Proteomic Signature of Muscle Atrophy in Rainbow Trout

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JOURNAL OF PROTEOMICS 73 (2010) 778– 789

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Proteomic signature of muscle atrophy in rainbow trout

Mohamed Salema, P. Brett Kenneya, Caird E. Rexroad IIIb, Jianbo Yaoa,⁎ aLaboratory of Animal Biotechnology and Genomics, Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506-6108, United States bNational Center for Cool and Cold Water Aquaculture, Kearneysville, WV 25430, United States

ARTICLE INFO ABSTRACT

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 tes [7,8],andsepsis[8], as well as muscle disuse and denervation [9,10]. Growth, development and degradation of skeletal muscle are Fish use mechanisms of muscle growth and degradation governed by dynamic processes involving orchestrated ex- that are distinct from mammalian mechanisms [11,12]. Fish pression of genes encoding contractile and regulatory proteins species, has two anatomically well-separated muscle fiber [1]. Molecular mechanisms that regulate mammalian muscle types, red (aerobic) and white (anaerobic). This separation degradation have received substantial interest in the literature facilitates study of physiology and biochemistry of muscle [2–5]. Under a variety of physiological and pathological growth and degeneration in each muscle fiber types. This conditions, distinct cellular stimuli activate unique cellular situation provides an ideal experimental model in contrast to responses causing muscle wasting as seen in response to mammals, where study is much more difficult because fiber starvation [6], diseases including cancer, renal failure, diabe- 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 JOURNAL OF PROTEOMICS 73 (2010) 778– 789 779 differences in gene expression were reported between white and red muscle [14,15]. In addition, fish are ectothermic 2. Experimental procedures animals that rapidly use proteins as oxidative substrates [16] and have a lower metabolic rate than endothermic animals. 2.1. Fish and muscle sampling Fish can acclimate to a considerable range of environmental stressors, in particular, during the sexual maturation and Mature fertile (diploid) and sterile (triploid) female rainbow reproductive cycle, females rainbow trout orchestrate metab- trout, Oncorhynchus mykiss (500 g), were collected from Flowing olism to support the dominant process of oocyte develop- Springs Trout Farm (Delray, WV) during the spawning season in ment. Previously, we reported that fertile fish at spawning had early October. Fish were cultured in identical raceways receiving less muscle mass and less muscle protein compared to sterile water from a common spring at 13±3 °C. Fish were fed ad libitum fish and post-spawning fertile fish [11]. Consequently, this (Zeiglar Gold; Zeigler Bros., Gardeners, PA) via demand feeders. species in this scenario represents an ideal model for No difference in feed consumption was noticed between groups, elucidating the functional genomics of muscle growth, and fish had access to feed when sampled. As confirmed by degeneration/regeneration and fillet quality [12,17]. dissection, fertile fish were gravid with a gonado-somatic index Molecular mechanisms that regulate fish muscle degener- (GSI=ovary weight/fish weight×100) of 15.8±0.3 (n=5),andthe ation/regeneration have not received enough interest in the GSI of sterile fish was 0.3±0.2 (n=5). White muscle samples (20 g) literature. Some studies have examined individual genes/ from five fish of each group were collected from the dorsal mechanisms that control muscle atrophy [18–22]. Only few musculature and flash frozen in liquid nitrogen and stored at studies have dealt with fish muscle atrophy at the genome- -80 °C for proteomic analysis. Following muscle sample remov- wide level [12,17,23,24]. We used cDNA microarray to charac- al, fish were eviscerated, and ribs and vertebral column were terize transcriptomic responses to atrophy of fast-twitch removed to yield a butterfly fillet. This fillet was trimmed and muscles from gravid females compared to sterile females as skin was removed to generate boneless, skinless portions. This the control. This study identified an expression pattern that boneless, skinless muscle and initial muscle samples were closely resembles the mammalian muscle atrophy paradigm combined and expressed as a percentage of the whole fish [12]. Subsequently, we used high-density oligonucleotide weight. Collectively, this sum was used as the indication of array to corroborate our findings [17]. separable muscle. A portion of the muscle was used for Although much attention has been placed on changes in proximate composition [30]. transcriptional regulation, gene expression is regulated at several levels, all of which must be studied together to obtain a 2.2. MS and data analysis complete picture of a physiological or cellular process. Transcriptomic measurements do not necessarily reflect the MS and data analysis were done at Monarch Lifesciences amount of change in the active mature protein. In fact, the final (Indianapolis, Indiana) as previously described [27,31]. Tissue concentration of the active gene product, e.g. protein or samples were thawed and homogenized in a hypotonic lysis enzyme, is the most relevant quantity to the phenotype. buffer (100 mL of freshly made 8 M urea, 10 mM DTT solution). Studies comparing mRNA and protein abundance on a Tissue lysates were reduced and alkylated by triethylpho- genome-wide scale indicate that mRNAs only partly correlate sphine and iodoethanol, and subsequently digested using with the corresponding protein concentrations. It has been trypsin. All steps were carried out in one tube without washing estimated that only 20–40% protein concentrations are deter- or filtering steps. Peptide concentration was determined by mined by the corresponding mRNA concentrations [25,26]. the Bradford Protein Assay [32]. Lysis buffer was used as a Mass spectrometry (MS)-based proteomics has become an blank reference for the protein assay and as the buffer for important tool for investigating posttranslational modification protein standards (BSA). of proteins and protein interactions [27,28]. It allows hundreds Peptides were prepared and subjected to LC/MS analysis as to thousands of proteins to be simultaneously monitored, thus, previously described [27]. Tryptic peptides (∼20 μg) were allowing global profiling of proteins in muscle under an analyzed using Thermo linear ion-trap mass spectrometer atrophying metabolic state. The objective of this study was to (LTQ) coupled with a Surveyor HPLC system (Thermo, Waltham, characterize the global proteomic profile in muscle of rainbow MA). A C-18, reverse phase column (i.d.=2.1 mm, length= trout relative to sexual maturation using a high throughput LC/ 50 mm) was used to separate peptides at a flow rate of 200 μL/ MS-based label-free protein quantification method. In parallel, min. Peptides were eluted with 5 to 45% acetonitrile gradient transcriptional changes were characterized using a microarray developed over 120 min, and data were collected in the triple- chip approach and published separately [12,17]. This proteo- play mode (MS scan, zoom scan, and MS/MS scan). The acquired mic profile study will differentiate between transcriptional and data were filtered and analyzed by a proprietary algorithm that post-transcriptional regulatory mechanisms that control fish was developed by Higgs and coworkers [28,33]. Database muscle atrophy. Important genes affecting meat quality traits searches against the NCBI trout database were carried out have been identified and tested for potential improvement of using both the X!Tandem and SEQUEST algorithms. Protein muscle quality in breeding programs [29]. Hence, this study quantification was carried out using a proprietary protein will help in identifying genetic markers of improved muscle quantification algorithm licensed from Eli Lilly and Company growth and quality in rainbow trout. These candidate genes [28]. Briefly, once the raw files were acquired from the LTQ, all can subsequently be assessed for use in marker-assisted extracted ion chromatograms (XIC) were aligned by retention selection of rainbow trout with superior muscle growth and time. To be used in the protein quantification procedure, each fillet quality traits. aligned peak must match parent ion, charge state, daughter ions 780 JOURNAL OF PROTEOMICS 73 (2010) 778– 789

