Influence of myosin heavy- and light chain isoforms on early postmortem glycolytic rate and pork quality

This study addressed the influence of the content of myosin heavy- (MHC) and light chain (MLC) isoforms on early postmortem glycolytic rate and meat quality traits in the porcine longissimus muscle. The fast-glycolysing group showed lower contents of MHC slow and MLC 1s isoforms (P<0.05), and higher MHC fast isoform contents than the normal-glycolysing group (P<0.05). The MHC fast/slow ratio was correlated with lactate content (r=0.41) and early postmortem muscle pH (r=-0.51), and the content of the MLC 1s isoform was negatively correlated with lactate content and glycolytic potential (r=-0.38 and -0.36, respectively). Hence, both the MHC and MLC isoforms did influence metabolite contents, thus also affecting glycolytic rate, and suggested that the myosin isoforms, in particular the MHC isoforms, might also have some bearing on the extent of protein denaturation and pork quality during the early postmortem period.     

Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles

The present study investigated the relationship between muscle type and components of the caspase protease system in porcine trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) muscles. Muscles were classified according to slow and fast myosin heavy chain (MHC) content determined by western blotting. MHC slow, but not MHC fast protein expression was significantly different between muscles (p<0.001). Protein levels of caspases 3, 8 and 12 and the caspase inhibitor apoptosis repressor with caspase recruitment domain (ARC) were determined. In addition the level of caspase 3 mRNA and activity levels of caspase 3/7 were determined. There was a significant difference in protein levels and activity between muscles (p<0.01), although no difference was observed in mRNA abundance. The data show that multiple components of the caspase system are expressed in porcine skeletal muscle and that their levels are variable, but there is not a distinct association of expression with a particular muscle.     

Effects of myosin heavy chain isoforms on meat quality, fatty acid composition, and sensory evaluation in Berkshire pigs

The main objective of this study was to investigate the effects of myosin heavy chain (MHC) isoforms on meat and sensory quality in Berkshire pigs. A total of 85 pigs were evaluated, and muscle samples were taken for the analyses of MHC isoform, meat quality, fatty acid composition, and sensory evaluation. Content of the MHC slow isoform was significantly correlated with pH(24h) (r=0.26, P<0.05) and drip loss (r=-0.32, P<0.01), although the content of MHC isoforms showed limited relationships with individual fatty acids. In the case of sensory evaluation of meat by a trained panel test, the MHC fast/slow ratio was correlated with the juiciness (r=-0.33, P<0.01), off-flavor (r=0.34, P<0.01), tenderness attributes (r=-0.43 to -0.47). These results imply that the content of MHC isoforms can influence various aspects of quality including pork and sensory quality in Berkshire pigs.     

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.     

Presence of an unidentified myosin isoform in certain bovine foetal muscles

Genetic variation in the establishment of bovine muscle fibre types was studied by comparing muscle differentiation at 210 days of foetal life in normal cattle and in ‘culard’ animals, which have muscular hypertrophy. The different fibre types were determined by histochemical and immunohistochemical analyses with monoclonal antibodies specific to different myosin heavy chain isoforms. The isoforms were separated by electrophoresis and quantified by the ELISA method. Four muscles with different contractile and metabolic characteristics were studied: Semitendinosus, Longissimus thoracis, Masseter (slow) and Cutaneus trunci (fast). Muscle fibres recognized by none of the antibodies used were observed in ‘culard’ foetuses in all the muscles studied and also in the Cutaneus trunci of normal animals. Electrophoretic analysis showed no particular myosin isoform in these muscles. It is possible therefore that the fibres contained a mysosin isoform until now unidentified in cattle, with a molecular weight the same as that of known isoforms. This newly observed isoform seems to be specific to muscles rich in IIB fibres such as Cutaneus trunci and to the muscles of adult ‘culard’ cattle.     

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.     

Myosin heavy chain isoforms expressed in bovine skeletal muscles

Nucleotide sequences including the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from bovine adult skeletal muscles. The deduced amino acid sequences were 1940, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. Like other mammalian MyHC isoforms, the bovine MyHC isoforms had homologous sequences except for substitutions concentrated on the loop 1, loop 2, and light chain binding regions. RT-PCR amplifications showed that the adult bovine skeletal muscles expressed the MyHC-2a, -2x, and -slow isoforms but no -2b isoform. The absence of the MyHC-2b isoform and substitutions on the loop2 region could explain some differences in meat quality between beef and pork.     

