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Protein expression in pectoral skeletal muscle of chickens as influenced by dietary methionine1 W. Zhai,* L. F. Araujo,* S. C. Burgess,† A. M. Cooksey,† K. Pendarvis,† Y. Mercier,‡ and A. Corzo*2,3 *Department of Poultry Science, and †Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University, Mississippi State 39762; and ‡Adisseo France S.A.S. Commentry, 03 600, Commentry, France ABSTRACT Effects of dietary methionine (Met) on pectoralis muscle development and the effect that Met as a nutritional substrate has on protein expression of skeletal muscle cells of pectoralis muscle of chickens were evaluated in this study. Broiler chickens received a common pretest diet up to 21 d of age and were subsequently fed either a low (LM) or high Met (HM) diet (0.41 vs. 0.51% of diet) from 21 to 42 d of age. Dietary deficiency was shown in vivo judging by the depression in breast meat weight and yield when broilers were fed the LM diet. Global protein expression was analyzed by quantitative high-performance liquid chromatography nanospray ionization tandem mass spectrometry. Up- and downregulated proteins were analyzed via Ingenuity Pathways Analysis to identify the metabolic pathways affected. Four canonical pathways related to muscle development were identified as being differentially regulated between LM- and HM-fed chickens. These pathways included the citrate cycle and calcium, actin cytoskeleton, and clathrin-mediated endocytosis signaling. The HM diet may have allowed for increased muscle growth by an increased availability of nutrients to muscle cells. Although the Met supplementation was associated with enhanced breast muscle growth, contraction fiber concentrations in muscles decreased and were associated with a lower calcium transportation rate and sensitivity and with a lower energy supply. It is further suggested that increased muscle protein deposition, that was induced by Met supplementation, may have been largely due to sarcoplasmic rather myofibrillar hypertrophy. Key words: broiler, methionine, pectoral skeletal muscle, protein expression 2012 Poultry Science 91:2548–2555 http://dx.doi.org/10.3382/ps.2012-02213 INTRODUCTION Methionine (Met) is typically the first limiting amino acid among common feed ingredients used in poultry diets. In addition to being a structural component of nucleic acids and proteins in the chicken, Met is also recognized as a precursor in the biosynthesis of various metabolites (sarcosine, betaine, and choline via transmethylation) and as an intermediary in the conversion to cystine or cysteine (via homocysteine). The pectoralis (breast) major and minor muscles of chickens have been subject to intensive genetic selection for maximal breast meat tissue deposition (Scheuermann et al., 2003). Although among all essential amino ©2012 Poultry Science Association Inc. Received February 9, 2012. Accepted June 19, 2012. 1 Approved for publication as Journal article no. J-12103 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. 2 Corresponding author: [email protected] 3 Present address: Elanco Animal Health, 2500 Innovation Way, PO Box 708, Greenfield, IN 46140. acids, the concentration of Met within these muscles is the lowest (Murphy, 1994); a dietary deficiency of Met has been shown to hinder breast muscle development (Hickling et al., 1990; Schutte and Pack, 1995). Consequently, levels of Met in the diets of broiler chickens has been extensively evaluated in recent years to further understand its role in the metabolism and development of rapidly growing chickens (NRC, 1994; Schutte and Pack, 1995; Kalinowski et al., 2003). Recently, Corzo et al. (2006) modeled the expression of pectoral skeletal muscle protein in chickens in response to dietary Met inadequacy. After Met-adequate or -deficient diets were fed to broiler chickens, the global protein expression of their breast muscle was analyzed using 2-dimensional liquid chromatography (LC) tandem mass spectrometry (MS). At that time, only limited methodologies and experimental techniques were available for the analysis of differential protein expression in tissues. Therefore, only a small number of proteins were identified that were affected by dietary Met. However, the more recent availability of LC MS-based proteomics methods and systems has greatly improved 2548 DIETARY METHIONINE INFLUENCES PROTEIN EXPRESSION chromatographic resolution and sensitivity. This has involved the conversion of micro-flow to nano-flow in HPLC, an upgrading of the ESI source to nanoESI, and other improvements in mass spectrophotometers. Many more mass spectra can be generated for analysis using these new systems (Sokale et al., 2011). Therefore, the current study, using updated technology, was designated to replicate the in vivo model provided by Corzo et al. (2006). Furthermore, differential