(MS/MS data) and retention time (within a one-minute window). had less muscle mass compared to samples collected 4 After alignment, area-under-the-curve (AUC) for each individ- months later. On the other hand, sterile fish reared under ually aligned peak from each sample was measured, normal- identical conditions showed no change in muscle mass [11]. ized, and these areas were compared for relative abundance. All Current studies in our lab using six samples collected over peak intensities were transformed to a log2 scale before quantile 8 months pre/post spawning confirmed our previous results normalization [34]. Quantile normalization is a method of (to be published elsewhere). This response is a suitable model normalization that essentially ensures that every sample has to investigate mechanisms of muscle degradation/regenera- a peptide intensity histogram of the same scale, location and tion in fish and to identify genetic markers for muscle growth shape. This normalization removes trends introduced by and quality for aquaculture applications [11,12,17]. In the sample handling, sample preparation, total protein differences current study, the global proteomic profile in degenerating and changes in instrument sensitivity while running multiple muscle of rainbow trout was analyzed using the response samples. If multiple peptides have the same protein identifica- associated with vitellogenesis-induced muscle atrophy. Pro- tion, then their quantile normalized log2 intensities were files of atrophying fast-twitch muscles collected from gravid averaged to obtain log2 protein intensities. The log2 protein rainbow trout at spawning season were compared with sterile intensity is the final quantity that is fit by a separate ANOVA fish muscle as the control. Atrophying muscle of fertile fish (Analysis of Variance) statistical model for each protein: had 11% less muscle mass and protein content compared to non-atrophying muscle of sterile fish, indicating extensive ð Þ ð Þ log2 Intensity = overallmean + groupeffect fixed muscle atrophy (Fig. 1, A and B; P<0.05) [11,12]. ð Þ + sampleeffect random Using a label-free LC/MS-based protein quantification + replicateeffectðrandomÞ: method, we identified 146 differentially expressed unique proteins in atrophying muscle (cut-off value: ±1.2 fold change). For the previous equation, group effect refers to the effect Differentially expressed proteins had 1.5 average fold change caused by the experimental conditions or treatments being and 3.2 maximum fold change values. Changes noticed at the evaluated. Sample effect represents the random effects from transcriptomic levels had average fold change value about 2.6 individual biological samples. It also includes random effects [12]. Proteins with gene ontology (GO) identifiers were grouped from sample preparation. The replicate effect refers to the according to their functions as given below. Moderate changes random effects from replicate injections of the same sample. of several proteins that are well-regulated in specific path- A total of 10 injections (5 from atrophying muscle [fertile fish] ways/mechanisms are connected with the spawning-associ- and 5 from non-atrophying muscles [sterile fish] were ated muscle degradation in rainbow trout. analyzed in this study. All of the injections were randomized and the instrument was operated by the same operator. The 3.2. Glycolysis/ATP production inverse log2 of each sample mean was calculated to determine the fold change between samples. Atrophying muscle showed decreased abundance of three successive enzymes in the intermediate steps of the glycolytic pathway, fructose-bisphosphate aldolase, triosephosphate 3. Results and discussion isomerase and glyceraldehyde-3-phosphate dehydrogenase. This reduction suggests reduced glucose utilization in atro- 3.1. Muscle atrophy in response to vitellogenesis phying muscle cells (Table 1). On the other hand the hexokinase enzyme, catalyzing the first step in glycolysis; Our previous studies showed that constraints of sexual phosphorylation of glucose to glucose-6-phosphate, showed maturation and spawning in rainbow trout cause significant up-regulated expression. This enzyme acts to trap glucose loss in muscle mass. At spawning season, rainbow trout fish 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). JOURNAL OF PROTEOMICS 73 (2010) 778– 789 781

