) and Thr(45)-Ala(46), respectively, and that a sTnT isoform is cleaved specifically at Glu(23)-Glu(24).     

Difference in postmortem degradation pattern among troponin T isoforms expressed in bovine longissimus, diaphragm, and masseter muscles

The postmortem degradation of troponin T (TnT) in bovine longissimus (LT), diaphragm (DP), and masseter (MS) was analyzed. A 28.3kDa (conventional 30kDa) fragment of fast-type TnT isoforms showed the highest content in both LT and DP, where a 35.4kDa isoform had the highest expression among the other fast isoforms. Meanwhile, a 26.0kDa fragment was found to be the most highly produced among the fast TnT fragments in MS, where the expression of 36.5 and 32.8kDa isoforms was higher than that of 35.4 and 34.8kDa isoforms. Thus, the compositions of both the intact TnT isoform proteins and the postmortem fragments differed among the muscles examined, indicating that each TnT isoform degrades into a specific fragment in each muscle. Among the muscles, the LT muscle showed a high extent of TnT degradation and the highest expression of fast TnT isoforms containing a taste-related peptide sequence.     

Influence of myosin heavy chain isoform expression and postmortem metabolism on the ATPase activity of muscle fibers

The objective of this study was to determine the effects of postmortem muscle pH and temperature declines on the actomyosin ATPase activity of muscle fibers expressing different MyHC isoforms. Using a quantitative histochemical procedure to determine ATPase activity, the maximum actomyosin ATPase activity was determined on individual fibers classified by MyHC expression. Samples were collected from the red (RST) and white (WST) semitendinosus muscles at 3 min and 24 h postmortem from electrically stimulated (ES) and control (NS) pork carcasses. In samples taken at 3 min postmortem, type I fibers had the lowest ATPase activity staining and type 2X and 2B had the highest activity staining, with type 2A fibers intermediate. Postmortem time and carcass treatment did not influence the ATPase activity staining of type I muscle fibers. ATPase activity staining of 2A fibers was lower (p<0.001) in 24 h samples than in 3 min samples from ES carcasses. In 3 min and NS-24 h samples, RST type 2A fibers had lower (p<0.05) activities than type 2A fibers from the WST. In type 2X fibers, ATPase activity staining decreased (p<0.01) from 3 min to 24 h postmortem in ES carcasses. This decrease was more severe in WST 2X fibers compared to RST 2X fibers. ATPase activity staining in type 2B fibers did not decrease from 3 min to 24 h postmortem in NS carcasses. In ES carcasses, activity staining of 2B fibers decreased (p<0.0001) with time postmortem. The results of the experiment indicate that fibers expressing fast MyHC isoforms have a higher ATPase activity early postmortem than slow muscle fibers but are more prone to inactivation by a rapid pH decline.     

Effect of fiber type on postmortem proteolysis in longissimus muscle of Landrace and Korean native black pigs

The current study was conducted to characterize objective meat quality, fiber type and their relations to postmortem proteolysis in longissimus muscle of Landrace and Korean native black (KNP) pigs. Longissimus muscles from each 10 market-weighted male pigs were removed after conventional slaughtering and chilling procedures, and aged for 1 or 7 days at 4 °C to determine WB-shear force, objective meat color, proportion of myosin heavy chain I (MyHC I), intramuscular fat content and rate of proteolysis by a proteomics approach. KNP had a significantly (p < 0.05) higher content of MyHC I, and that concurred with greatly (p < 0.05) higher intramuscular fat content and Hunter a^* value, and significantly (p < 0.05) lower drip loss than those seen in Landrace. One-dimension SDS-PAGE indicated that GAPDH, troponin I and creatine kinase were prominent proteolytic products during chiller ageing. By applying a gel-based proteome analysis, 26 proteins were identified, which showed different degradation properties during ageing between the breeds. Biopsied sample revealed that myosin regulatory light chain 2, myosin light chain isotype v/sb, fatty acid-binding protein and albumin were expressed at a greatly higher level for KNP, but their relation to fiber type (or genetic background) is unclear. It was particular noticeable that different actin isoforms showed various degradation behavior during ageing time.     

