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J O U RN A L OF P R O TE O MI CS 7 3 (2 0 1 0 ) 7 7 8–7 8 9

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Proteomic signature of muscle atrophy in rainbow trout Mohamed Salem a , P. Brett Kenney a , Caird E. Rexroad III b , Jianbo Yao a,⁎ a

Laboratory of Animal Biotechnology and Genomics, Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506-6108, United States b National Center for Cool and Cold Water Aquaculture, Kearneysville, WV 25430, United States

AR TIC LE I N FO

ABS TR ACT

Article history:

Muscle deterioration arises as a physiological response to elevated energetic demands of

Received 3 September 2009

fish during sexual maturation and spawning. Previously, we used this model to characterize

Accepted 31 October 2009

the transcriptomic mechanisms associated with fish muscle degradation and identified potential biological markers of muscle growth and quality. However, transcriptional measurements do not necessarily reflect changes in active mature proteins. Here we report the characterization of proteomic profile in degenerating muscle of rainbow trout in relation to the female reproductive cycle using a LC/MS-based label-free protein quantification method. A total of 146 significantly changed proteins in atrophying muscles (FDR < 5%) was identified. Proteins were clustered according to their gene ontology identifiers. Muscle atrophy was associated with decreased abundance in proteins of anaerobic respiration, protein biosynthesis, monooxygenases, follistatins, and myogenin, as well as growth hormone, interleukin-1 and estrogen receptors. In contrast, proteins of MAPK/ERK kinase, glutamine synthetase, transcription factors, Stat3, JunB, Id2, and NFkappaB inhibitor, were greater in atrophying muscle. These changes are discussed in light of the mammalian muscle atrophy paradigm and proposed fish-specific mechanisms of muscle degradation. These data will help identify genes associated with muscle degeneration and superior flesh quality in rainbow trout, facilitating identification of genetic markers for muscle growth and quality. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

Growth, development and degradation of skeletal muscle are governed by dynamic processes involving orchestrated expression of genes encoding contractile and regulatory proteins [1]. Molecular mechanisms that regulate mammalian muscle degradation have received substantial interest in the literature [2–5]. Under a variety of physiological and pathological conditions, distinct cellular stimuli activate unique cellular responses causing muscle wasting as seen in response to starvation [6], diseases including cancer, renal failure, diabe-

tes [7,8], and sepsis [8], as well as muscle disuse and denervation [9,10]. Fish use mechanisms of muscle growth and degradation that are distinct from mammalian mechanisms [11,12]. Fish species, has two anatomically well-separated muscle fiber types, red (aerobic) and white (anaerobic). This separation facilitates study of physiology and biochemistry of muscle growth and degeneration in each muscle fiber types. This situation provides an ideal experimental model in contrast to mammals, where study is much more difficult because fiber types are mixed in a given muscle [13] and significant

⁎ Corresponding author. Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506-6108, United States. Tel.: +1 304 293 2631x4414; fax: +1 304 293 2232. E-mail address: [email protected] (J. Yao). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.10.014

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differences in gene expression were reported between white and red muscle [14,15]. In addition, fish are ectothermic animals that rapidly use proteins as oxidative substrates [16] and have a lower metabolic rate than endothermic animals. Fish can acclimate to a considerable range of environmental stressors, in particular, during the sexual maturation and reproductive cycle, females rainbow trout orchestrate metabolism to support the dominant process of oocyte development. Previously, we reported that fertile fish at spawning had less muscle mass and less muscle protein compared to sterile fish and post-spawning fertile fish [11]. Consequently, this species in this scenario represents an ideal model for elucidating the functional genomics of muscle growth, degeneration/regeneration and fillet quality [12,17]. Molecular mechanisms that regulate fish muscle degeneration/regeneration have not received enough interest in the literature. Some studies have examined individual genes/ mechanisms that control muscle atrophy [18–22]. Only few studies have dealt with fish muscle atrophy at the genomewide level [12,17,23,24]. We used cDNA microarray to characterize transcriptomic responses to atrophy of fast-twitch muscles from gravid females compared to sterile females as the control. This study identified an expression pattern that closely resembles the mammalian muscle atrophy paradigm [12]. Subsequently, we used high-density oligonucleotide array to corroborate our findings [17]. Although much attention has been placed on changes in transcriptional regulation, gene expression is regulated at several levels, all of which must be studied together to obtain a complete picture of a physiological or cellular process. Transcriptomic measurements do not necessarily reflect the amount of change in the active mature protein. In fact, the final concentration of the active gene product, e.g. protein or enzyme, is the most relevant quantity to the phenotype. Studies comparing mRNA and protein abundance on a genome-wide scale indicate that mRNAs only partly correlate with the corresponding protein concentrations. It has been estimated that only 20–40% protein concentrations are determined by the corresponding mRNA concentrations [25,26]. Mass spectrometry (MS)-based proteomics has become an important tool for investigating posttranslational modification of proteins and protein interactions [27,28]. It allows hundreds to thousands of proteins to be simultaneously monitored, thus, allowing global profiling of proteins in muscle under an atrophying metabolic state. The objective of this study was to characterize the global proteomic profile in muscle of rainbow trout relative to sexual maturation using a high throughput LC/ MS-based label-free protein quantification method. In parallel, transcriptional changes were characterized using a microarray chip approach and published separately [12,17]. This proteomic profile study will differentiate between transcriptional and post-transcriptional regulatory mechanisms that control fish muscle atrophy. Important genes affecting meat quality traits have been identified and tested for potential improvement of muscle quality in breeding programs [29]. Hence, this study will help in identifying genetic markers of improved muscle growth and quality in rainbow trout. These candidate genes can subsequently be assessed for use in marker-assisted selection of rainbow trout with superior muscle growth and fillet quality traits.