protein expressions in pectoral skeletal muscle samples were compared which elucidate the anabolic mechanisms by which Met influences breast muscle deposition in chickens. MATERIALS AND METHODS Chickens and Diets In total, 216 one-day-old male broiler chicks (Ross × Ross 708) were obtained (Aviagen North America, Huntsville, AL) and placed in 18 floor pens (0.9 × 1.2 m/pen) after equalization of BW across pens (±2 g/ chick). Water and feed were supplied for ad libitum intake with a 23 h of light and 1 h of dark lighting program. Temperatures were set for the birds to achieve thermoneutrality throughout the study (Aviagen North America, 2005). All animal procedures were approved by the Mississippi State University Institutional Animal Care and Use Committee. To ensure accurate diet formulation, samples of corn and soybean meal were analyzed before formulation for total amino acids (AOAC International, 2006; 985.28, 994.12) and crude protein composition (AOAC International, 2006; 968.06). Upon analyses, the nutrient matrix of the feed ingredients used was updated and all the test diets were then formulated using linear programming that solved for energy, amino acids, and mineral and vitamin needs for optimal growth. A common pretest diet in crumbled form, formulated to satisfy all nutrient recommendations (NRC, 1994), was fed to all the broilers in the study from placement until 21 d of age (Table 1). Subsequently, the test diet was fed from 21 to 42 d of age (Table 1) and was formulated to satisfy all nutrient recommendations for the 21- to 42-d period, with the exception of Met (NRC, 1994; Kalinowski et al., 2003). Because poultry can efficiently utilize dietary Met in both d- and l-isoforms, dl-methionine (99%) was supplemented at the expense of an inert filler to achieve a low dietary Met level (LM) and fed to half of the pens, whereas the other half received the high Met (HM) test diet (total Met: 0.41 vs. 0.51% of diet). Composite samples of both dietary treatments were later analyzed for amino acid composition by HPLC (AOAC International, 2006; 985.28, 994.12). To prevent sulfur amino acid degradation during hydrolysis of the raw feed ingredients (before formulation of the Table 1. Experimental diet composition (%, as-is) Item Ingredient Corn Soybean meal Poultry oil Dicalcium phosphate Calcium carbonate Sodium chloride dl-Methionine Premix1 Coccidiostat2 l-Lysine-HCl Filler3 l-Threonine Calculated composition ME (kcal/kg) CP Calcium Available phosphorus Digestible sulfur amino acids Digestible methionine Total methionine4 Digestible lysine 2549 Common starter (0–21 d) Experimental grower (21–42 d) 52.891 39.558 3.989 1.841 0.619 0.487 0.265 0.250 0.050 0.050 — — 3,100 22.5 0.94 0.47 0.96 0.55 0.60 1.31 65.990 27.421 2.871 1.713 0.715 0.493 0.105 0.250 0.05 — 0.350 0.043 3,150 20.2 0.86 0.43 0.69 0.37 0.41 1.10 1The vitamin and mineral premix contained per kilogram of diet: retinyl acetate, 2,654 μg; cholecalciferol, 110 μg; dl-α-tocopherol acetate, 9.9 mg; menadione, 0.9 mg; B12, 0.01 mg; folic acid, 0.6 μg; choline, 379 mg; d-pantothenic acid, 8.8 mg; riboflavin, 5.0 mg; niacin, 33 mg; thiamin, 1.0 mg; d-biotin, 0.1 mg; pyridoxine, 0.9 mg; ethoxyquin, 28 mg; manganese, 55 mg; zinc, 50 mg; iron, 28 mg; copper, 4 mg; iodine, 0.5 mg; selenium, 0.3 mg. 2Dietary inclusion of coccidiostat provides 60 g of salinomycin sodium per 907.2 kg of feed. 3Inert filler included in the experimental grower diet and used as an inert filler to supplement dl-methionine. 4Analyzed total methionine dietary concentrations were in close agreement with calculated values. Calculated and analyzed (in parentheses) total methionine values were low methionine: 0.41% (0.42%); high methionine: 0.51% (0.50%). 2550 Zhai et al. diets) and the test diets, oxidation was initiated using performic acid to derive stable products of Met and cystine. The formed products, methionine sulphone and cysteic acid, were then determined by chromatography and calculated back to the Met and cystine content in the experimental diets in accordance with AOAC International (2006; 985.28, 994.12). Mean bird BW in all pens was recorded at the initiation (21 d) and termination (42 d) of the experimental phase. Processing Measurements At 42 d of age, 10 birds per pen were randomly selected, tagged, and individually weighed and placed in chicken coops 12 h before processing. Birds were processed at a pilot processing plant. Electrical stunning was performed by applying 11.5 V (<0.05 mA, AC to DC current) for 30 s to each broiler. Broiler carcasses were scalded, picked, and eviscerated automatically using commercial prototype equipment. Birds were then chilled for 4 h, at which point all birds were manually deboned and weights were obtained for their pectoralis muscles. Absolute and relative pectoralis muscles weights were then