Differences in molecular structure among the porcine myosin heavy chain-2a, -2x, and -2b isoforms

Full coding regions for fast type myosin heavy chain (MyHC) isoforms were sequenced from a porcine skeletal muscle to analyze sequence diversity relating to the contractile properties of muscle fibers. An approximately 6-kb fragment for each MyHC was amplified through RT-PCR using isoform type-specific primers, which were designed in the 5' and 3' non-coding regions of the porcine MyHCs. The lengths of deduced amino acid sequences were 1939, 1939, and 1937 for the porcine MyHC-2a,-2x, and-2b, respectively. The entire amino acid sequences were highly conserved among the three MyHCs, except for the 50/20 k junction region (loop 2) which would weakly bind actin molecules. The porcine MyHC-2b possessed different amino acids from MyHC-2a and-2x, in loop1 and ELC binding region. The sequence data suggested the diversity of contractile properties among the porcine MyHC isoforms.     

Enzymological investigations on the causes for the PSE-syndrome, I. Comparative studies on pyruvate kinase from PSE- and normal pig muscles

A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with enzyme from normal muscle, pyruvate kinase isolated from PSE-muscle shows a five times lower K(m) for phosphoenol pyruvate and a more than ten times higher k (cat)K (m) value. The pH-dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lower K(m) value and a 100-fold increased k (cat)K (m) value for phosphoenol pyruvate compared to isoform 1. The presence of isoform 3 in PSE-muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE-muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE-muscles.     

Evidence for expression of IIb myosin heavy chain isoform in some skeletal muscles of Blonde d’Aquitaine bulls

In cattle, expression of the IIb myosin heavy chain (MyHC) isoform has been demonstrated only in extraocular muscles. In this study, we demonstrated for the first time its expression in the Semitendinosus and Longissimus thoracis muscles of a French beef breed, Blonde d’Aquitaine. Several techniques were used: RT-PCR, electrophoresis, western blotting, histochemistry with ATPase staining and immunohistochemistry using a combination of anti MyHC antibodies on serial sections. We found that MyHC IIb was expressed at the mRNA level in the two muscles of the cattle studied. However, the protein was observed at the tissue and cellular levels in only five of the 22 young bulls analysed, suggesting complex regulation of its expression. Using immunohistochemistry we demonstrated the presence of this MyHC isoform in pure fibres and also in hybrid fibres in co-expression with other MyHC.     

Specificity of different anti-myosin heavy chain antibodies in bovine muscle

Muscle fibre types play an important role in bovine meat tenderness, and conditions the speed of ageing. Two generations of myotubes appear during foetal life. Their differentiation results in several types of fibres differing in their contractile and metabolic properties, namely type I fibres and type II fibres (IIA and IIB). Fibre types were identified using antibodies by immunohistochemistry and western-blotting techniques. The aim was to test different antibodies on foetal and adult muscles by these two techniques. Ten monoclonal antibodies reported to be specific to different myosin heavy chain (MHC) isoforms in other species were tested. Only three antibodies were selected: BF.35, specific for I and IIa MHCs, F1.652 specific for foetal MHC and SC.71 an anti II MHCs (IIa and IIb) antibody. The use of these antibodies will enable a more precise characterisation of muscle fibres and will have applications in the study of bovine muscle ontogenesis.     

Alteration of the electrophoretic pattern of myofibrillar proteins in fish mince during frozen storage

A study was made of the variations in the electrophoretic profile of myofibrillar proteins in the muscles of blue whiting (Micromesistius poutassou R.), horse mackerel (Trachurus trachurus L.) and mackerel (Scomber scombrus L.). It was shown that all species presented different deterioration patterns during frozen storage. The fish were caught at two separate times of the year (winter and summer) and were stored frozen at -18°C for 1 year. The results indicate that during frozen storage, electrophoretic patterns varied according to species. Comparison of myosin heavy chain/actin (MHC/A) ratios indicates that blue whiting is the species that undergoes most alteration, and that this is more intense in fish caught in the summer than in the winter. Alteration of the MHC/A ratio was similar in horse mackerel and mackerel caught in the winter, whereas in the summer horse mackerel proved to be the most stable species. In all cases, the reduction of the MHC/A ratio was due essentially to alteration of the MHC, an effect which was particularly marked in blue whiting. Tropomyosin remained stable throughout the storage period in all three species.     

