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ANIMAL PRODUCTS |
* Department of Food Science, Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark;
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Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense; and
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Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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, elongin B, and phosphohistidine phosphatase 14. The changes observed on MLC II and MLC III could be a consequence of enzymatic cleavage in the neck region of the globular myosin head domain that causes the release of MLC II and MLC III from the actomyosin complex. It has previously been hypothesized that compensatory growth results in an increased postmortem proteolysis; thus it was presumed that the intensity of some protein fragments would be affected by compensatory growth. However, the peptides that were found to be affected at 48 h postmortem were all full-length proteins. The 14-3-3 protein has been proposed to play a role in the contraction of muscle during rigor and may thereby have an effect on meat tenderness. This study reveals some very interesting changes in the muscle proteome affected by compensatory growth, which may be useful in understanding the relationship among compensatory growth, protein turnover, and meat tenderness.
Key Words: compensatory growth • feeding strategy • meat quality • muscle growth • proteome analysis • sarcoplasmic protein
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Studies in cattle have shown that compensatory muscle growth is due firstly to a larger difference between the rate of muscle protein synthesis and the rate of muscle protein degradation and, secondly, due to the fact that the rate of protein turnover is elevated at certain periods during compensatory growth (Jones et al., 1990).
Studies of pigs revealed that female pigs with compensatory growth have increased muscle protein synthesis and in vitro protein degradation at slaughter compared with controls (Therkildsen et al., 2004). It was reported that compensatory growth also resulted in more tender meat compared with meat from pigs fed ad libitum (Kristensen et al., 2004). However, the relationship between compensatory growth and meat tenderness is still unclear. It has been hypothesized that the improved tenderness is a result of increased postmortem protein degradation as a consequence of elevated protein turnover during compensatory growth (Kristensen et al., 2002).
The aim of the current study was to evaluate the influence of compensatory growth on the muscle proteome postmortem with the use of a proteomics approach to increase the understanding of the linkage between compensatory growth and meat tenderness. During recent years there has been a huge development in proteomics that enables the monitoring hundreds of different proteins simultaneously and that makes it possible to study the complex patterns of protein and gene expression in cells and tissues. Furthermore, it has been shown that proteomics is a useful tool to study the mechanism of meat tenderization (Lametsch and Bendixen, 2001; Hwang et al., 2005).
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Sixteen female pigs (Duroc x Landrace x Large White), 2 from each of 8 litters, were divided into 2 groups, 1 pig from each litter in each group. The groups had either free access to the diet (control group) from d 28 to 140 or were fed the diet at a restricted level (60% of ad libitum feeding) from d 28 to 80 and then had free access to the diet (compensatory growth group) until slaughter at d 140. All pigs were penned individually and were fed diets according to their age. From weaning to d 56 they were fed a starter diet, from d 57 to 77 a grower diet 1, from d 78 to 112 a grower diet 2, and finally a finishing diet from d 113 to 140 as described in Therkildsen et al. (2004). This experiment was approved by and the animals treated in accordance with the guidelines outlined by the Danish Inspectorate of Animal Experimentation.
Slaughtering and Muscle Sampling
The pigs were slaughtered litterwise at d 140 using CO2 stunning at the experimental slaughterhouse at Research Center Foulum, Denmark. The slaughter procedure is described in detail in Therkildsen et al. (2004). Samples for 2-dimensional gel electrophoresis (2DE) were collected within 15 min postmortem and 48 h postmortem from M. longissimus dorsi, snap-frozen in liquid nitrogen, and stored at –80°C.
Sample Preparation for 2-Dimensional Gel Electrophoresis
One gram of muscle tissue was homogenized in 6 mL of ice-cold buffer [0.1 M Tris, pH 8.0, with added protease inhibitor (complete, Roche, Huidoure, Denmark)] on ice with an Ultra Turrax T25 (Ika Laboratechnik, Staufen, Germany) twice for 30 s each at 9,500 rpm followed by twice for 30 s each at 13,500 rpm. The samples were centrifuged at 4°C for 20 min at 25,000 x g, and the supernatant was removed for 2DE and stored at –80°C until use. The protein concentration was determined using the BCA assay (Pierce Chemical Company, Rockford, IL).