calculated. Protein Expression Analyses One bird from each pen (n = 9 per diet) was randomly selected at 41 d of age and euthanized via cervical dislocation. One cubic centimeter from each bird was aseptically dissected from the cranial region of the left pectoralis major postmortem and immediately frozen at −80°C. A 150-mg sample of pectoralis major from each bird was processed by differential detergent fractionation and trypsin digested as outlined by McCarthy et al. (2005). Following digestion, samples were desalted using a peptide macrotrap (TR1/25108/52; Michrom BioResources, Auburn, CA), followed by a strong cation exchange (Michrom TR1/25108/53) step to remove any detergents and other polymers that can interfere with MS/MS analysis. Each sample was loaded on a BioBasic C18 reversed phase column (Thermo 72105–100266) and flushed for 20 min with 5% acetonitrile (ACN) and 0.1% formic acid to remove salts. Peptide separation was achieved using a Thermo Surveyor MS pump with a 655-min nano-HPLC method consisting of a gradient from 5% to 50% ACN for 620 min, followed by a 20-min wash with 95% ACN and subsequent equilibration with 5% ACN for 15 min (all solvents contain 0.1% formic acid as a proton source). Ionization of peptides was achieved via nanospray ionization using a Thermo Finnigan nanospray source type I operated at 1.85 kV with 8 μm internal diameter silica tips (New Objective FS360– 75–8-N-20-C12). High voltage was applied using a tconnector with a gold electrode in contact with the HPLC solvent. A Thermo LCQ DECA XP Plus ion trap mass spectrometer was used to collect data over the 655 min of duration of each HPLC run. Precursor mass scans were performed using repetitive MS scans, each immediately followed by 3 MS/MS scans of the 3 most intense MS peaks. Dynamic exclusion was enabled for a duration of 2 min with 2 repeat counts. Once a mass is measured twice, it is added to a list to be excluded from further analysis for a predetermined amount of time, which was 2 min in the current trial. This allows the MS to collect data on different masses. In the meantime, the mass will have eluted from the column. Dynamic exclusion allows for a more efficient, deeper depth of sample coverage (Zhang et al., 2009b). Statistical Analyses Data collected and analyzed for pectoral breast meat muscle weight and yield were evaluated by one-way ANOVA in a completely randomized design. Pen was used as the experimental unit for analysis. Data were analyzed using the GLM procedure of SAS Institute (2004). Treatment effects (P ≤ 0.05) were separated using a t-test. Database searches of mass spectrometry data for protein expression were performed using the SEQUEST (Yates et al., 1995) algorithm in Bioworks 3.3. The database for peptide spectral matching was the Chicken Affymetrix nucleotide database. Decoy searches were performed using a randomized version of the target database. Search results were filtered using a decoy-based, statistical method in which a probability of being a false-positive match is assigned to each peptide (Pendarvis et al., 2009). Proteins containing peptides with a probability of 0.05 or less were evaluated for differential expression using Monte Carlo resampling statistics (Pendarvis et al., 2009). Proteins with a probability of differential expression of 0.05 or less were selected for further modeling. Expression fold changes were calculated as described by Old et al. (2005). Ingenuity Pathways Analysis Data were analyzed through the use of Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity. com). Canonical pathways analysis identified the pathways from the Ingenuity Pathways Analysis library of canonical pathways that were most significant to the data set. Molecules from the data set that were differentially regulated between LM- and HM-fed chickens and were associated with a canonical pathway in Ingenuity’s Knowledge Base were considered for the analysis. The significance of the association between the data set and the canonical pathway was measured by the following 2 ways: 1) a ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway displayed; 2) Fisher’s exact test and B-H multiple testing correction (Benjamini and Hochberg, multiple testing correction which is for controlling the false discovery rate) was used to calculate a P-value determining the probability that the association between the genes in the data set and the canonical pathway is 2551 DIETARY METHIONINE INFLUENCES PROTEIN EXPRESSION explained by chance alone. Pathways were considered statistically significantly associated with the data set if the P-value for the B-H was 0.05 or less. RESULTS Growth and Pectoralis Muscle Accretion All broiler chicks being raised on a common diet from placement until 21 d of age grew at an adequate growth rate (866 ± 27.2 g/bird) before being fed the experimental diets. Calculated