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 ion. 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 com- petence 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 1.7 3 0.006 GO:0006096 P Glycolysis ref|NP_001135182 1.3 16 0.026 GO:0003824 F Catalytic activity ref|NP_001133174 1.8 60 0.007 GO:0006096 P Glycolysis ref|NP_001117033 1.7 1 0.004 GO:0000166 F Nucleotide binding gb|AAZ17382 1.9 1 0.001 GO:0005739 C Mitochondrion ref|YP_961364 1.4 2 0.002 GO:0006839 F Mitochondrial transport ref|NP_001118043 1.2 18 0.027 GO:0000166 F Nucleotide binding ref|NP_001118187 1.6 1 0.021 GO:0005524 F ATP binding ref|NP_001135114 1.4 2 0.014 GO:0000166 F Nucleotide binding ref|NP_001133546 2.7 4changes in the 0.011 kinetics GO:0005524 F of mRNA ATP binding and enzymes of ref|NP_001117775 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]. There- fore, transcriptomic and proteomic data support suppression Protein name Symbol Fold Change No. unique peptides qValue GO:ID GO aspect Gene ontology/function NCBIof acc# protein synthesis as a mechanism whereby muscle atrophy occurs [7,37]. Protein synthesis accounts for a high percentage Differential expression of glycolysis/ATP production proteins. GO aspects are C, cellular component; P, biological process and F, molecular funct

– 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 Table 1 Fructose-bisphosphate aldolase C ALD-B Triosephosphate isomerase 1b TPI Glyceraldehyde-3-phosphate dehydrogenase GAPDH GlucokinaseATP synthase beta-subunitGastric H+/K+-ATPase alpha subunit ATP4a ATPsyn-B 1.3 GK 1.3 6 0.016 2 GO:0008553 F 0.002 H-exporting ATPase GO:0004340 activity dbj|BAE45286 F Glucokinase activity ref|NP_001117721 NADH dehydrogenase subunit 4L MT-ND4L Uncoupling protein 2B UCP2B Creatine kinase CK1 78 kDa glucose-regulated protein GRP78 ATP-binding cassette, sub-family B (MDR/TAP) 3 ABCB3B(A) Nucleoside diphosphate kinase NDPK minimize ATP demands under unfavorable conditions [38,39]. 782