2.

Experimental procedures

2.1.

Fish and muscle sampling

779

Mature fertile (diploid) and sterile (triploid) female rainbow trout, Oncorhynchus mykiss (500 g), were collected from Flowing Springs Trout Farm (Delray, WV) during the spawning season in early October. Fish were cultured in identical raceways receiving water from a common spring at 13± 3 °C. Fish were fed ad libitum (Zeiglar Gold; Zeigler Bros., Gardeners, PA) via demand feeders. No difference in feed consumption was noticed between groups, and fish had access to feed when sampled. As confirmed by dissection, fertile fish were gravid with a gonado-somatic index (GSI = ovary weight/fish weight× 100) of 15.8 ± 0.3 (n = 5), and the GSI of sterile fish was 0.3 ± 0.2 (n = 5). White muscle samples (20 g) from five fish of each group were collected from the dorsal musculature and flash frozen in liquid nitrogen and stored at -80 °C for proteomic analysis. Following muscle sample removal, fish were eviscerated, and ribs and vertebral column were removed to yield a butterfly fillet. This fillet was trimmed and skin was removed to generate boneless, skinless portions. This boneless, skinless muscle and initial muscle samples were combined and expressed as a percentage of the whole fish weight. Collectively, this sum was used as the indication of separable muscle. A portion of the muscle was used for proximate composition [30].

2.2.

MS and data analysis

MS and data analysis were done at Monarch Lifesciences (Indianapolis, Indiana) as previously described [27,31]. Tissue samples were thawed and homogenized in a hypotonic lysis buffer (100 mL of freshly made 8 M urea, 10 mM DTT solution). Tissue lysates were reduced and alkylated by triethylphosphine and iodoethanol, and subsequently digested using trypsin. All steps were carried out in one tube without washing or filtering steps. Peptide concentration was determined by the Bradford Protein Assay [32]. Lysis buffer was used as a blank reference for the protein assay and as the buffer for protein standards (BSA). Peptides were prepared and subjected to LC/MS analysis as previously described [27]. Tryptic peptides (∼ 20 μg) were analyzed using Thermo linear ion-trap mass spectrometer (LTQ) coupled with a Surveyor HPLC system (Thermo, Waltham, MA). A C-18, reverse phase column (i.d. = 2.1 mm, length = 50 mm) was used to separate peptides at a flow rate of 200 μL/ min. Peptides were eluted with 5 to 45% acetonitrile gradient developed over 120 min, and data were collected in the tripleplay mode (MS scan, zoom scan, and MS/MS scan). The acquired data were filtered and analyzed by a proprietary algorithm that was developed by Higgs and coworkers [28,33]. Database searches against the NCBI trout database were carried out using both the X!Tandem and SEQUEST algorithms. Protein quantification was carried out using a proprietary protein quantification algorithm licensed from Eli Lilly and Company [28]. Briefly, once the raw files were acquired from the LTQ, all extracted ion chromatograms (XIC) were aligned by retention time. To be used in the protein quantification procedure, each aligned peak must match parent ion, charge state, daughter ions

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(MS/MS data) and retention time (within a one-minute window). After alignment, area-under-the-curve (AUC) for each individually aligned peak from each sample was measured, normalized, and these areas were compared for relative abundance. All peak intensities were transformed to a log2 scale before quantile normalization [34]. Quantile normalization is a method of normalization that essentially ensures that every sample has a peptide intensity histogram of the same scale, location and shape. This normalization removes trends introduced by sample handling, sample preparation, total protein differences and changes in instrument sensitivity while running multiple samples. If multiple peptides have the same protein identification, then their quantile normalized log2 intensities were averaged to obtain log2 protein intensities. The log2 protein intensity is the final quantity that is fit by a separate ANOVA (Analysis of Variance) statistical model for each protein: log2 ðIntensityÞ = overall mean + group effect ðfixedÞ + sample effect ðrandomÞ + replicate effect ðrandomÞ: For the previous equation, group effect refers to the effect caused by the experimental conditions or treatments being evaluated. Sample effect represents the random effects from individual biological samples. It also includes random effects from sample preparation. The replicate effect refers to the random effects from replicate injections of the same sample. A total of 10 injections (5 from atrophying muscle [fertile fish] and 5 from non-atrophying muscles [sterile fish] were analyzed in this study. All of the injections were randomized and the instrument was operated by the same operator. The inverse log2 of each sample mean was calculated to determine the fold change between samples.

3.

Results and discussion

3.1.