and analyzed total Met levels of the experimental diets were in close agreement, therefore, validating the experimental dietary treatments fed to the broiler chickens (Table 1; footnote 4). Table 2 shows the effect that feeding the LM diet had on pectoral muscle development of broilers when compared with HM-fed birds. Feeding the LM diet resulted in a 10% decrease in pectoralis muscle weight and a 0.72 percentage point drop in breast meat yield (% of live BW), and both of these parameters were found to be statistically different from those of chickens fed HM diets. The drop in breast meat yield was found to be 1 percentage point when the pectoral muscles were expressed relative to the carcass weight. Protein Identification A total of 5,016 unique proteins was identified from biological replicates of LM chickens, and 4,296 unique proteins were identified from biological replicates of HM chickens. Of these proteins, 1,468 were differentially regulated (593 upregulated; 875 downregulated) between LM and HM chickens. Six canonical pathways were differentially regulated between LM- and HM-fed chickens. Those pathways were citrate cycle, calcium signaling, actin cytoskeleton signaling, clathrin-mediated endocytosis signaling, germ cell-sertoli cell junction signaling, and cellular effects of sildenafil (Figure 1). The differentially regulated proteins of the first 4 pathways (citrate cycle, calcium signaling, actin cytoskeleton signaling, clathrin-mediated endocytosis signaling) have been listed in Table 3. The germ cell-sertoli cell junction signaling and cellular Figure 1. Protein expression difference in pectoralis muscles of broilers fed a low (LM) or high (HM) dietary methionine diet from 21 to 42 d of age. Color version available in the online PDF. effects of sildenafil pathways were not presented due to their indistinct function on breast muscle development and growth. DISCUSSION Methionine is encoded by a single codon (AUG) in the standard genetic code. The codon AUG is also the “start” message for a ribosome that signals the initiation of protein translation from mRNA. As a consequence, Met is incorporated into the N-terminal position of all proteins in eukaryotes and archaea during translation (Berg et al., 2002). In the current study, dietary treatments imposed to these broiler chickens resulted in markedly different pectoral muscle development, thus allowing for a valid proteome comparison to obtain a better understanding of dietary Met-induced metabolic and physiological differences. Protein expression was affected by Met supplementation, as indicated by higher absolute and relative muscle weights, as well as changes in 6 canonical pathways. However, 2 pathways (Germ cell-sertoli cell junction signaling and cellular effects of sildenafil) are important only in the male reproductive system. Their functions in muscle development remain obscure and should require further Table 2. Body weight gain, feed conversion rate, pectoralis muscles weight and yield of broilers fed a low or high dietary methionine diet from 21 to 42 d of age1 Pectoralis muscles Treatment BW gain (g) Feed conversion2 0.41% Methionine 0.51% Methionine SEM P-value 1,972 2,013 52.5 0.17 1.92 1.85 0.051 0.32 a,bValues Weight (g) Yield3 Yield4 552b 606a 11.5 0.002 19.9b 20.6a 0.16 0.0004 29.7b 30.7a 0.22 0.0008 within a column with different superscripts differ significantly. means are calculated from 10 carcasses from each experimental unit. 2Values represent the feed conversion of birds for the experimental phase (21 to 42 d of age) after being corrected for mortality weight. 3Yield data expressed relative to the live weight. 4Yield data expressed relative to the carcass weight. 1Treatment 2552 Zhai et al. Table 3. Protein or peptide that has been differentially regulated in the 4 pathways Pathway Symbol Entrez gene name Citrate cycle ACLY ATP5G3 ATP citrate lyase ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C3 (subunit 9) Malate dehydrogenase 2, NAD (mitochondrial) Phosphoenolpyruvate carboxykinase 1 (soluble) Aconitase 2, mitochondrial Isocitrate dehydrogenase 3 (NAD+) α Succinate dehydrogenase complex, subunit B, iron sulfur (Ip) Succinate dehydrogenase complex, subunit D, integral membrane protein Succinate-CoA ligase, α subunit Succinate-CoA ligase, GDP-forming, β subunit Glutamate receptor, ionotropic, AMPA 1 Histone deacetylase 4 Myocyte enhancer factor 2A Troponin C type 1 (slow) Actin, α 1, skeletal muscle ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 ATPase, Ca++ transporting, ubiquitous cAMP responsive element binding protein 1 Histone deacetylase 9 Myosin, light chain 1, alkali; skeletal, fast Myosin, light chain 3, alkali; ventricular, skeletal, slow Protein phosphatase 3, regulatory subunit B, α Troponin C type 2 (fast) Troponin I type 2 (skeletal, fast) Tropomyosin 3 ARP2 actin-related