Table 2 – Differential expression of protein biosynthesis and modification proteins. GO aspects are C, cellular component; P, biological process and F, molecular function. Protein name Symbol Fold No. unique qValue GO:ID GO Gene ontology/function NCBI acc# Change peptides aspect

Eukaryotic translation initiation factor 2s 1 EIF2S1 −2.4 1 0.002 GO:0006412 P Translation ref|NP_001133655 − Elongation factor 1-alpha EF1A 1.4 1 0.003 GO:0003746 F Translation elongation factor activity ref|NP_001135381 778 (2010) 73 PROTEOMICS OF JOURNAL Mitochondrial 28S ribosomal protein S33 MRPS33 −1.9 1 0.004 GO:0005840 C Ribosome gb|ACM09556 60S ribosomal protein L27a RL27A −1.3 1 0.003 GO:0003735 F Structural constituent of ribosome ref|NP_001135307 Putative 40S ribosomal protein RPS20 −1.3 2 0.017 GO:0003723 F RNA binding ref|NP_001117836 60S ribosomal protein L4-A RPL-4 1.4 1 0.004 GO:0003735 F Structural constituent of ribosome ref|NP_001135255 Heat shock 90 kDa protein 1 beta a HSP90BA −1.4 2 0.001 GO:0005524 F ATP binding ref|NP_001117703 Heat shock protein 47 HSP47 −2.5 1 0.003 GO:0004867 F Serine-type endopeptidase inhibitor ref|NP_001117706 Alpha 2 type I collagen COL1A2 −2.3 1 0.036 GO:0005201 F Extracellular matrix structural constituent ref|NP_001117679 Keratin, type II cytoskeletal 8 K2C8 −1.5 1 0.033 GO:0005198 F Structural molecule activity ref|NP_001133687 K18, simple type I keratin KRT18 1.2 1 0.002 GO:0005198 F Structural molecule activity ref|NP_001118196 M-calpain CAPN2 1.4 1 0.007 GO:0004197 F Cysteine-type endopeptidase activity ref|NP_001117701 Proteasome subunit, beta9a LMP2 −1.6 1 0.003 GO:0004175 F Endopeptidase activity ref|NP_001117730 Oocyte protease inhibitor-1 OPI-1 −1.4 1 0.008 GO:0005520 F Insulin-like growth factor binding ref|NP_001117917 Myosin light chain 1 MYL1 1.2 35 0.000 GO:0005509 F Calcium ion binding ref|NP_001117763 Glutamine synthetase GS 1.4 1 0.001 GO:0003824 F Catalytic activity ref|NP_001117786 Cystathionine-beta-synthase CBS −1.3 1 0.008 GO:0008652 P Amino acid anabolism ref|NP_001118158 Alpha-2,8-polysialyltransferase IV ST8SIA IV 1.3 1 0.018 GO:0006486 P Protein amino acid glycosylation ref|NP_001117688 –

VHSV-induced protein-3 LOC100135995 1.3 1 0.002 GO:0006464 P Protein modification process ref|NP_001117804 789 SH3 and PX domain-containing protein 2A SPD2A −1.4 1 0.002 GO:0005515 F Protein binding ref|NP_001139826 Glial fibrillary acidic protein GFAP 1.2 1 0.002 GO:0005882 F Intermediate filament gb|AAO13017 Plasminogen PLG 1.2 1 0.001 GO:0003824 F Catalytic activity ref|NP_001117863 Otolith matrix macromolecule-64 LOC100170213 −1.3 2 0.006 Collagen associated matrix protein ref|NP_001123464 Outer dense fiber of sperm tail protein 3 ODF3 −1.4 1 0.007 GO:0001520 C Outer dense fibre ref|NP_001117978 JOURNAL OF PROTEOMICS 73 (2010) 778– 789 783