Muscle atrophy in response to vitellogenesis

Our previous studies showed that constraints of sexual maturation and spawning in rainbow trout cause significant loss in muscle mass. At spawning season, rainbow trout fish

had less muscle mass compared to samples collected 4 months later. On the other hand, sterile fish reared under identical conditions showed no change in muscle mass [11]. Current studies in our lab using six samples collected over 8 months pre/post spawning confirmed our previous results (to be published elsewhere). This response is a suitable model to investigate mechanisms of muscle degradation/regeneration in fish and to identify genetic markers for muscle growth and quality for aquaculture applications [11,12,17]. In the current study, the global proteomic profile in degenerating muscle of rainbow trout was analyzed using the response associated with vitellogenesis-induced muscle atrophy. Profiles of atrophying fast-twitch muscles collected from gravid rainbow trout at spawning season were compared with sterile fish muscle as the control. Atrophying muscle of fertile fish had 11% less muscle mass and protein content compared to non-atrophying muscle of sterile fish, indicating extensive muscle atrophy (Fig. 1, A and B; P < 0.05) [11,12]. Using a label-free LC/MS-based protein quantification method, we identified 146 differentially expressed unique proteins in atrophying muscle (cut-off value: ±1.2 fold change). Differentially expressed proteins had 1.5 average fold change and 3.2 maximum fold change values. Changes noticed at the transcriptomic levels had average fold change value about 2.6 [12]. Proteins with gene ontology (GO) identifiers were grouped according to their functions as given below. Moderate changes of several proteins that are well-regulated in specific pathways/mechanisms are connected with the spawning-associated muscle degradation in rainbow trout.

3.2.

Glycolysis/ATP production

Atrophying muscle showed decreased abundance of three successive enzymes in the intermediate steps of the glycolytic pathway, fructose-bisphosphate aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. This reduction suggests reduced glucose utilization in atrophying muscle cells (Table 1). On the other hand the hexokinase enzyme, catalyzing the first step in glycolysis; phosphorylation of glucose to glucose-6-phosphate, showed up-regulated expression. This enzyme acts to trap glucose inside the cell and maintain high G6P concentration within

Fig. 1 – Effect of vitellogenesis-associated muscle atrophy on percentages of extractable muscle (A) and total protein (B). Different letters (a, b) indicate a significant difference (P < 0.05, means ± SE, n = 5).

ref|NP_001135182 ref|NP_001133174 ref|NP_001117033 ref|NP_001117721 dbj|BAE45286 gb|AAZ17382 ref|YP_961364 ref|NP_001118043 ref|NP_001118187 ref|NP_001135114 ref|NP_001133546 ref|NP_001117775 Glycolysis Catalytic activity Glycolysis Glucokinase activity H-exporting ATPase activity Nucleotide binding Mitochondrion Mitochondrial transport Nucleotide binding ATP binding Nucleotide binding ATP binding P F P F F F C F F F F F GO:0006096 GO:0003824 GO:0006096 GO:0004340 GO:0008553 GO:0000166 GO:0005739 GO:0006839 GO:0000166 GO:0005524 GO:0000166 GO:0005524 3 16 60 2 6 1 1 2 18 1 2 4 ALD-B TPI GAPDH GK ATPsyn-B ATP4a MT-ND4L UCP2B CK1 GRP78 ABCB3B(A) NDPK Fructose-bisphosphate aldolase C Triosephosphate isomerase 1b Glyceraldehyde-3-phosphate dehydrogenase Glucokinase ATP synthase beta-subunit Gastric H+/K+-ATPase alpha subunit NADH dehydrogenase subunit 4L Uncoupling protein 2B Creatine kinase 78 kDa glucose-regulated protein ATP-binding cassette, sub-family B (MDR/TAP) 3 Nucleoside diphosphate kinase

− 1.7 − 1.3 − 1.8 1.3 1.3 − 1.7 −1.9 − 1.4 − 1.2 − 1.6 − 1.4 − 2.7

0.006 0.026 0.007 0.002 0.016 0.004 0.001 0.002 0.027 0.021 0.014 0.011

Gene ontology/function GO aspect GO:ID qValue No. unique peptides Fold Change Symbol Protein name

Table 1 – Differential expression of glycolysis/ATP production proteins. GO aspects are C, cellular component; P, biological process and F, molecular function.

NCBI acc#

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781

the cell. This function maintains continuous transport of glucose through the plasma membrane and prevents glucose from leaking out since cells lacks transporters for G6P [35]. Together these results point to a glycolytic deficit perhaps due to reduced glucose availability as energetic demands elevate during vitellogenesis and thus egg maturation. These results directly support our microarray results that demonstrated a well orchestrated and substantial decrease in the expression of the glycolytic pathway enzymes [12]. Similar changes in glycolytic enzymes were also reported in catabolic states of mammalian and fish muscle degradation, thus representing a common feature of muscle atrophy in mammals and fish [7,9,24,36]. Several genes belonging to the oxidative phosphorylation and ATP buffering processes were differentially expressed in atrophying muscle cells (Table 1). Expression of the ATP synthase beta-subunit was up-regulated while a homologue of the gastric H+/K+-ATPase alpha subunit was down-regulated. NADH dehydrogenase subunit 4 L, an enzyme belonging to the electron transport pathway, and the uncoupling protein 2B, function to deplete the body of ATP to generate heat, was down-regulated. Creatine kinase, which plays a central role in ATP buffering to maintain constant levels during large and fluctuating energy demands of muscle, was down-regulated in atrophying muscle. Our previous microarray studies showed that muscle deterioration is associated with enhanced competence for aerobic ATP production, buffering, and utilization [12,17]. Similarly, muscle wastage associated with salmon migration triggered shift from anaerobic glycolysis to oxidative phosphorylation [24]. Current results point to conflicting changes at transcriptional and posttranscriptional levels and suggest that observations of altered aerobic respiration and ATP buffering may represent temporal changes. A general trend of suppressed mitochondrial energy production was reported in catabolic states of mammalian muscle [7,9]. Additional time-dependent studies are currently underway in our lab; we anticipate that they will help to clarify temporal changes in the kinetics of mRNA and enzymes of the aerobic respiration process.