Two-Dimensional Gel Electrophoresis
Eleven-centimeter, pH 4 to 7, immobilized pH gradients strips were used for the first dimension and 40 µg of protein was loaded for the analytical gels, whereas 75 µg was loaded for the preparative gels during the rehydration step. In the second dimension, an 8 to 16% gel (Criterion, BioRad) was used. The analytical gels were silver stained according to Lametsch and Bendixen (2001), and the preparative gels were stained using an imidazolezinc reverse staining according to Fernandez-Patron et al. (1995).
Image and Statistical Analysis
The gels were scanned using a UMAX PowerLook 1120 scanner (UMAX Technologies, Freemont, CA), and ImageMaster 2D Platinum software v5 (Amersham Bioscience, Uppsala, Sweden) was used for image analysis. The spot intensity was measured as the relative volume within a gel and represents normalized values that remain relatively independent of irrelevant variations between the gels. The gels were analyzed in 2 groups, one with the samples removed at slaughter and the other with samples removed 48 h after slaughter.
Data were analyzed for the effect of compensatory growth in a complete randomized block (litter) design using the mixed model procedure of SAS (SAS Inst., Inc., Cary, NC) with the fixed effects of treatment. Litter origin was included in the model as a random effect, and weaning weight was used as a covariate. The spots that were found to be significantly (P < 0.05) affected by compensatory growth were individually inspected and confirmed.
In-Gel Digestion of Protein Spots
The spots of interest were cut out of the preparative gels and submitted to in-gel trypsin digestion as described by Jensen et al. (1998). The excised gel plugs were washed 3 times for 15 min each in 30 µL of 50% acetonitrile/50 mM NH4CO3, pH 7.8, and then shrunk in 30 µL of 100% acetonitrile. The proteins were reduced and alkylated with 30 µL of 50 mM NH4CO3, 100 mM dithiothreitol, followed by 30 µl of 50 mM H4CO3, 150 mM iodoacetamide. The gel piece was washed with 30 µL of 50% acetonitrile/50 mM NH4CO3, pH 7.8, and 30 µL of 100% acetonitrile and dried by lyophilization. Fifteen microliters of trypsin (sequencing grade, Roche, Mannheim, Germany), dissolved in 50 mM of NH4CO3, pH 7.8 (10 ng of trypsin/µL), was added to the dry gel pieces, and they were left to reswell on ice. After 1 h, the supernatant was removed, and 30 µL of 50 mM NH4CO3, pH 7.8, was added, and the digestion was incubated overnight at 37°C.
Desalting and Concentration
Custom-made chromatographic columns were used for desalting and concentration of the peptide mixture before mass spectrometric analysis (Gobom et al., 1999). A column consisting of 100 to 300 nL of Poros reverse phase R2 material (20 µm bead size, PerSeptive Biosystems, Framingham, MA) was packed in a constricted GeLoader tip (Eppendorf, Hamburg, Germany). A 10-mL syringe was used to force the liquid through the column. Twenty microliters of the tryptic protein digests was acidified by trifluoroacetic acid (TFA), loaded onto the column, and washed with 20 µL of 0.1% TFA. For analyses by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), the peptides were eluted with 0.5 µL of matrix solution (15 to 20 g/L of 
-cyano-4-hydroxycinnamic acid in 70% acetonitrile/0.1% TFA) directly onto the MALDI target in very small droplets.
Peptide-Mass Mapping by MALDI-TOF-TOF-MS
The proteins of interest were identified with the use of a MALDI-TOF-TOF instrument (4700 Proteomics analyzer, Applied Biosystems, Foster City, CA). Protein identification was performed using the Mascot database search program. The peptide-mass maps and protein identifications were evaluated as described by Jensen et al. (1998).
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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. The pigs that were feed-restricted from d 28 to 80 followed by a period with free access to the diet showed compensatory growth, which was expressed as a greater absolute and fractional rate of growth from d 81 to 140. Although the restricted pigs were on average 6 kg lighter at d 80, the compensatory growth response resulted in similar carcass weight, meat percentage, and fat thickness across treatments at slaughter, indicating complete compensation at this time. Tyrosine release from incubated muscle strips and RNA and elongation factor 2 concentrations in muscle tissue were increased in pigs showing compensatory growth at slaughter (Therkildsen et al., 2004), indicating increased protein turnover and suggesting that the muscle was still in a state of compensatory growth.