protein 2 homolog (yeast) v-crk sarcoma virus CT10 oncogene homolog (avian)-like Fibronectin 1 Phosphoinositide-3-kinase, catalytic, α polypeptide Radixin Vinculin Actin, α 1, skeletal muscle Actin, β Actin, gamma 1 Cell division cycle 42 (GTP binding protein, 25 kDa) Myosin, light chain 1, alkali; skeletal, fast Myosin, light chain 3, alkali; ventricular, skeletal, slow Neuroblastoma RAS viral (v-ras) oncogene homolog Protein phosphatase 1, regulatory (inhibitor) subunit 12A ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) Slingshot homolog 1 (Drosophila) WAS protein family, member 2 ARP2 actin-related protein 2 homolog (yeast) Clathrin, heavy chain (Hc) Huntingtin interacting protein 1 related Numb homolog (Drosophila) Phosphoinositide-3-kinase, catalytic, α polypeptide Actin, α 1, skeletal muscle Actin, β Actin, gamma 1 Amphiphysin Cell division cycle 42 (GTP binding protein, 25 kDa) Heat shock 70 kDa protein 8 Protein phosphatase 3, regulatory subunit B, α RAB5C, member RAS oncogene family RAB7A, member RAS oncogene family ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) Ubiquitin specific peptidase 9, X-linked Calcium signaling Actin cytoskeleton signaling Clathrin-mediated endocytosis MDH2 PCK1 ACO2 IDH3A SDHB SDHD SUCLG1 SUCLG2 GRIA1 HDAC4 MEF2A (includes EG:4205) TNNC1 ACTA1 ATP2A2 ATP2A3 CREB1 HDAC9 (includes EG:9734) MYL1 MYL3 PPP3R1 TNNC2 TNNI2 TPM3 ACTR2 CRKL FN1 PIK3CA RDX VCL ACTA1 ACTB ACTG1 CDC42 MYL1 MYL3 NRAS PPP1R12A RAC1 SSH1 WASF2 ACTR2 CLTC HIP1R NUMB PIK3CA ACTA1 ACTB ACTG1 AMPH CDC42 HSPA8 PPP3R1 RAB5C RAB7A RAC1 USP9X 1Positive Fold changes1 5.50 7.97 1.13 4.72 −5.41 −0.57 −3.67 −4.51 −4.54 −6.41 4.57 5.37 5.31 8.19 −0.47 −0.32 −4.62 −3.60 −1.80 −3.56 −0.98 −7.03 −0.49 −1.94 −0.34 6.07 2.66 5.52 5.37 5.69 0.67 −0.47 −4.39 −0.25 −1.03 −3.56 −0.98 −3.68 −4.44 −2.61 −0.78 −2.40 6.07 5.74 1.36 3.41 5.37 −0.47 −4.39 −0.25 −8.35 −1.03 −0.52 −7.03 −5.56 −3.99 −2.61 −4.23 numbers mean upregulated; negative numbers mean downregulated. investigation. Therefore, only 4 of the 6 canonical pathways that were differentially regulated between LM and HM chickens are addressed in this manuscript. Citrate Cycle The citrate cycle is a series of enzyme-catalyzed chemical reactions that converts carbohydrates, fats, and proteins into carbon dioxide and water to generate usable energy within mitochondria (Berg et al., 2002). In the current study, supplementary dietary Met decreased the overall expression of several proteins in the citrate cycle. Adenosine triphosphate (ATP) citrate lyase expression was upregulated (Figure 2; Table 3). Adenosine triphosphate citrate lyase catalyzes the for- DIETARY METHIONINE INFLUENCES PROTEIN EXPRESSION Figure 2. Simplified citrate cycle that shows the enzyme expression difference in pectoralis muscles of broilers fed a low (LM) or high (HM) dietary methionine diet from 21 to 42 d of age. Symbols: ↑ means upregulated and ↓ means downregulated, ◊ are enzymes and □ are substrates. Color version available in the online PDF. mation of acetyl-CoA and oxaloacetate from citrate and CoA with a concomitant hydrolysis of ATP, which is in opposition to ATP synthesis that is occurring in the TCA cycle. The expression of various enzymes that catalyze various chemical reactions of the citrate cycle was downregulated in HM groups (Figure 2). For example, aconitase (ACO2), isocitrate dehydrogenase (IDH3A), succinate-CoA ligase (SUCLG1 and SUCLG2), and succinate dehydrogenase complex (SDHB and SDHD) were all downregulated (Figure 2; Table 3). Isocitrate dehydrogenase (IDH3A) catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate and is a ratelimiting step of the citrate cycle. These enzyme level changes could lead to a reduction in mitochondrial ATP production and an accumulation of oxaloacetate. The increase in oxaloacetate may also have been used for gluconeogenesis, as indicated by the increase of phosphoenolpyruvate carboxykinase (PCK1), a main control point for the regulation of gluconeogenesis. Phosphoenolpyruvate carboxykinase (PCK1) catalyzes the formation of phosphoenolpyruvate from oxaloacetate, and various amino acids in the body are metabolized through the citrate cycle. Thus, a balanced amino acid composition in the HM diet may have lowered the rate of amino acid metabolism in the citrate cycle, allowing more amino acids to be preserved for muscle growth rather than being entered into the citrate cycle for energy production. Calcium Signaling As a second messenger, Ca2+ activates a protein kinase which translocates to the cell nucleus, where it activates a cAMP responsive element binding protein 2553 (CREB). The CREB in turn acts as a DNA-binding protein that plays an important role in cellular growth and development (Lehrmann et al., 2002). In the current study, CREB expression was found to be downregulated in HM-fed chickens. In association with the decrease in CREB, several proteins of the fast muscle fiber contraction complex (dominate in broiler breast muscle) including actin-α, myosin, tropomyosin, and troponins were downregulated. These findings concur with a previous report by Corzo et al. (2006), in which several myosin-related proteins were found downregulated in Met-supplemented groups. In addition, 2 isoforms (ATP2A2 and ATP2A3) of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-2, were observed to be downregulated. The current results were similar to a previous report in which SERCA was also downregulated in HM-fed chickens (Corzo et al., 2006). Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) catalyzes the hydrolysis of ATP coupled with the translocation of calcium from the cytosol into the sarcoplasmic reticulum lumen and is involved in the regulation of muscular contraction and relaxation. Downregulation of the contraction complex and SERCA suggest that HM birds may exhibit a lower muscle contraction capability. In addition, calcineurin (regulation of Ca2+ sensitivity; Groenendyk et al., 2004) was downregulated in HM chickens. The downregulation of calcineurin further suggests that muscle contraction was lowered in HM chickens. Actin Cytoskeleton Signaling The main functions of the actin cytoskeleton are to mediate cell motility and cell shape, for cytoplasmic organization, and to generate mechanical forces within the cell (Schmidt and Hall, 1998). Similar to the changes in the calcium signaling pathway, contraction fibers (myosin and F-actin) were downregulated in HM chickens. The expression of 2 myosin isoforms (MYL1 and MYH6) was downregulated. Myosins are a large family of motor proteins that share common features, including ATP hydrolysis, actin binding, and kinetic energy transduction. They are involved in muscle contraction through cyclic interactions with actin-rich thin filaments, creating a contractile force. Methionine supplementation downregulated the expression level of various isoforms of F-actin: ACTA1, which is a major constituent of the contractile apparatus; ACTB and ACTG1, which are involved in the regulation of cell motility. Results of the current study were similar to a study conducted by Yalçin et al. (2010), in which a high protein diet decreased myofiber density and increased sarcoplasmic protein solubility in broiler muscle. The interaction of myosin and actin is regulated by phosphorylation and intracellular Ca2+ concentrations. Calcium also activates pyruvate dehydrogenase, IDH3A, and α-ketoglutarate dehydrogenase to increase reaction rate (Denton et al., 1975). Supplementation of Met decreased IDH3A in this study. 2554 Zhai et al. The expression of 2 other proteins (slingshot homolog and α actinins) involved in the formation of actin was also downregulated. Slingshot homolog plays a role in actin dynamics to stabilize actin filaments (Niwa et al., 2002). In skeletal muscle, α actinins are localized to the Z-disc and analogous dense bodies, where they help anchor the myofibrillar actin filaments (Luther, 2009). Associated with the decrease of contraction fibers, the expression of Rho-like GTPases, such as RAC1 and CDC42 was downregulated. Rho-like GTPases are key regulators in signaling pathways that link extracellular growth signals or intracellular stimuli to the assembly and organization of the actin cytoskeleton (Schmidt and Hall, 1998). Although the contraction components (proteins that lead to actin polymerization and actomyosin assembly contraction) were downregulated, the 2 proteins for anchoring and linking (Radixin and Vinculin) involved in focal complex assembly and focal adhesion assembly, were found to be upregulated in HM-fed chickens. Focal adhesions and adherens junctions are membrane-associated complexes that serve as nucleation sites for actin filaments and as cross-linkers between the cell exterior, the plasma membrane, and the actin cytoskeleton (Yamada and Geiger, 1997; Schmidt and Hall, 1998). Additionally, fibronectin (FN1) involved in cell adhesion and migration processes (Pankov and Yamada, 2002), was also upregulated in HM chickens. Clathrin-Mediated Endocytosis Signaling Clathrin-mediated endocytosis is the major route for endocytosis in most cells. It is mediated by the molecule clathrin (CLTC), which plays a major role in the formation of coated vesicles. Clathrin-mediated endocytosis is required for a large number of essential cell functions, including nutrient uptake, cell-cell communication, and modulation of membrane composition (Ramanan et al., 2011). Clathrin expression was upregulated in HM-fed chickens. It is hypothesized that increased clathrin expression could increase the number of clathrin-coated pits, quicken endocytosis, and enhance recycling and absorption of nutrients from outside of the cell. Because more nutrients were taken into the