Structural differences between myofibrillar protein, paratropomyosin, and tropomyosin as revealed by high-performance liquid chromatography.

Paratropomyosin (PTM) composes myofibril functions to weaken the rigor linkages formed between actin and myosin during postmortem aging of muscles. PTM has the similar physico-chemical properties as tropomyosin (TM) that is a regulatory protein of myofibrils. So far, it is unclear whether PTM is definitely different from TM, because the primary structure of PTM has not been determined yet. The aim of this study was to clarify structural difference of PTM from TM. PTM was prepared by column chromatography immediately after slaughter from broiler breast muscle, and purified by high-performance liquid chromatography (HPLC). Purified PTM was successfully separated from TM, and the recovered PTM molecule was reduced with dithiothreitol to separate again by HPLC. Two subunits were obtained and peptides from each digested subunit by V8 protease were recovered by HPLC, and then amino acid sequences of the peptides were analyzed by protein sequencing. As a result, some amino acid residues were replaced from that of TMα1 isoform which is the major isoform of TM, and also was different between the two subunits. Therefore, it is concluded that PTM clearly differs from TM and it is suggested that functional difference in PTM from TM is attributed to amino acid replacements in subunits composing PTM.

Hypoxanthine enhances the cured meat taste.

We evaluated the enhancement of cured meat taste during maturation by sensory analysis. We focused on the heat-stable sarcoplasmic fraction (HSSF) to identify the factors related to cured meat taste. Because the dry matter of HSSF contained more than 30% nitrogen, nitrogen compounds such as free amino acids, small peptides and adenosine triphosphate-related compounds seemed to be the important components of HSSF. The samples cured with HSSF for 2 h exhibited the same taste profile as ones cured without HSSF for 168 h. Therefore, the changes in the amount and fractions of nitrogen compounds were examined in HSSF during incubation from 0 to 168 h. The concentration of hypoxanthine (Hx) gradually increased, while inosine-5'-monophosphate decreased during the incubation. The samples cured with pickles containing various concentrations of Hx were subjected to sensory analysis. The addition of Hx, in a dose-dependent fashion, enhanced cured meat taste by maturation for 2 h. It was concluded that Hx is essential for the enhancement of cured meat taste.

Role of Heavy Meromyosin in Heat-Induced Gelation in Low Ionic Strength Solution Containing L-Histidine.

The gelation of myosin has a very important role in meat products. We have already shown that myosin in low ionic strength solution containing L-histidine forms a transparent gel after heating. To clarify the mechanism of this unique gelation, we investigated the changes in the nature of myosin subfragments during heating in solutions with low and high ionic strengths with and without L-histidine. The hydrophobicity of myosin and heavy meromyosin (HMM) in low ionic strength solution containing L-histidine was lower than in high ionic strength solution. The SH contents of myosin and HMM in low ionic strength solution containing l-histidine did not change during the heating process, whereas in high ionic strength solution they decreased slightly. The heat-induced globular masses of HMM in low ionic strength solution containing L-histidine were smaller than those in high ionic strength solution. These findings suggested that the polymerization of HMM molecules by heating was suppressed in low ionic strength solution containing L-histidine, resulting in formation of the unique gel.

The importance of subfragment 2 and C-terminus of myosin heavy chain for thick filament assembly in skeletal muscle cells.

In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM.

Effects of meat species on the postprandial thermic effect in rats.

We examined animal species differences in the postprandial thermic effect of meat and investigated the postprandial thermic effect of mutton in rats. After intake of experimental diets containing each meat, body temperatures of rats fed mutton or venison were significantly higher than that of rats fed rabbit meat. After intake of experimental diets containing fractionized mutton, the body temperatures of rats fed diets containing lean mutton protein were higher than those of rats fed diets without lean mutton protein. In a two-dimensional fluorescence difference gel electrophoresis study of brown adipose tissue, it was shown that the intake of mutton up-regulated the expression of many signaling molecules that are involved in energy metabolism. The postprandial thermic effect of mutton seems to be due not to catecholamine and adrenocorticotropic hormone but to thyroid hormones. The results suggest that intake of lean mutton protein stimulates the secretion of thyroid hormones and facilitates energy metabolism in rats.

Effect of liquid whey feeding on fecal microbiota of mature and growing pigs.