The meat quality traits of the pigs used in this study are described in detail in Kristensen et al. (2004). In summary, the sensory analysis showed that female pigs exhibiting compensatory growth produced meat with increased tenderness compared with control pigs (P < 0.05). Although not significant, similar results were obtained on the Warner Bratzler shear force. Desmin and troponin-T degradation and the myofibrillar fragmentation index were not affected by treatment. No dietary treatment effects on the activity of µ-calpain, m-cal-pain, or calpastatin at the time of slaughter were observed.
The muscle samples used for proteome analysis were fractionated, and only the sarcoplasmic proteins fraction was analyzed with 2DE. This resulted in highly reproducible gels containing more spots compared with gels containing nonfractionated protein samples because the muscle proteins of major abundance such as myosin heavy chain, actin, titin, and nebulin, which make up nearly 80% of the total muscle protein, were removed. Only spots that were well separated and were represented in 75% of the gels were used for data analysis. The proteins identified are listed in Table 1 together with the database accession number, the calculated molecular weight, the estimated molecular weight, and the score of the database search.
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, and the mean spot intensity of the 2 treatments for the proteins that were found to be affected by compensatory growth are shown in Figure 2A.
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; Mccomb and Spurlock, 1997). Furthermore, it is believed that HSC70 together with BAG-1 and CHIP play a role in the regulation of the proteolytic pathways, such as the proteasome complex, during muscle development and regeneration (Liu and Steinacker, 2001; Alberti et al., 2003; McDonough and Patterson, 2003). The HSC70 is required to regulate the fate of aberrantly folded or damaged proteins and thus has a critical role in regulating the proteasome pathway. Protein degradation by the proteasome pathway is a dominant mechanism involved in myofibrillar protein degradation in vivo and may also be involved in the control of myoblast proliferation through the selective degradation of MyoD and regulatory proteins of the cell cycle such as cyclins (Hasselgren et al., 2002).
Two spots of HSP27 were affected by compensatory growth. The major functions of the small heat shock protein HSP27 include stabilization of microfilaments and cytokine signal transduction (Liu and Steinacker, 2001). Several studies have revealed that HSP27 show abundant constitutive expression in skeletal muscle (Kato et al., 1992; Sugiyama et al., 2000), and its role in organizing and protecting the myofibril structure has been suggested by the demonstration of the localization of HSP27 on specific sarcomeric structures such as Z-or I-bands (Sugiyama et al., 2000). The HSP27 also showed regulated expression in pig muscle during development and in C2C12 cells during differentiation (Sugiyama et al., 2000; Ito et al., 2001; Tallot et al., 2003). The different functions that have been described for the 2 members of the heat shock family HSC70 and HSP27 indicate that the 2 proteins may have an important role in the hypertrophic muscle growth observed during compensatory growth.
The enolase family is part of the glycolytic pathway and is considered to be a useful marker in the study of differentiation and pathological alteration. It has been shown that protein expression and activity of enolase 3 is related to regeneration and development of the muscle (Merkulova et al., 2000). Glycerol-3-phosphate dehydrogenase is also part of the glycolytic pathway and has been described as a marker for the glycolytic potential (White et al., 2000). The spot intensity of both enolase 3 and GPDH were decreased in pigs showing compensatory growth, which indicates a decrease in the glycolytic potential.
Bisphosphoglycerate mutase belongs to a family of glycolytic housekeeping enzymes. The main function of the enzyme is to regulate the oxygen affinity of hemoglobin through synthesis of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin (Wang et al., 2004). Aldehyde dehydrogenase catalyzes the pyridine nucleotide-dependent oxidation of aldehydes to acid and is categorized as critical for normal development (Sladek, 2003). Here we found the expression of bisphosphoglycerate mutase, and aldehyde dehydrogenase E2 and E3, to be decreased by compensatory growth, but the role of these proteins in compensatory growth is unclear.