cell, the overall endocytosis may have been increased. Furthermore, the expression of 2 other related proteins (numb and HIP1R, huntingtin interacting protein 1 related) was found to be upregulated in HM-fed chickens. Numb (an endocytic adaptor) has been shown to be associated with Eps15, which is involved in cell growth regulation (Gulino et al., 2010) and plays an important role in the assembly of clathrin-coated pits (Naslavsky and Caplan, 2005). Huntingtin interacting protein (HIP1R) is a component of clathrin-coated pits and vesicles that may link the endocytic machinery to the actin cytoskeleton (Boettner et al., 2011). However, not all endocytosis procedures were upregulated. The expression of 2 proteins (CDC42 and RAC1) that are responsible for clathrin-mediated endocytosis of virus- es and actin polymerization (Li et al., 1998; Van den Broeke et al., 2010) was downregulated. This may suggest that substrates or nutrients were selectively taken up by the cell. Even the overall rate of endocytosis would presumably be upregulated in HM chickens, as indicated by higher clathrin and HIP1R expression levels. However, the disassembly of the clathrin-coated vesicles would not be increased because of the downregulation of the following 3 proteins: heat shock 70-kDa protein (responsible for disassembly of the vesicle; Eisenberg and Greene, 2007), RAB5C, and RAB7A (RAS oncogene family responsible for docking or fusion of vesicle and vesicular transport; Han et al., 1996; Zhang et al., 2009a). Conclusion Despite the report of an increase in the absolute and relative weights of pectoralis muscle in HM-fed chickens, F-actin and myosin (2 main contractile proteins) were found to be downregulated. There are 2 types of muscle hypertrophy: myofibrillar hypertrophy and sarcoplasmic hypertrophy. Myofibrillar hypertrophy is characterized by an increase in contractile proteins, such as actin and myosin, which subsequently increases the strength of muscle contraction and is associated with physical activity such as exercise. Sarcoplasmic hypertrophy is characterized by the growth of sarcoplasm (a semifluid interfibrillar substance lacking contractile proteins) without contribution to the production of muscle force. The results of this study would suggest that sarcoplasmic hypertrophy predominated over myofibrillar hypertrophy. A predominance of sarcoplasmic hypertrophy would lead to a decrease in muscle fiber density, whereas muscle fiber cross-sectional area would increase, without an accompanying increase in muscle strength (Zatsiorsky and Kraemer, 2006). In the current study, the dietary supplementation of Met to levels considered to be adequate led to an increase in breast muscle development. However, the contraction fiber concentration in the muscle was found to be downregulated, as suggested by the lower calcium transportation rate, sensitivity, and energy supply. More nutrients may have been taken up into the muscle cells and were preserved for growth instead of being converted to energy for contraction. The increase of muscle protein deposition that was induced by Met supplementation might have been caused mainly by sarcoplasm hypertrophy rather than by the myofibrillar hypertrophy. Maltin et al. (1997), Shackelford et al. (1997), and Rehfeldt et al. (2000) reported that an increase in muscle mass, largely as a result of myofibrillar hypertrophy, may decrease the tenderness of pig and lamb muscle and thereby affect its meat quality. Sarcoplasm hypertrophy via Met supplementation in the current study suggests that further investigations into the effects of dietary Met supplementation on meat tenderness and meat quality are needed. DIETARY METHIONINE INFLUENCES PROTEIN EXPRESSION REFERENCES AOAC International. 2006. Official Methods of Analysis (985.28, 968.06, and 994.12). 18th ed. AOAC Int., Arlington, VA. Aviagen North America. 2005. Broiler Management Guide. Huntsville, AL. Berg, J. M., J. L. Tymoczko, and L. Stryer. 2002. Biochemistry. 5th ed. W. H. Freeman and Company, New York, NY. Boettner, D. R., H. Friesen, B. Andrews, and S. K. Lemmon. 2011. Clathrin light chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin. Mol. Biol. Cell 22:3699–3714. Corzo, A., M. T. Kidd, W. A. Dozier III, A. Shack, and S. C. Burgess. 2006. Protein expression of pectoralis major muscle in chickens in response to dietary methionine status. Br. J. Nutr. 95:703–708. Denton, R. M., P. J. Randle, B. J. Bridges, R. H. Cooper, A. L. Kerbey, H. T. Pask, D. L. Severson, D. Stansbie, and S. Whitehouse. 1975. Regulation of mammalian pyruvate dehydrogenase. Mol. Cell. Biochem. 9:27–53. Eisenberg, E., and L. E. Greene. 2007. Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis. Traffic 8:640–646. Groenendyk, J., J. Lynch, and M. Michalak. 2004. Calreticulin, Ca2+, and calcineurin—Signaling from the endoplasmic reticulum. Mol. Cells 17:383–389. Gulino, A., L. Di Marcotullio, and I. Screpanti. 