The effect of liquid whey feeding on fecal bacteria and their metabolites was assessed in five pregnant sows and 66 growing pigs. Sows were fed a control diet for 4 weeks (control period) followed by the same diet but with whey feeding (5 L/day/pig) for 4 weeks (whey period). One group of growing pigs was given 267 L of whey per pig (whey group), while the other group was not (control group). In both cases, liquid whey was given separately from control diet. Sows in the whey period had feces showing lower pH, lower ammonia concentration, and larger population sizes of total bacteria, lactobacilli and bifidobacteria. The bacterial gene library analysis indicated that Mitsuokella and Megasphaera were more frequently detected, while Clostridium disporicum were detected less frequently in the whey period. Feces from whey-fed growing pigs showed lower pH than that from control pigs in the early stage of growing. Also, larger populations of total bacteria, lactobacilli and bifidobacteria were recorded in the whey group. From the bacterial gene library analysis, the detection frequency of Lactobacillus reuteri tended to be higher in the whey group. These results indicate that whey feeding influences the hindgut microbiota of pigs, possibly leading to a fermentation shift that is favorable for animal health.

In vitro measurement of post-natal changes in proliferating satellite cell frequency during rat muscle growth.

Satellite cells, resident myogenic stem cells found in postnatal skeletal muscle, are most abundant during early postnatal development and sharply decline in frequency thereafter to adult levels in mice and rats. Therefore, postnatal changes in satellite cell mitotic activities are important aspects for further understanding a muscle growth strategy. In large meat-production animals, however, the traditional in vivo proliferation assay may be less realistic because it requires intra-peritoneal (ip) injection of huge dosage of mutagenic nucleosides, (3)H-labeled thymidine or bromodeoxyuridine (BrdU), at each age-time of sacrifice. We report in the present pilot study using rats that in vivo proliferation activity of satellite cells can be evaluated by an in vitro BrdU-incorporation assay in early cultures. Briefly, satellite cells were prepared from upper hind-limb and back muscles and maintained for 24 h with imposing by BrdU addition for the last 2 h, followed by the regular immunocytochemistry for determining BrdU-incorporated cell percentage. This in vitro assay demonstrated a rapid decrease in proliferating satellite cell frequency to the adult level during about 3-month period after birth, and yielded a high correlation to the measurements by the in vivo BrdU ip-injection method during the postnatal period examined from day-2 to month-11. The in vitro proliferation assay may be further adaptable for large domestic animals by the combination with a muscle biopsy technique that enables age-interval sampling from the same growing animals.

Nitric oxide inhibits the formation of zinc protoporphyrin IX and protoporphyrin IX.

The aim of this study was to elucidate the mechanism by which curing agents, especially nitrite, inhibit the formation of zinc protoporphyrin IX (ZPP) in dry-cured hams such as Parma ham. The oxidation-reduction potential of model solutions was increased by the addition of nitrite, but it was not clear whether the formation of ZPP is inhibited by the oxidizing property of nitrite. The effect of nitric oxide (NO) produced from nitrite on the formation of ZPP was examined. The amount of ZPP formed was decreased by the addition of NO donors. The amount of protoporphyrin IX (PPIX), which is the precursor of ZPP, was also decreased by the addition of NO donors. It is concluded that NO produced from nitrite inhibited the formation of PPIX and ZPP was therefore not formed in cured meat products with the addition of nitrite or nitrate.

Interaction between myostatin and extracellular matrix components.

Myostatin, a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle mass. We have recently demonstrated that decorin binds to myostatin in vitro, and that immobilized decorin within the collagen matrix prevents myostatin-mediated inhibition of myoblast proliferation. However, little is known about other ECM molecules that bind to myostatin and modulate its activity. Thus, in the present study, we investigated the interaction of several other ECM molecules with myostatin. We here show that fibromodulin, fibronectin and laminin bind to myostatin in the presence of Zn(2+) with a dissociation constant (K(D)) of 10(-10) approximately 10(-8) mol/L. Fibromodulin shows the highest affinity for myostatin among them. These results suggest that these ECM molecules may modulate myostatin activity like decorin does.

Formation of zinc protoporphyrin IX in Parma-like ham without nitrate or nitrite.