3.3.

Protein biosynthesis and modification

Atrophying muscle had a reduced abundance of proteins involved in protein biosynthesis; additionally, synthesis of posttranslational chaperonins, and a few structural proteins was reduced (Table 2). The list of proteins includes eukaryotic translation initiation and elongation factors, several ribosomal proteins, heat shock proteins, collagen alpha 2 type I and Keratin, type II. Our previous transcriptomic studies showed that accumulation of protein biosynthesis transcripts is impaired in vitellogenesis-induced degeneration of trout muscle [12]. Similarly, muscle loss associated with salmon migration triggered massive protein turnover [19,24]. Therefore, transcriptomic and proteomic data support suppression of protein synthesis as a mechanism whereby muscle atrophy occurs [7,37]. Protein synthesis accounts for a high percentage of the animal's metabolic costs. Therefore, down-regulation of protein synthesis may be used to limit energy expenditures during muscle wastage. Protein synthesis is reduced to minimize ATP demands under unfavorable conditions [38,39].

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Table 2 – Differential expression of protein biosynthesis and modification proteins. GO aspects are C, cellular component; P, biological process and F, molecular function. Symbol

Fold Change

No. unique peptides

qValue

GO:ID

GO aspect

Gene ontology/function

NCBI acc#

Eukaryotic translation initiation factor 2s 1 Elongation factor 1-alpha Mitochondrial 28S ribosomal protein S33 60S ribosomal protein L27a Putative 40S ribosomal protein 60S ribosomal protein L4-A Heat shock 90 kDa protein 1 beta a Heat shock protein 47 Alpha 2 type I collagen Keratin, type II cytoskeletal 8 K18, simple type I keratin M-calpain Proteasome subunit, beta9a Oocyte protease inhibitor-1 Myosin light chain 1 Glutamine synthetase Cystathionine-beta-synthase Alpha-2,8-polysialyltransferase IV VHSV-induced protein-3 SH3 and PX domain-containing protein 2A Glial fibrillary acidic protein Plasminogen Otolith matrix macromolecule-64 Outer dense fiber of sperm tail protein 3

EIF2S1 EF1A MRPS33 RL27A RPS20 RPL-4 HSP90BA HSP47 COL1A2 K2C8 KRT18 CAPN2 LMP2 OPI-1 MYL1 GS CBS ST8SIA IV LOC100135995 SPD2A GFAP PLG LOC100170213 ODF3

− 2.4 − 1.4 − 1.9 − 1.3 − 1.3 1.4 − 1.4 − 2.5 − 2.3 − 1.5 1.2 1.4 − 1.6 − 1.4 1.2 1.4 − 1.3 1.3 1.3 − 1.4 1.2 1.2 −1.3 − 1.4

1 1 1 1 2 1 2 1 1 1 1 1 1 1 35 1 1 1 1 1 1 1 2 1

0.002 0.003 0.004 0.003 0.017 0.004 0.001 0.003 0.036 0.033 0.002 0.007 0.003 0.008 0.000 0.001 0.008 0.018 0.002 0.002 0.002 0.001 0.006 0.007

GO:0006412 GO:0003746 GO:0005840 GO:0003735 GO:0003723 GO:0003735 GO:0005524 GO:0004867 GO:0005201 GO:0005198 GO:0005198 GO:0004197 GO:0004175 GO:0005520 GO:0005509 GO:0003824 GO:0008652 GO:0006486 GO:0006464 GO:0005515 GO:0005882 GO:0003824

P F C F F F F F F F F F F F F F P P P F F F

GO:0001520

C

Translation Translation elongation factor activity Ribosome Structural constituent of ribosome RNA binding Structural constituent of ribosome ATP binding Serine-type endopeptidase inhibitor Extracellular matrix structural constituent Structural molecule activity Structural molecule activity Cysteine-type endopeptidase activity Endopeptidase activity Insulin-like growth factor binding Calcium ion binding Catalytic activity Amino acid anabolism Protein amino acid glycosylation Protein modification process Protein binding Intermediate filament Catalytic activity Collagen associated matrix protein Outer dense fibre

ref|NP_001133655 ref|NP_001135381 gb|ACM09556 ref|NP_001135307 ref|NP_001117836 ref|NP_001135255 ref|NP_001117703 ref|NP_001117706 ref|NP_001117679 ref|NP_001133687 ref|NP_001118196 ref|NP_001117701 ref|NP_001117730 ref|NP_001117917 ref|NP_001117763 ref|NP_001117786 ref|NP_001118158 ref|NP_001117688 ref|NP_001117804 ref|NP_001139826 gb|AAO13017 ref|NP_001117863 ref|NP_001123464 ref|NP_001117978