Comparative studies of pyruvate kinase from PSE and normal pig muscles

A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with the enzyme from normal muscle, pyruvate kinase isolated from PSE muscle shows a five times lower Michaelis constant,K _m, for phosphoenol pyruvate and a more than ten times higherk _cat/K _m value. The pH dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lowerK _m value and a 100-fold increasedk _cat/K _m value for phosphoenol pyruvate as compared to isoform 1. The presence of isoform 3 in PSE muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE muscles.     

Porcine sperm motility is regulated by serine phosphorylation of the glycogen synthase kinase-3alpha

Sperm functions are critically controlled through the phosphorylation state of specific proteins. Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase with two different isoforms (alpha and beta), the enzyme activity of which is inhibited by serine phosphorylation. Recent studies suggest that GSK3 is involved in the control of bovine sperm motility. Our aim was to investigate whether GSK3 is present in porcine spermatozoa and its role in the function of these cells. This work shows that both isoforms of GSK3 are present in whole cell lysates of porcine sperm and are phosphorylated on serine in spermatozoa stimulated with the cAMP analog, 8Br-cAMP. A parallel increase in serine phosphorylation of the isoform GSK3alpha, but not in the isoform GSK3beta, is observed after treatments that also induce a significant increase in porcine sperm velocity parameters. Therefore, a significant positive correlation among straight-line velocity, circular velocity, average velocity, rapid-speed spermatozoa, and GSK3alpha serine phosphorylation levels exists. Inhibition of GSK3 activity by alsterpaullone leads to a significant increase in the percentage of rapid- and medium-speed spermatozoa as well as in all sperm velocity parameters and coefficients. Moreover, pretreatment of porcine spermatozoa with alsterpaullone significantly increased the percentage of capacitated porcine spermatozoa and presents no effect in the number of acrosome-reacted porcine spermatozoa. Our work suggests that the isoform GSK3alpha plays a negative role in the regulation of porcine sperm motility and points out the possibility that sperm motile quality might be modulated according the activity state of GSK3alpha.     

Characteristics of impala (Aepyceros melampus) skeletal muscles

The aim of this study was to determine citrate synthase (CS), phosphofructokinase (PFK) activities and myosin heavy chain (MHC) isoform contents in four muscle groups (semimembranosus (S), deltoideus (D), longissimus lumborum (LL), and psoas major (PM)) of impala (n=6). All four muscle groups expressed predominantly MHC IIa (means of 55±22-93±12%). MHC IIx was only expressed in D. In D, positive correlations were found between MHC I and age (r=0.93; P<0.05) and the weight of the animals (r=0.94; P<0.01). PFK (means of 175±179-374±181), CS (means of 100±23-126±38 μmol/min/gdw) and MHC content indicated that energy provision in the impala is produced to a large extent via oxidative pathways and fibre types vary with animal characteristics.     

Influence of different feeding systems on the growth performance and muscle development of Japanese Black steers

This study investigated the growth performance and gene expression for muscle development between grass hay-fed (GH) and concentrate-fed (CT) steers. Daily gain and energy intake during the fattening period of the GH group were lower than those of the CT group. Analysis of C/EBPα, PPARγ2, myosin heavy chain (MHC), and myostatin gene expressions was performed by real-time PCR. Expressions of C/EBPα and myostatin in semitendinosus and longissimus lumborum (LL) muscles were higher in the CT group than in the GH group at the end of fattening. In LL muscle, MHC expression at the end of fattening was greater in the GH group than in the CT group. These results suggest that regulation of adipogenesis and myogenesis by the expression of genes involved in muscle development might have occurred in the skeletal muscle of the GH group by the feeding of grass hay and/or because of the low energy intakes.     