The relationship between muscle fiber characteristics, postmortem metabolic rate, and meat quality of pig longissimus dorsi muscle

The aim of this study was to investigate the histochemical parameters of muscle fibers, and to estimate the correlation of muscle fiber characteristic to postmortem metabolic rate and meat quality traits in pigs. A total of 231 crossbred pigs were evaluated. Samples of the longissimus dorsi muscle were taken to evaluate the histochemical characteristics, postmortem metabolic rate and meat quality. Fiber type composition was mainly related to postmortem metabolic rate and meat quality traits among various muscle fiber characteristics. The percentage of type IIb fiber was negatively related to pH(45min) (r=-0.33) and positively to R-value (r=0.32). Drip loss was negatively related to fiber area percentages of type I and IIa (r=-0.25 and -0.26, respectively) and positively related to type IIb percentage (r=0.39). A similar tendency was found between lightness and fiber area percentage. In conclusion, increasing the percentage of type IIb fiber is related to increasing the postmortem metabolic rate, and is related to the deterioration of meat quality.     

Postmortem changes in bovine troponin T isoforms on two-dimensional electrophoretic gel analyzed using mass spectrometry and western blotting: The limited fragmentation into basic polypeptides

To comprehend postmortem changes in troponin T (TnT), whole beef proteins were developed on a two-dimensional electrophoretic gel. Multiple TnT-related spots were identified by both western blotting and MALDI-TOF MS utilizing bovine TnT isoform mRNA sequences. More than 10 TnT fast-type isoform spots (pI 5.7-9.6<) and the two slow-type isoform spots (pI 5.6-5.7) were observed at slaughter. All the isoforms were degraded exclusively into basic spots (pI 9.6<) at day 14 postmortem. Some TnT-related phosphorylated spots present at slaughter had disappeared by day 14, suggesting that the phosphorylated N-terminal region was cut off during beef aging. The intact isoforms and the fragments were identified by the MS with sequence coverage of 20.8-62.7%, and two of the fragments included the cutting site peptide of a conventional 30kDa or of a slow TnT-derived fragment. These results revealed that all of the TnT isoforms are cut exclusively in the glutamic acid-rich amino-terminal region during postmortem aging.     

DEGRADATION OF ACTIN BY CATHEPSINS IN BEEF FIBERS STORED AT 20C

The degradation of actin throughout 48 h of postmortem storage of muscle fibers at 4 and 20C was studied by means of SDS-gel electrophoresis and immunoblotting using a commercial monoclonal antibody. Electrophoretic patterns showed an enhanced proteolysis of meat held at 20C, as revealed by the increased number and intensity of degradation peptides in the region of 30 kDa, as well as by the appearance of a new peptide of a molecular weight between those of actin and troponin T. Western blotting of actin demonstrated its degradation at 20C, while not at 4C; at least a peptide originating from actin was evident at the former temperature. When fibers were incubated at 20C in the presence of cathepsin inhibitors E-64 and pepstatin, which do not inhibit calpains, the immunoblots showed no degradation of actin. This clearly demonstrated that cathepsins are effectively involved in actin degradation in fibers stored at 20C.     

Difference in tenderness and pH decline between water buffalo meat and beef during postmortem aging

The objective of this research was to determine the difference in tenderness and some characteristics of water buffalo meat and beef during postmortem aging. Five female crossbred water-buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native), were finished on the same diet for 6 months and slaughtered at 30 months of age. The muscle pH was measured at 40min, 3h, 7h, 24h, and 48h postmortem. Longissimus thoracis (LT) and semimembranosus (SM) muscles were excised at 2d postmortem, and shear force was measured at 2, 4, 7, and 14d postmortem. Glycogen and lactate concentrations were determined from 0, 2, and 4d LT samples, and myosin heavy chain type of buffalo and cattle LT was determined by ELISA methods. Myofibrillar protein degradation was also observed by SDS-PAGE and Western blotting of fast-type troponin T. Results showed that the buffalo meat had significantly lower shear force values compared to beef for LT and SM muscles, which was supported by a difference in troponin T degradation. Postmortem pH decline of buffalo meat was significantly slower than that of beef, which was confirmed by lactic acid concentrations, but was not explained by glycogen content. In addition, there was no significant difference in the ratio of slow to fast type muscle fibers in buffalo and cattle, indicating that myosin heavy chain type was not responsible for the difference in pH decline and tenderness between the buffalo meat and beef. This study demonstrated that the tenderness of water buffalo meat was superior to that of Brahman beef, which may have been due to the difference in pH decline and the subsequent effect on muscle protease activity.     

Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle

The objective was to determine the effect of three different protease inhibitors, caspase-6 specific inhibitor VEID-CHO (N-Acetyl-Val-Glu-Ile-Asp-al), calpain inhibitor leupeptin or calpain inhibitor EGTA on protein degradation, ultrastructure of myofibrils and calpain activity during postmortem (PM) aging of chicken muscle. Results showed that proteolysis of nebulin, troponin-T and desmin during 14-days postmortem storage were inhibited significantly by leupeptin. Inhibitive effects of VEID-CHO and EGTA on these protein degradations were significant only during 1-day postmortem storage. The activities of calpains were inhibited noticeably by leupeptin and EGTA, but not by VEID-CHO. Samples treated with VEID-CHO, leupeptin and EGTA retarded structural disruption of chicken muscle fibers. These results demonstrate that calpain is a major contributor to PM tenderization; while caspase-6 plays, if any, a minimal role in the conversion of chicken muscle to meat.     

The Effect of µ/m‐Calpain on Protein Degradation of Chicken Breast Meat

This study was designed to determine the effects of µ/m‐calpain on the degradation of cytoskeletal proteins in pectoralis major. Four chickens were slaughtered and the breasts were removed and stored for 12 hr at 4 °C. Each sample was divided into three groups and respectively immersed in control reagent, calpain inhibitor, and caspase inhibitor at 4 °C. The samples were used to evaluate troponin‐T and desmin degradation, calpain activity, and myofibril ultrastructure at 12 hr, day 1, day 3, and day 7. Casein zymography revealed that µ‐calpain could not be detected in all samples after 12 hr postmortem. The calpain inhibitor inhibited µ/m‐calpain activity and reduced troponin‐T and desmin degradation during 7 day postmortem. The caspase inhibitor inhibited µ/m‐calpain activity and, troponin‐T and desmin degradation before day 3 postmortem. The findings suggest that, µ/m‐calpain had an effect on cytoskeletal protein degradation after 12 hr postmortem.     

Separation and amino acid composition of three troponin components from bovine muscle

Three troponin components were isolated from bovine skeletal muscle, and their molecular weights and amino acid composition were studied. Crude troponin prepared from bovine muscle was purified by DEAE-Toyopearl chromatography. The purified troponin was dissociated in the order of tropopins C, I and T by CM-Toyopearl chromatography in the presence of 6 M urea. The molecular weights of troponins C, I and T were 19,500, 23,300 and 40,400, respectively, as determined with SDS-PAGE. The separation of troponin into three components was also achieved using reverse-phase HPLC; however, the elution order of troponins T and C was contrary to that of the cation-exchange chromatography described above. In this study, the amino acid composition of the three troponin components from bovine skeletal muscle was first determined. The amino acid composition of the three troponin components among bovine, rabbit and chicken skeletal muscles showed stronger similarity than that between bovine skeletal and cardiac muscle with a different muscle type. We considered that this method of troponin preparation from bovine muscle must be a very useful technique for investigating the changes in troponin components, especially troponin T, during ageing of beef.     

Role of the ubiquitin‐proteasome pathway on proteolytic activity in postmortem proteolysis and tenderisation of sheep skeletal muscle

The objective of this study was to investigate the effects of ubiquitin‐proteasome pathway on meat tenderisation. The sheep muscle longissimus lumborum was injected with or without PYR‐41 (inhibitor of ubiquitination) or MG‐132 (inhibitor of proteasome). Muscle samples were collected at 6, 15, 24 and 48 h after injection. Myofibrillar protein degradation, muscle ultrastructure and sarcomere length were determined. Results showed that inhibition of proteasome or ubiquitination affected sarcomere length at 48 h after treatments. Destruction of muscle ultrastructure in both treatments was reduced when compared to control. Inhibition of proteasome produced different fragments of myofibrillar proteins in comparison with control at 48 h. In conclusion, ubiquitin‐proteasome plays a role in postmortem proteolysis and might contribute to meat tenderisation.     