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Protein name

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Increased proteolysis has also been suggested as a mechanism to cause muscle atrophy in mammals [7,40,41] and fish [11,12,17,19]. Surprisingly, this study did not show substantial increase in any of the major proteolytic pathways; the membrane-bound lysosomal enzymes, calpain proteinases and the ubiquitin–proteasome enzymes. A single exception was up-regulated abundance of m-calpain (Table 2). In our previous work, we did not observe significant changes in calpain activity associated with vitellogenesisinduced muscle degeneration [11]. Therefore, an active role of the calpain pathway in vitellogenesis-induced muscle degeneration is unlikely. The proteasome subunit, beta9a protein was down-regulated in atrophying muscle. This result is consistent with our previous reports that showed no change in activity and down-regulation of several proteasome genes in response to vitellogenesis-induced muscle atrophy [11,12]. These results support previous reports suggesting that the rainbow trout, proteasome pathway, unlike mammals, has no significant role in piscine protein turnover [11,42]. Our previous studies reported that muscle deterioration during spawning was associated with greater mRNA accumulation and elevated activity of cathepsin-L. Unfortunately, cathepsin-L was not detected in this experiment, perhaps, due to lack of tryptic cleavage site. A substantial decrease in transcripts of myofibrillar/structural proteins was noticed in our previous microarray study. Consequently, large decreases in myofibrillar proteins were anticipated. Although several myosin and actin isoforms were identified in this study, none of them was significantly changed. Only two structural proteins, collagen alpha 2 type I and Keratin, type II, were down-regulated in atrophying muscle (Table 2). Whereas, K18, simple type I keratin, and myosin light chain-1 showed increased abundances, possibly due to exchange of protein isoforms caused by sarcomeric remodeling, in atrophying muscle [43]. These results indicate that myofibril/structural protein concentrations only partially correlate with the corresponding mRNAs concentrations. Mammalian studies of muscle atrophy were contradictory for transcriptional/pos-transcriptional suppression of the myofibrillar genes in multiple types of atrophying muscles [7,44]. Further studies are required to resolve this contradiction. Glutamine synthetase was more abundant in atrophying muscle (Table 2). This result is consistent with our previous report demonstrating induction of glutamine synthetase transcript in degenerating muscle [12]. In addition, studies using mammalian models reported dramatic increases in glutamine synthetase in degenerating muscle associated with conditions such as starvation, cancer cachexia, diabetes, uremia and exposure to glucocorticoids [7,45,46]. Glutamine synthetase catalyzes de novo glutamine synthesis from glutamate and ammonia maintaining glutamine homeostasis under conditions of increased glutamine demand by other tissues for gluconeogenesis or as a result of its limited supply [47]. Muscle atrophy induces glutamine efflux, thereby depleting muscle glutamine stores [48]. These effects suggest that glutamine synthetase may be a suitable marker for monitoring muscle catabolism during vitellogenesis and spawning in rainbow trout. Further studies are needed to determine the effect of glutamine feed supplementation on prevention of fish muscle atrophy during vitellogenesis.

3.4.

783

Inflammatory/immune response

Atrophying muscle had 21 differentially expressed proteins associated with mechanisms of the inflammatory/immune response (Table 3). The inhibitor of nuclear factor kappa B alpha (NFkBIA) was up-regulated in atrophying muscle. NFkBIA plays a central role in activation of inflammatory genes. It inactivates the transcription factor, nuclear factor kappaB (NFkB) in the cytoplasm. Upon encountering diverse stimuli, including TNF-α and IL-1, protein kinases phosphorylate and promote degradation of NFkBIA allowing translocation of NFkB to the nucleus and activation of the inflammatory responses [49,50]. Simultaneously and with up-regulation of NFkBIA, the level of two IL-1 receptors, essential for activation of inflammatory pathways, was reduced, while IL-20 receptor alpha, highly expressed in human skin and up-regulated in psoriasis, was more abundant in atrophying muscle. Collectively, these data suggest down-regulated expression of the NFkB inflammatory pathway. Systemic inflammation has been reported as the primary cause of muscle atrophy associated with aging and chronic disorders but not during starvation, cachexia, diabetes or uremia [7,50]. Therefore, an active role of the cytokine signaling of the inflammatory responses in the vitellogenesis-induced muscle atrophy in rainbow trout is not likely. Our muscle degradation model also revealed a downregulated protein component of the innate immune responses during atrophy (Table 3). Major Histocompatibility Complex I (MHC) class-I heavy chain protein and the nonclassical MHC class-I antigen were down-regulated while an MHC class-I antigen was up-regulated in atrophying muscle. Additionally, three up-regulated and two down-regulated proteins of the complement component pathway, in addition to an LPS binding protein, were observed. Many immune response proteins were down-regulated in atrophying muscle, including Immunoglobulin mu heavy chain, liver-expressed antimicrobial peptide 2B, chemokine receptor-like protein-1 and Tcell receptors (V and B chain). NFkB also controls the expression of genes encoding molecules important for immune responses and T-cell activation [51]. Consequently, down-regulation of the immune responses is consistent with the down-regulated expression of the NFkB inflammatory pathway.

3.5.

Signal transduction/transcription regulation

Atrophying muscle exhibited well-regulated expression of numerous arrays of signal transduction cascades that affect muscle growth (Table 4). The differentially expressed proteins list includes two isoforms of growth hormone receptors, GHR1 and GHR2. Growth hormone, GH, is the major peptide hormone stimulating somatic growth in fish. The GH/IGF-I axis is a critical mediator of skeletal muscle growth and adaptation [52]. Growth hormone action involves binding to membrane receptors in many tissues including skeletal muscle [53]. Nutritional status had a major influence on the animal's somatotrophic axis, and regulation at the level GH receptors is significant [54]. Therefore, the reduced abundance of GHR is consistent with insufficient calories available for growth resulting from elevated energetic demands of

784

Table 3 – Differential expression of inflammatory/immune response proteins. GO aspects are C, cellular component; P, biological process and F, molecular function. Symbol