Phosphorylation and glycosylation isoforms of bovine κ-casein variant E in homozygous Swedish Red cow milk detected by liquid chromatography-electrospray ionization mass spectrometry

Variations in the phosphorylation and glycosylation patterns of the common κ-casein (CN) variants A and B have been explored, whereas studies on variant E heterogeneity are scarce. This study reports for the first time the detailed phosphorylation and glycosylation pattern of the κ-CN variant E in comparison with variants A and B. Individual cow milk samples representing κ-CN genotype EE (n = 12) were obtained from Swedish Red cows, and the natural posttranslational modifications of its κ-CN were identified and quantified by liquid chromatography-electrospray mass spectrometry. In total, 12 unique isoform masses of κ-CN variant E were identified. In comparison, AA and BB milk consisted of 14 and 17 unique isoform masses, respectively. The most abundant κ-CN E isoform detected in the EE milk was the monophosphorylated, unglycosylated [1P 0G, ～70%; where P indicates phosphorylation from single to triple phosphorylation (1–3P), and G indicates glycosylation from single to triple glycosylation (1–3G)] form, followed by diphosphorylated, unglycosylated (2P 0G, ～12%) form, resembling known patterns from variants A and B. However, a clear distinction was the presence of the rare triphosphorylated, nonglycosylated (3P 0G, ～0.05%) κ-CN isoform in the EE milk. All isoforms detected in variant E were phosphorylated, giving a phosphorylation degree of 100%. This is comparable with the phosphorylation degree of variants A and B, being also almost 100%, though with very small amounts of nonphosphorylated, glycosylated isoforms detected. The glycosylation degree of variant E was found to be around 17%, a bit higher than observed for variant B (around 14%), and higher than variant A (around 7%). Among glycosylation, the glycan e was the most common type identified for all 3 variants, followed by c/d (straight and branched chain trisaccharides, respectively), and b. In contrast to κ-CN variants A and B, no glycan of type a was found in variant E. Taken together, this study shows that the posttranslational modification pattern of variant E resembles that of known variants to a large extent, but with subtle differences.     

Proteomic evaluation of myofibrillar carbonylation in chilled fish mince and its inhibition by catechin

The present study investigates the susceptibility of individual myofibrillar proteins from mackerel (Scomber scombrus) mince to undergo carbonylation reactions during chilled storage, and the antioxidant capacity of (+)-catechin to prevent oxidative processes of proteins. The carbonylation of each particular protein was quantified by combining the labelling of protein carbonyls by fluorescein-5-thiosemicarbazide (FTSC) with 1-D or 2-D gel electrophoresis. Alpha skeletal actin, glycogen phosphorylase, unnamed protein product (UNP) similar to enolase, pyruvate kinase, isoforms of creatine kinase, aldolase A and an isoform of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) showed elevated oxidation in chilled non-supplemented mince. Myosin heavy chain (MHC) was not carbonylated in chilled muscle, but an extensive MHC degradation was observed in those samples. The supplementation of catechin reduced protein oxidation and lipid oxidation in a concentration-dependent manner: control > 25 > 100 ≈ 200 ppm. Therefore, the highest catechin concentrations (100 and 200 ppm) exhibited the strongest antioxidant activity. Catechin (200 ppm) reduced significantly carbonylation of protein spots identified as glycogen phosphorylase, pyruvate kinase muscle isozyme, isoforms of creatine kinase. Conversely, catechin was ineffective to inhibit the oxidation of actin and UNP similar to enolase. These results draw attention to the inefficiency of catechin to prevent actin oxidation, in contrast to the extremely high efficiency of catechin in inhibiting oxidation of lipids and other proteins.     

Differential expression and secretion of alpha1-acid glycoprotein in bovine milk

alpha1-Acid glycoprotein (AGP) is a lipocalin that is produced mainly by the liver and secreted into plasma in response to infections and injuries. In this study, we evaluated AGP isoforms that can be detected in bovine milk. We found that milk-AGP content is made up of at least two isoform groups, a low MW group (44 kDa) that is produced in the mammary gland (MG-AGP), and a higher MW group (55-70 kDa), that is produced by somatic cells (SC-AGP). Identical SC-AGP isoforms can be found both in milk and blood PMN cells. Analysis of the mammary tissue cDNA showed that the sequence of the MG-AGP isoform is identical to that of plasma AGP. Each group contains several proteins with different MWs and different isoelectric points, as shown by 2D-electrophoresis. The glycosylation patterns of these isoforms were analysed by means of specific lectin binding, to evaluate the degree of sialylation, fucosylation and branching. The MG-AGP glycan pattern was identical to plasma AGP produced by the liver. Several differences were detected, however, between plasma and SC-AGP isoforms, the most evident being the strong degree of fucosylation and the elevated number of di-antennary glycans in SC-AGP. Immunohistochemistry showed that AGP is found in all tissues that make up the mammary gland, but that it is most likely produced for the main part by the alveoli.