2010. The multiple functions of Numb. Exp. Cell Res. 316:900–906. Han, H. J., K. Sudo, J. Inazawa, and Y. Nakamura. 1996. Isolation and mapping of a human gene (RABL) encoding a small GTP-binding protein homologous to the Ras-related RAB gene. Cytogenet. Cell Genet. 73:137–139. Hickling, D., W. Guenter, and M. E. Jackson. 1990. The effects of dietary methionine and lysine on broiler chicken performance and breast meat yield. Can. J. Anim. Sci. 70:673–678. Kalinowski, A., E. T. Jr. Moran, and C. L. Wyatt. 2003. Methionine and cystine requirements of slow- and fast-feathering broiler males from three to six weeks of age. Poult. Sci. 82:1428–1437. Lehrmann, H., L. L. Pritchard, and A. Harel-Bellan. 2002. Histone acetyltransferases and deacetylases in the control of cell proliferation and differentiation. Adv. Cancer Res. 86:41–65. Li, E., D. Stupack, G. M. Bokoch, and G. R. Nemerow. 1998. Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases. J. Virol. 72:8806–8812. Luther, P. K. 2009. The vertebrate muscle Z-disc: Sarcomere anchor for structure and signalling. J. Muscle Res. Cell Motil. 30:171–185. Maltin, C. A., C. C. Warkup, K. R. Matthews, C. M. Grant, A. D. Porter, and M. I. Delday. 1997. Pig muscle fibre characteristics as a source of variation in eating quality. Meat Sci. 47:237–248. McCarthy, F. M., S. C. Burgess, B. H. van den Berg, M. D. Koter, and G. T. Pharr. 2005. Differential detergent fractionation for non-electrophoretic eukaryote cell proteomics. J. Proteome Res. 4:316–324. Murphy, M. E. 1994. Amino acid compositions of avian eggs and tissues: Nutritional implications. J. Avian Biol. 25:27–38. Naslavsky, N., and S. Caplan. 2005. C-terminal EH-domain-containing proteins: Consensus for a role in endocytic trafficking, EH? J. Cell Sci. 118:4093–4101. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. 2555 Niwa, R., K. Nagata-Ohashi, M. Takeichi, K. Mizuno, and T. Uemura. 2002. Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108:233–246. Old, W. M., K. Meyer-Arendt, L. Aveline-Wolf, K. G. Pierce, A. Mendoza, J. R. Sevinsky, K. A. Resing, and N. G. Ahn. 2005. Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol. Cell. Proteomics 4:1487–1502. Pankov, R., and K. M. Yamada. 2002. Fibronectin at a glance. J. Cell Sci. 115:3861–3863. Pendarvis, K., R. Kumar, S. C. Burgess, and B. Nanduri. 2009. An automated proteomic data analysis workflow for mass spectrometry. BMC Bioinformatics 10(Suppl. 11):S17. Ramanan, V., N. J. Agrawal, J. Liu, S. Engles, R. Toy, and R. Radhakrishnan. 2011. Systems biology and physical biology of clathrin-mediated endocytosis. Integr. Biol. (Camb) 3:803–815. Rehfeldt, C., I. Fiedler, G. Dietl, and K. Ender. 2000. Myogenesis and postnatal skeletal muscle cell growth as influenced by selection. Livest. Prod. Sci. 66:177–178. SAS Institute. 2004. SAS User’s Guide. Version 9.1 ed. SAS Institute Inc., Cary, NC. Scheuermann, G. N., S. F. Bilgili, J. B. Hess, and D. R. Mulvaney. 2003. Breast muscle development in commercial broiler chickens. Poult. Sci. 82:1648–1658. Schmidt, A., and M. N. Hall. 1998. Signaling to the actin cytoskeleton. Annu. Rev. Cell Dev. Biol. 14:305–338. Schutte, J. B., and M. Pack. 1995. Effects of dietary sulphur containing amino acids on performance and breast meat deposition of broiler chicks during the growing and finishing phases. Br. Poult. Sci. 36:747–762. Shackelford, S. D., T. L. Wheeler, and M. Koohmaraie. 1997. Effect of callipyge phenotype and cooking method on tenderness of several major lamb muscles. J. Anim. Sci. 75:2100–2105. Sokale, A., E. D. Peebles, W. Zhai, K. Pendarvis, S. Burgess, and T. Pechan. 2011. Proteome profile of the pipping muscle in broiler embryos. Proteomics 11:4262–4265. Van den Broeke, C., M. Radu, J. Chernoff, and H. W. Favoreel. 2010. An emerging role for p21-activated kinases (Paks) in viral infections. Trends Cell Biol. 20:160–169. Yalçin, S., H. Ozkul, S. Ozkan, R. Gous, I. Yasa, and E. Babacanoglu. 2010. Effect of dietary protein regime on meat quality traits and carcase nutrient content of broilers from two commercial genotypes. Br. Poult. Sci. 51:621–628. Yamada, K. M., and B. Geiger. 1997. Molecular interactions in cell adhesion complexes. Curr. Opin. Cell Biol. 9:76–85. Yates, J. R. 3rd, J. K. Eng, A. L. McCormack, and D. Schieltz. 1995. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67:1426–1436. Zatsiorsky, V. M., and W. J. Kraemer. 2006. Pages 47–65 in Science and Practice of Strength Training. 2nd ed. Human Kinetics, Champaign, IL. Zhang, M., L. Chen, S. Wang, and T. Wang. 2009a. Rab7: Roles in membrane trafficking and disease. Biosci. Rep. 29:193–209. Zhang, Y., Z. Wen, M. P. Washburn, and L. Florens. 2009b. Effect of dynamic exclusion duration on spectral count based quantitative proteomics. Anal. Chem. 81:6317–6326.