Fold Change

No. unique peptides

qValue

GO:ID

GO aspect

Inhibitor of nuclear factor kappa B alpha Interleukin-1 receptor type 1 Interleukin-1 receptor type II Interleukin-20 receptor alpha MHC class I heavy chain Nonclassical MHC class I antigen MHC class I antigen Complement component C6 Complement factor B/C2-B Complement component C8 beta Complement component C3-4 Complement component C8 alpha chain Immunoglobulin mu heavy chain LBP (LPS binding protein)/BPI (bactericidal/ permeability-increasing protein)-1 Liver-expressed antimicrobial peptide 2B Chemokine receptor-like protein 1 T-cell receptor V-alpha5 chain T-cell receptor beta chain Myxovirus resistance 2 Precerebellin-like protein Tapasin-related

NFKBIA IL-1R1 IL-1RII IL20Ra ONMY-UA-B13 ONMY-LCA ONMY-UBA C6 BFC2-B C8B C3-4 C8A IGH-6 LBP/BPI-1

1.3 −1.5 −2.4 1.2 −1.8 −1.9 1.4 1.2 −1.6 1.3 1.8 −1.6 −1.3 −1.3

1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.005 0.005 0.003 0.001 0.003 0.002 0.001 0.002 0.003 0.017 0.007 0.001 0.047 0.045

GO:0042345 GO:0004872 GO:0004872 GO:0004872 GO:0006955 GO:0006955 GO:0006955 GO:0006955 GO:0006955 GO:0006955 GO:0006955 GO:0006955

P F F F P P P P P P P P

GO:0006953

P

LEAP2B CMKLR1 TCR V-ALPHA5 NITR1 RBTMX3 CBLNL TPSNR

−2.0 −2.0 −2.0 1.2 −1.9 1.4 1.3

1 1 1 1 1 1 1

0.002 0.006 0.001 0.019 0.001 0.001 0.004

GO:0042742 GO:0004930 GO:0004872 GO:0004872 GO:0005525

P F F F F

GO:0019885

P

Gene ontology/function

NCBI acc#

Regulation of NF-kappaB Receptor activity Receptor activity Receptor activity Immune response Immune response Immune response Immune response Immune response Immune response Immune response Immune response Immune response Acute-phase response

ref|NP_001117840 ref|NP_001117832 ref|NP_001138892 ref|NP_001118088 gb|AAB62228 gb|ABI21845 gb|AAK84490 ref|NP_001118093 ref|NP_001117673 ref|NP_001118079 gb|AAG40610 ref|NP_001118096 gb|ABR15659 ref|NP_001118057

Defense response to bacterium G-protein coupled receptor Receptor activity Receptor activity GTP binding Acute phase response Antigen processing

ref|NP_001117937 ref|NP_001117878 gb|AAA98475 emb|CAD57366 ref|NP_001117162 ref|NP_001117737 ref|NP_001118026

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Protein name

Table 4 – Differential expression of signal transduction/transcription regulation proteins. GO aspects are C, cellular component; P, biological process and F, molecular function. Symbol

Fold Change

No. unique peptides

qValue

GO:ID

GO aspect

Gene ontology/function

NCBI acc#

Growth hormone receptor isoform 1 Growth hormone receptor isoform 2 IGF binding protein 4 IGF binding protein 5 TMyogenin Pit-1 Follistatin Follistatin-like 3 glycoprotein Id2 protein MAPK /ERK kinase Signal transducer and activator of transcription 1 alpha Transcription factor jun-B Estrogen receptor beta 1 Estrogen receptor beta 2 Beta2-adrenergic receptor Rab protein Regulator of G-protein signalling 18 Corticotropin-releasing factor receptor type 1 Macrophage colony stimulating factor receptor-like Granulocyte colony stimulating factor receptor Mineralocorticoid receptor form B Toll-like leucine-rich repeat cyclin B2 homeobox protein HoxA4an Homeobox protein Hox-D4a Multidrug resistance associated protein 2 14 kDa transmembrane protein Synaptonemal complex protein 3

GHR1 GHR2 IGFBP4 IGFBP5 TMYOGENIN POU1F1 FST FSTL3 ID2 TMEK STAT3 JUNb ERB1 ERB2 ADRB2 RAB24 RGS18 CRHR1 M-CSFR CSF3R MRB TLR5 CCNB2 HOXA4AII HOXD4AI MRP2 I14K SCP3

−1.5 −1.5 1.3 −1.3 −1.4 −1.2 −1.6 −1.8 1.2 1.3 1.3 1.3 −2.2 −1.9 −1.8 −1.2 −1.4 −1.3 1.2 1.3 1.3 1.4 −1.9 1.2 1.3 −1.6 3.2 1.3

1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 3 1 1

0.002 0.043 0.017 0.002 0.005 0.031 0.001 0.002 0.006 0.001 0.006 0.003 0.005 0.001 0.001 0.046 0.017 0.001 0.001 0.007 0.001 0.009 0.002 0.000 0.021 0.000 0.008 0.036

GO:0004872 GO:0004872 GO:0005520 GO:0001558 GO:0003677 GO:0003677 GO:0005515 GO:0030514 GO:0005634 GO:0000166 GO:0003700 GO:0003677 GO:0006355 ] GO:0006355 ] GO:0004871 GO:0007264 GO:0004871 GO:0004871 GO:0004672 GO:0004872 GO:0004872 GO:0004872 GO:0007049 GO:0003677 GO:0003677 GO:0000166 GO:0009607 GO:0007049