Zinc protoporphyrin IX (ZPP) is a characteristic red pigment in meat products that are manufactured without the addition of a curing agent such as nitrate or nitrite. To examine the effects of impurities such as mineral components in sea salt on the formation of ZPP, we manufactured Parmatype dry-cured hams that were salted with refined salt or sea salt and examined the involvement of oxidation-reduction potential (ORP) in the formation of ZPP. The content of ZPP was increased drastically after 40 weeks. Microscopic observation showed strong fluorescence caused by ZPP muscle fiber after 40 weeks. Conversely, heme content varied considerably during processing. ORP increased during processing. However, there was no obvious difference between ham salted with refined salt and that salted with sea salt. Therefore, it was concluded that impurities in sea salt were not involved in the formation of ZPP.

Quantitative determination of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC.

We measured the contents of Zn protoporphyrin IX (ZPP), heme and protoporphyrin IX (PPIX) in Parma ham by simultaneous analysis using high-performance liquid chromatography (HPLC). Extraction with ethyl acetate-acetic acid (4:1) was suitable for the quantitative analysis of ZPP. The contents of heme, ZPP and PPIX in Parma ham were 15.0-29.9, 27.7-47.0 and 0.4-1.1µg/g, respectively, and total content of porphyrin was 43.7-76.6µg/g. The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60-70% of all porphyrins.

Inhibition of matrix metalloproteinases suppresses the migration of skeletal muscle cells.

Skeletal muscle satellite cells are quiescent stem cells that localized between the plasmalemma and the basement membrane of muscle fiber. When muscle is injured, satellite cells are activated, migrate to the injured site and contribute to the regeneration of muscle. However, little is known about the mechanism by which satellite cells migrate underneath the basement membrane. To clarify this, we investigated the effect of MMP inhibition on the migration of C2C12 muscle cells in vitro using a time-lapse imaging system. The migration speed of cells cultured with an MMP-inhibitor reagent was significantly lower (P<0.01) than the control cultured without an inhibitor reagent. The persistency index was significantly higher (P<0.01) in cells cultured with an inhibitor than in those without an inhibitor. Furthermore, MMP-3 knockdown cells migrated faster than control cells. These results strongly suggest that MMPs synthesized in skeletal muscle cells play an important role in the migration of these cells.

Decorin expression in quiescent myogenic cells.

Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells.

Decorin enhances the proliferation and differentiation of myogenic cells through suppressing myostatin activity.

Decorin, a small leucine-rich proteoglycan, plays an important role in the regulation of cell growth. Our recent study has shown that immobilized decorin in the collagen matrix sequesters myostatin into the extracellular matrix and prevents its inhibitory action to myoblast proliferation in vitro. However, it still remains unclear whether free decorin could affect the proliferation and differentiation of myogenic cells by regulating myostatin activity. In the present study, we generated stable clonal C2C12 myoblasts that were over-expressing decorin, and showed that decorin over-expressing cells had an increased rate of proliferation as compared to control cells. Decorin over-expressing cells formed multi-giant hypertrophic myotubes with an elongated morphology and larger size as compared to control cells, although the initiation of differentiation in decorin over-expressing cells was somewhat delayed as compared to control cells. Western blot analysis demonstrated that MyoD expression in decorin over-expressing cells was lower than that in control cells until 12 h after induction to differentiate. At 48-h differentiation, the expressions of MyoD, p21 and myogenin were dramatically increased in cells that over-expressed decorin. Furthermore, we revealed that over-expression of decorin suppressed the activity of myostatin endogenously synthesized in C2C12 myoblasts and attenuated the signaling of exogenous myostatin. Consistent with these results, knock-down of decorin impairs C2C12 myoblast growth by increasing the sensitivity to exogenous myostatin. These results clearly show that decorin enhances the proliferation and differentiation of C2C12 myoblasts through suppressing myostatin activity.

Spatiotemporal expression of decorin and myostatin during rat skeletal muscle development.