Figure 2A
shows that the proteins found to be affected by compensatory growth at slaughter all have lower mean spot intensity compared with control pigs. It was presumed that at least the intensity of some affected spots would be greater in muscle samples from pigs that exhibited a compensatory growth response. An explanation for the fact that all of the affected spots at slaughter have a lower mean intensity in the samples from the compensatory growth group compared with those of the controls could be that mainly myofibrillar proteins increase during compensatory growth, with no change in the sarcoplasmic proteins, and thus a change in the ratio between myofibrillar and sarcoplasmic proteins. However, compensatory growth did not affect the protein concentration of the sarcoplasmic proteins extracted from the muscle, and analysis of the sarcoplasmic protein fractions with 1D gel electrophoresis showed no effect on the relatively small amount of actin and myosin (data not shown).
The proteome analysis of muscle samples removed 48 h postmortem showed that 7 different proteins increased in staining intensity in pigs showing compensatory growth: myosin light chain (MLC) II, MLC III, sulfite oxidase, chloride intracellular channel 1, elongin B, phosphohistidine phosphatase, and 14-3-3 protein . In addition, one was downregulated in the compensatory growth group: GPDH. The positions of the proteins identified are illustrated in Figure 3, and the mean spot intensity of the 2 treatments for the proteins found to be affected by compensatory growth is shown in Figure 2B.
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). Therefore, it was presumed that the intensity of some protein fragments would be affected by compensatory growth in the samples removed 48 h postmortem. Previous studies using 2DE have revealed that a large number of protein fragments increase in intensity postmortem as a consequence of postmortem proteolysis (Lametsch et al., 2002; Morzel et al., 2004; Hwang et al., 2005). However, the peptides found to be affected by compensatory growth at 48 h postmortem were all full-length proteins. It was found that the intensity of both MLC II and MLC III was increased by compensatory growth 48 h after slaughter.
The MLC is the part of myosin that has an important role in the structure of the muscle fiber. It has previously been reported that the postmortem intensity of MLC II may be related to tenderness (Lametsch et al., 2003). However, the impact of MLC on tenderness is unclear. Recently, it was reported that MLC is dephosphorylated postmortem, and that the dephosphorylation is related to the postmortem metabolism (Morzel et al., 2004). On the other hand, it has been reported that myosin is cleaved in the neck region of the globular myosin head domain (Lametsch et al., 2002), which also contains the binding site for MLC II and III (Rayment et al., 1993), and it can be speculated that this cleavage would release MLC II and III from the actomysin complex into the sarcoplasmic fraction. If this is the case, then the greater intensity of the MLC II and III in the samples from the pig showing compensatory growth indicates a greater proteolytic activity postmortem in these pigs. This is in agreement with the hypothesis that the influence of compensatory growth on meat tenderness is a consequence of increased postmortem protein degradation.
The 14-3-3 protein is member of the 14-3-3 protein family, which are conserved regulatory proteins that bind a multitude of functionally diverse signaling proteins including kinases and phophatases (Fu et al., 2000). It has been proposed that the 14-3-3 proteins have a role in the regulation of myosin light chain kinase that becomes phosphorylated during muscle contraction, and it can be speculated that 14-3-3 protein may play a role in the muscle contraction during rigor (Haydon et al., 2002). However, it is unclear why 14-3-3 protein is affected by compensatory growth and if it has any relationship with meat tenderness. There was no significant effect of compensatory growth on the sarcomere length (Kristensen et al., 2004), which could indicate that 14-3-3 protein , through an effect on sarcomere length, could affect meat tenderness. The 14-kDa phosphohistidine phosphatase was found to have a greater intensity in samples from pigs showing compensatory growth.
The mitochondrial protein sulfite oxidase, and the 2 nuclear proteins elongin B and chloride intracellular channel 1, were increased in intensity 48 h after slaughter in the compensatory growth group. An explanation of these observations could be that the number of mitochondria and satellite cells is increased as a consequence of increased protein turnover and that the proteins from these 2 organelles leaked into sarcoplasm postmortem, or that the membrane stability of mitochondria and satellite cells is affected by compensatory growth leading to a quicker release of sulfite oxidase, elongin B, and chloride intracellular channel 1.
1 Corresponding author: rla{at}kvl.dk
Received for publication April 26, 2005. Accepted for publication November 7, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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