F F F P F F F F C F F F F F F P F F F F F F P F F F P

Receptor activity Receptor activity Insulin-like growth factor binding Regulation of cell growth DNA binding DNA binding Protein binding Inhibition of BMP signaling Nucleus Nucleotide binding Transcription factor activity DNA binding Regulation of transcription Regulation of transcription Signal transducer activity Small GTPase mediated signaling Signal transducer activity Signal transducer activity Protein kinase activity Receptor activity Receptor activity Receptor activity Cell cycle DNA binding DNA binding Nucleotide binding Response to biotic stimulus Cell cycle

ref|NP_001118007 ref|NP_001118203 ref|NP_001133058 ref|NP_001117121 ref|NP_001118199 ref|NP_001118118 ref|NP_001153960 ref|NP_001153959 ref|NP_001118195 ref|NP_001117896 ref|NP_001117126 ref|NP_001133373 ref|NP_001118225 ref|NP_001118042 ref|NP_001117912 ref|NP_001117845 ref|NP_001118112 gb|AAT38872 ref|NP_001118211 ref|NP_001117874 ref|NP_001118212 ref|NP_001117163 ref|NP_001118131 ref|NP_001133041 ref|NP_001134844 ref|NP_001118127 ref|NP_001136191 ref|NP_001117979

J O U RN A L OF P R O TE O MI CS 73 ( 20 1 0 ) 7 7 8–7 8 9

Protein name

785

786

Table 5 – Differential expression of miscellaneous function proteins. GO aspects are C, cellular component; P, biological process and F, molecular function. Symbol

Fold Change

No. unique peptides

qValue

GO:ID

GO aspect

Gene ontology/function

NCBI acc#

Cytochrome P450 21-hydroxylase Cytochrome b-245, beta polypeptide Cytochrome P450 2K5 Aryl hydrocarbon receptor 2 delta 11-beta-hydroxylase Fatty acid binding protein H-FABP Delta 6-desaturase Vitellogenin Zona radiata structural protein Rh30-like3 Bile salt export pump Potassium channel TSK3 Inwardly-rectifying channel, subfamily J, 12 Voltage-gated sodium channel alpha type IV Neuronal-type voltage-gated calcium channel Cav2 Potassium voltage-gated channel subfamily H2 Calcium channel, voltagedependent, L type, alpha 1D Anion exchanger Transport-associated protein Transferrin Terminal deoxynucleotidyl transferase Hypothetical protein LOC100136074

CYP21 CYBB CYP2K5 AHR2D CYP11B FABP3 FD6D VTG1 ZP2.3 RH30 ABCB11 LOC100135972 KIR2.2 SCN4AA CACNA1B KCNH2 CACNA1D LOC100136955 TAP1 TRF DNTT LOC100136074

− 1.9 − 1.5 − 1.3 − 1.3 1.3 1.7 − 1.3 − 1.9 − 2.3 1.3 1.2 − 1.3 − 2.3 − 1.5 − 1.3 1.2 1.3 1.2 1.3 − 1.2 1.3 −1.7

1 1 1 1 1 6 35 2 1 1 3 1 2 4 3 1 1 1 1 2 1 2

0.002 0.000 0.003 0.003 0.007 0.000 0.000 0.007 0.002 0.002 0.006 0.045 0.004 0.000 0.006 0.010 0.004 0.023 0.003 0.006 0.001 0.004

GO:0004497 GO:0005506 GO:0004497 GO:0003677 GO:0004497 GO:0005215 GO:0005506 GO:0005319 GO:0006869 GO:0016021 GO:0005215 GO:0005216 GO:0005216 GO:0001518 GO:0005216 GO:0000155 GO:0005216 GO:0005215 GO:0000166 GO:0005576 GO:0006260 GO:0005576

F F F F F F F F P C F F F C F F F F F C F C

Monooxygenase activity Iron ion binding Monooxygenase activity DNA binding Monooxygenase activity Transporter activity Iron ion binding Lipid transporter activity Lipid transport Integral to membrane Transport activity Ion channel activity Ion channel activity Voltage-gated sodium channel complex Ion channel activity Two-component sensor activity Ion channel activity Transporter activity Nucleotide binding Extracellular region DNA replication Extracellular region

gb|ABX10835 ref|NP_001138891 ref|NP_001118214 ref|NP_001117015 ref|NP_001117736 ref|NP_001118185 ref|NP_001117759 dbj|BAH10127 ref|NP_001118072 ref|NP_001118135 ref|NP_001118128 ref|NP_001117784 gb|ABE02699 ref|NP_001118204 ref|NP_001118101 ref|NP_001118148 ref|NP_001117800 ref|NP_001118213 ref|NP_001117145 ref|NP_001118024 ref|NP_001118178 ref|NP_001117853