Decorin, a small leucine-rich proteoglycans, plays an important role in tissue morphogenesis through the regulation of collagen fibrillogenesis and the modulation of some growth factors. Our recent study has shown that decorin binds to myostatin, a negative regulator of skeletal muscle mass, and modulates its inhibitory action to myogenic cell growth in vitro. However, it still remains unclear whether decorin binds to myostatin in vivo during the development of skeletal muscle. To clarify this, we investigated the spatiotemporal expression of decorin and myostatin in rat skeletal muscle by RT-PCR and immunohistochemistry. Decorin mRNA abundance in fetal skeletal muscle was significantly higher than those in neonates and adults (P<0.05). Decorin mRNA expression decreased drastically at birth, and thereafter gradually up to 9 weeks of age. The mRNA expression pattern of myostatin was quite similar to that of decorin during prenatal and postnatal development of rat skeletal muscle. Immunohistochemical analysis demonstrated that myostatin was located in the muscle fibers, and that decorin was located in the periphery of muscle fibers in fetal rat skeletal muscle. Taken together with our previous data, these results suggest that decorin binds myostatin and sequesters it in the ECM during the development of rat skeletal.

Zn protoporphyrin IX is formed not from heme but from protoporphyrin IX.

We examined the effects of exogenous myoglobin, a bivalent chelator, and nitrite on Zn protoporphyrin IX (ZPP) formation by using model systems. ZPP was formed in a model solution without addition of exogenous myoglobin. After incubation, the amount of ZPP in a model solution was increased but that of heme was not decreased compared with the amounts before incubation. Protoporphyrin IX (PPIX) instead of ZPP also accumulated in a model solution with addition of EDTA, but the amount of heme was not reduced. These results suggested that ZPP was not formed by the Fe-Zn substitution in heme but was formed by the insertion of Zn into PPIX, which was formed independently. The fact that the effects of various factors in model systems with/without addition of a bivalent chelator were similar suggested that ZPP formation was strongly affected by PPIX formation. Inhibition of PPIX formation by nitrite might be the reason for the low levels of ZPP in cured meats.

Direct demonstration of the presence of zinc in the acetone-extractable red pigment from Parma ham.

We studied the presence of zinc in the red pigment extracted from Parma ham by scanning electron microscopy/energy dispersive X-ray microanalysis. The large peak of about 8.6keV and the small peaks at about 1.1keV and 9.5keV were derived from K(α), L(α1) and K(ß) X-ray of zinc, respectively. Thus, the results suggested that zinc existed in the red pigment extracted from Parma ham. In contrast, the K(α) X-ray peak of iron (6.4keV) was not detected and the K(α) X-ray peak of magnesium (1.3keV) was hardly detected. These results revealed that the red pigment contained not iron but zinc. In addition to the results of mass analysis in previous studies, the red pigment was demonstrated to be zinc protoporphyrin IX.

Low-pH preparation of skeletal muscle satellite cells can be used to study activation in vitro.

When skeletal muscle is stretched or injured, satellite cells are activated to enter the cell cycle, and this process could be mediated by hepatocyte growth factor (HGF) and nitric oxide (NO) as revealed by primary culture technique. In this system, which was originally developed by Allen et al. [Allen, R. E., Temm-Grove, C. J., Sheehan, S. M., & Rice, G. (1997). Skeletal muscle satellite cell cultures. Methods Cell Biol., 52, 155-176], however, some populations of satellite cells would receive activation signals during the cell isolation procedure; the high baseline level of activation diminishes the magnitude of the observed effect of HGF and NO. In this study, we modified the cell isolation procedure by lowering pH of muscle and isolation media from 7.2 (original) to 6.5. This modification was designed to block the activation signal generation, based on our previous observations that the satellite cell activation in response to mechanical stimulation only occurred between pH 7.1 and 7.5. Satellite cells prepared at low-pH showed a low baseline level of activation in bromodeoxyuridine incorporation and MyoD expression assays on control cultures, and demonstrated a large activation response to mechanical stretch, exogenous HGF and NO donor. Cell yield and myogenic purity were not affected by the modifications. The low-pH procedure could provide an improved satellite cell model for in vitro activation experiments.

Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor.

In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration-dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer N(G)-nitro-D-arginine methyl ester HCl (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which can activate satellite cells.

Decorin binds myostatin and modulates its activity to muscle cells.

Myostatin, a member of TGF-beta superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-beta and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between myostatin and decorin by surface plasmon assay. Decorin interacted with mature myostatin in the presence of concentrations of Zn(2+) greater than 10microM, but not in the absence of Zn(2+). Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K(D)) of 2.02x10(-8)M and 9.36x10(-9)M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap myostatin and modulate its activity to myogenic cells in the ECM.