The postmortem μ‐calpain activity,protein degradation and tenderness of sheep meat from Duolang and Hu breeds

This study aimed to investigate the differences in meat tenderisation between two Chinese native sheep breeds, Duolang and Hu. The tenderness of longissimus thoracis (LT) muscle, μ‐calpain autolysis and proteolysis of myofibrillar protein was measured at 1‐ and 7‐days postmortem storage at 4 °C. At 7‐days postmortem ageing, meat from Duolang sheep was more tender compared to that from Hu sheep. The Warner–Bratzler shear force of Duolang and Hu sheep was reduced by 55.20% and 41.51% at 7 days compared with 1 day, respectively. In Duolang LT, a higher proportion of 80‐kDa μ‐calpain was autolyzed than in the Hu samples at 1‐day postmortem ageing, but they did not differ significantly at 7 days. More titin, desmin and troponin‐T were degraded in Duolang sheep compared to Hu sheep. Additionally, pH values at the different ageing time were not significantly different, indicating that pH may be not a determinant factor contributing to the tenderness difference between the two breeds. Our data suggest the earlier activation of μ‐calpain and a larger extent of proteolysis of key structural proteins may contribute to the higher rate of tenderisation of Duolang compared to Hu sheep during postmortem ageing.     

Comparison of postmortem changes in goose cardiac and breast muscles at 5 °C

BACKGROUD: The tenderness of goose heart is an important consideration in its utilization as a popular meat product. It is generally thought that postmortem degradation of myofibrillar proteins may improved meat tenderness. Little information, however, is available regarding the postmortem changes in goose cardiac muscle. Therefore, the postmortem proteolysis between goose cardiac and breast muscles at 5 °C is compared. RESULTS: The pH is higher (P < 0.05) in cardiac samples than in breast samples. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots results indicate that postmortem degradation of titin and desmin and the appearance of the 28 and 30 kDa components are faster in breast muscle than in cardiac muscle. CONCLUSION: Our results may suggest that goose postmortem proteolysis occurs more rapidly in breast muscle than in cardiac muscle at 5 °C. Copyright © 2008 Society of Chemical Industry     

Protein denaturing conditions in beef deep semimembranosus muscle results in limited μ-calpain activation and protein degradation

The objective was to determine the effect of muscle location on protein solubility and protein degradation in deep (DSM) and superficial (SSM) portion of beef semimembranosus. At 24 h postmortem, the semimembranosus was removed from beef carcasses (n = 10), packaged in high-oxygen modified atmosphere (80% O₂ + 20% CO₂), and displayed for 7 d at 1 °C. DSM had higher (P < 0.05) L*, a*, b*, and hue values than SSM throughout display. DSM had significantly higher protein denaturation and less protein concentration than SSM. Western blotting for μ-calpain autolysis revealed that DSM maintained more (P < 0.05) unautolyzed μ-calpain than SSM. This result coincided with less desmin and troponin-T degradation in samples from the DSM. These results confirm the hypothesis that increased protein denaturation in DSM results in minimal proteolysis by negatively affecting μ-calpain activation. This demonstrates a potential to alter progression of proteolysis and improvement in tenderness associated with postmortem storage.     

Muscle fiber type characterization and myosin heavy chain (MyHC) isoform expression in Mediterranean buffaloes

This study aimed to evaluate myosin heavy chain (MyHC) isoform expression and muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) in Mediterranean buffaloes and possible fibers muscles modulation according to different slaughter weights. The presence of MyHC IIb isoforms was not found. Only three isoforms of MyHC (IIa, IIx/d and I) were observed and their percentages did not vary significantly among slaughter weights. The confirmation of the presence of hybrid muscles fibers (IIA/X) in LD and ST muscles necessitated classifying the fiber types into fast and slow according to their contractile activity, by m-ATPase assay. For both muscles, the muscle fiber frequency was higher for fast than for slow fibers in all weight groups. There was a difference (P<0.05) in the frequency of LD and ST muscle fiber types according to slaughter weights, which demonstrate that the slaughter weight influences the profile of muscle fibers from buffaloes.     