J O U RN A L OF P R O TE O MI CS 7 3 (2 0 1 0 ) 7 7 8–7 8 9

Protein name

J O U RN A L OF P R O TE O MI CS 73 ( 20 1 0 ) 7 7 8–7 8 9

reproduction. Actions of GH are mediated via stimulation of IGF-1 synthesis in the liver for systemic release and in skeletal muscle to elicit a local effect [55]. IGF binding proteins (IFGBPs) play an important role in regulating the availability of IGF 1 and thus, its action. Skeletal muscle produces four isotypes of the IGFBP family; IGFBP-3 through 6 [56]. Relative abundances of these IGFBPs can modulate IGF-1 bioavailability. IGFBP-4 inhibits IGF-1 [57]; whereas IGFBP-5 can either activate [58] or inhibit IGF-1 actions [59]. In our results, IGFBP5 was downregulated and IGFBP4 was up-regulated in atrophying muscle (Table 4). These changes suggest inhibition of IGF actions that is possibly coordinated with down-regulation of GHR1&2 in atrophying muscle. Evidence exits supporting a relationship between declining GH and IGF-1 levels and the age-related decline in human muscle mass [55]. Consequently, changes in GHRs and IGFBPs suggest an important role of the somatotrophic axis in regulating the vitellogenesis-associated muscle atrophy, perhaps through inhibiting protein synthesis [60]. Degenerating muscle also had under-expressed abundances of myogenin, a muscle-specific transcription factor that can induce myogenesis. This was coordinated with up-regulated expression of Id2 protein that block transcription and induces degradation of myogenin [61] (Table 4). Myogenin is IGF-1sensitive; reduced expression has been observed in mammalian loss of muscle mass [62,63]. A change in myogenin expression is consistent with the signal transduction cascade role of GH/IGF in mediating vitellogenesis-associated muscle atrophy. Atrophying muscle of spawning fish exhibited differential expression of other important arrays of transcription factors (Table 4). Abundance of two follistatin proteins, FST and FSTL3, was reduced in degenerating muscle. Follistatin is a potent positive regulator of muscle growth, and it binds and inhibits several negative regulators of muscle growth, including myostatin and activin [64]. In addition, STAT3, JunB and MAPK/ERK kinase were more abundant in degenerating muscle. In IL-6-induced, skeletal muscle atrophy, IL-6 induced activation, via phosphorylation, of STAT3 [52]. In atrogin1induced muscle atrophy, atrogin-1 increased phosphorylation of JNK (STAT3 activator) and c-Jun through a mechanism that involved degradation of MAPK phosphatase. Together, these changes suggest a shift in the balance of transcription factors to favor a more catabolic state in muscle. Jun was down-regulated in our microarray expression studies of muscle atrophy [7] suggesting posttranscriptional regulation of Jun in trout muscle atrophy. JunB and STAT3 have been identified as hub proteins in the general muscle atrophy network [65]. Additional studies are warranted to characterize roles of these transcription factors, and thus potential as markers of muscle growth in fish. The abundance of estrogen receptors, beta1 and 2, was lower in atrophying muscle (Table 4). Estrogen effects on muscle growth and development are conflicting. Some studies report that estrogen therapy can reduce contraction-induced and disuse muscle damage, but other studies indicated no effect [66–68]. Fish, throughout gonadal maturation and spawning, require large quantities of lipids and proteins to be drawn from body stores for oocyte development [69]. In teleosts, estrogen increases during gonadal maturation and is the principal ovarian steroid responsible for the hepatic yolk precursor production [70,71]. Therefore, a decreased abun-

787

dance of the estrogen receptors on the cell appears to permit catabolism of macromolecules in muscle while maintaining a high level of plasma estrogen. Three proteins of the G-protein signaling mechanism, beta2-adrenergic receptor (ADRB2), Rab protein (RAB24) and regulator of G-protein signaling 18 (RGS18) were reduced in atrophying muscle. Binding of catecholamine to the Beta2-adrenergic receptor increases muscle accretion through inhibition of protein catabolism, thereby moderating fish muscle atrophy [72].

3.6.

Miscellaneous functions

In addition to the aforementioned protein clusters, differential expression of several proteins involved in a broad array of functions was observed (Table 5). A well organized downregulated expression of five members of the cytochrome P450 superfamily of enzymes was noted. The cytochrome P450 proteins are monooxygenases that catalyze many reactions involved in xenobiotics metabolism and synthesis of cholesterol, steroids and other lipids. Further study is necessary to characterize the role(s) of monooxygenases in fish muscle degeneration.

4.

Conclusion

Outcomes of this study indicate that changes in protein expression are generally consisted with corresponding changes at the transcriptional level; atrophying muscle tends to have reduced enzymes of anaerobic respiration and protein biosynthesis. Other changes including the inflammatory/immune response and signal transduction/ transcription regulation appear to represent changes that are regulated at post transcriptional level. Proteomic and transcriptomic data allow a rational interpretation of the adaptive changes of muscle metabolism to support the fish reproductive cycle, in general, and oocyte growth and maturation, specifically. Muscle degradation is induced primarily as a consequence of imbalanced protein turnover described by decreased protein synthesis and increased protein degradation and resulting from the caloric demands of the rainbow trout reproductive cycle. Rainbow trout are ectothermic animals that rapidly use proteins as oxidative substrates [16]. Trout can acclimate to a considerable range of environmental stressors. During sexual maturation and reproductive cycle, females orchestrate metabolism to support the dominant process of oocyte development. Specific muscle regulatory mechanisms are involved including the GH/IGF-I axis and the muscle regulatory proteins myogenin and follistatin.

Acknowledgements This project was supported by National Research Initiative Competitive Grant No.2007-35205-17914 from the USDA Cooperative State Research, Education, and Extension Service; and USDA-ARS Cooperative Agreement No. 58-1930-5-537. It is published with the approval of the Director of the West Virginia Agriculture and Forestry Experiment Station as scientific paper No. 3053.

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