Effects of Postmortem Storage and Temperature on Muscle Protein Degradation: Analysis by SDS Gel Electrophoresis

The proteolytic breakdown of the major contractile proteins of bovine longissimus muscle was examined during postmortem storage by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Samples of muscle stored at 4°C for 14 days exhibited little proteolysis of the major contractile proteins; however, samples stored at 37°C for 1 day showed significant degradation of myosin heavy chains and almost complete proteolysis of this protein by day 14. Major degradation products of the myosin heavy chains included a series of polypeptides having molecular weights between 145,000 and 125,000. These experiments demonstrate that substantial degradation of the myosin heavy chain and other muscle proteins can occur during the storage of meat, and this phenomenon was highly temperature dependent.     

Skeletal muscle fiber type and myofibrillar proteins in relation to meat quality

Although numerous studies have reported the relationships among muscle fiber characteristics, lean meat content and meat quality, controversial perspectives still remain. Conventional histochemical classifications may be involved in a high level of error, subjectivity and it could not clearly explain variety of myofibrillar protein isoforms. Therefore, more information is needed on how different factors, such as species, breeds, gender, nutrient conditions, physiological state of animals, and environment factors, affect ultimate meat quality in order to evaluate these uncertainness. Unfortunately, there is little information that completely covers with relationship among the muscle fiber types, myofibrillar proteins and enzymatic proteolysis. In addition to the perspective of postmortem metabolism, protein quality control in skeletal muscle and proteolytic degradation of muscle proteins during postmortem period could help to clarify this relationship. Therefore, the present review will focus on muscle fiber types, typing methods, muscle proteins and meat quality, and will summarize aspects of enzymatic view of proteasome.     

Comparisons of longissimus muscle metabolic enzymes and muscle fiber types in Korean and western pig breeds

We compared differentially expressed genes and muscle fiber types in the longissimus muscles of Korean native pigs (KNP) and the western meat-producing breeds Landrace and Yorkshire. The KNP breed exhibited a higher muscle fat content and more red meat color as determined by the a^* (redness) value (P < 0.01) and b^* (yellowness) value (P < 0.05) compared to the western breeds. Using differential display RT-PCR, we detected two genes that were differentially expressed in skeletal muscle among the pig breeds. These genes were identified as NADH dehydrogenase subunit 1 and ATPase subunit 6 by cloning and sequencing analysis. Both of these genes are involved oxidative phosphorylation and therefore energy metabolism. The genes were more highly expressed in the KNP breed than in the other breeds, indicating that KNPs exhibit more oxidative metabolism than do the western breeds. We also analyzed the mRNA levels of myosin heavy-chain isoforms such as type I (oxidative), type IIb (glycolytic), and types IIa and IIx (intermediate) fibers using real-time RT-PCR. The mRNA levels of oxidative and intermediate fibers were elevated in the KNP breed, whereas the glycolytic fibers were more highly expressed in the Landrace and Yorkshire pigs. These results suggest that the elevated expression of the oxidation-related metabolism genes NADH dehydrogenase and ATPase is related to meat quality as indicated by a higher content of oxidative fibers and muscle fat, as well as redder meat color.     

Ultrastructural changes in bovine Longissimus muscle during postmortem ageing

The ultrastructural changes in type 1 (red) and type 2 (white) myofibres of the longissimus muscle were studied during in situ postmortem ageing in a group of young Hereford bulls, 14-16 months old. Type 1 myofibres were defined as those having wide, dense and distinct Z-discs and type 2 myofibres were those with narrower and less distinct Z-discs. Type 1 myofibres also had numerous intermyofibrillar mitochondria and type 2 myofibres had few intermyofibrillar mitochondria. Three time periods were studied-1 h, 48 h and 216 h postmortem. Type 1 myofibre Z-discs were essentially unaltered at all three postmortem time periods studied. Type 2 myofibres showed limited degradation (loss) of Z-disc material at 1 h postmortem, but degradation had increased at 48 h. However, little additional Z-disc degradation occurred between 48 h and 216h. Both type 1 and type 2 myofibres underwent myofibrillar fragmentation at the level of the Z-disc and I-band which was independent of Z-disc degradation. The most tender longissimus muscles tended to have more fragmentation at the Z-disc-I-band junction than the toughest muscle samples.