Increased proteolysis has also been suggested as a 3.4. Inflammatory/immune response mechanism to cause muscle atrophy in mammals [7,40,41] and fish [11,12,17,19]. Surprisingly, this study did not show Atrophying muscle had 21 differentially expressed proteins substantial increase in any of the major proteolytic pathways; associated with mechanisms of the inflammatory/immune the membrane-bound lysosomal enzymes, calpain protei- response (Table 3). The inhibitor of nuclear factor kappa B nases and the ubiquitin–proteasome enzymes. A single alpha (NFkBIA) was up-regulated in atrophying muscle. exception was up-regulated abundance of m-calpain NFkBIA plays a central role in activation of inflammatory (Table 2). In our previous work, we did not observe significant genes. It inactivates the transcription factor, nuclear factor changes in calpain activity associated with vitellogenesis- kappaB (NFkB) in the cytoplasm. Upon encountering diverse induced muscle degeneration [11]. Therefore, an active role of stimuli, including TNF-α and IL-1, protein kinases phosphor- the calpain pathway in vitellogenesis-induced muscle degen- ylate and promote degradation of NFkBIA allowing transloca- eration is unlikely. The proteasome subunit, beta9a protein tion of NFkB to the nucleus and activation of the inflammatory was down-regulated in atrophying muscle. This result is responses [49,50]. Simultaneously and with up-regulation of consistent with our previous reports that showed no change NFkBIA, the level of two IL-1 receptors, essential for activation in activity and down-regulation of several proteasome genes of inflammatory pathways, was reduced, while IL-20 receptor in response to vitellogenesis-induced muscle atrophy [11,12]. alpha, highly expressed in human skin and up-regulated in These results support previous reports suggesting that the psoriasis, was more abundant in atrophying muscle. Collec- rainbow trout, proteasome pathway, unlike mammals, has no tively, these data suggest down-regulated expression of the significant role in piscine protein turnover [11,42].Our NFkB inflammatory pathway. Systemic inflammation has previous studies reported that muscle deterioration during been reported as the primary cause of muscle atrophy spawning was associated with greater mRNA accumulation associated with aging and chronic disorders but not during and elevated activity of cathepsin-L. Unfortunately, cathep- starvation, cachexia, diabetes or uremia [7,50]. Therefore, an sin-L was not detected in this experiment, perhaps, due to lack active role of the cytokine signaling of the inflammatory of tryptic cleavage site. responses in the vitellogenesis-induced muscle atrophy in A substantial decrease in transcripts of myofibrillar/struc- rainbow trout is not likely. tural proteins was noticed in our previous microarray study. Our muscle degradation model also revealed a down- Consequently, large decreases in myofibrillar proteins were regulated protein component of the innate immune responses anticipated. Although several myosin and actin isoforms were during atrophy (Table 3). Major Histocompatibility Complex I identified in this study, none of them was significantly (MHC) class-I heavy chain protein and the nonclassical MHC changed. Only two structural proteins, collagen alpha 2 type I class-I antigen were down-regulated while an MHC class-I and Keratin, type II, were down-regulated in atrophying antigen was up-regulated in atrophying muscle. Additionally, muscle (Table 2). Whereas, K18, simple type I keratin, and three up-regulated and two down-regulated proteins of the myosin light chain-1 showed increased abundances, possibly complement component pathway, in addition to an LPS due to exchange of protein isoforms caused by sarcomeric binding protein, were observed. Many immune response remodeling, in atrophying muscle [43]. These results indicate proteins were down-regulated in atrophying muscle, includ- that myofibril/structural protein concentrations only partially ing Immunoglobulin mu heavy chain, liver-expressed antimi- correlate with the corresponding mRNAs concentrations. crobial peptide 2B, chemokine receptor-like protein-1 and T- Mammalian studies of muscle atrophy were contradictory for cell receptors (V and B chain). NFkB also controls the transcriptional/pos-transcriptional suppression of the myofi- expression of genes encoding molecules important for im- brillar genes in multiple types of atrophying muscles [7,44]. mune responses and T-cell activation [51]. Consequently, Further studies are required to resolve this contradiction. down-regulation of the immune responses is consistent with Glutamine synthetase was more abundant in atrophying the down-regulated expression of the NFkB inflammatory muscle (Table 2). This result is consistent with our previous pathway. report demonstrating induction of glutamine synthetase transcript in degenerating muscle [12]. In addition, studies 3.5. Signal transduction/transcription regulation using mammalian models reported dramatic increases in glutamine synthetase in degenerating muscle associated with Atrophying muscle exhibited well-regulated expression of conditions such as starvation, cancer cachexia, diabetes, numerous arrays of signal transduction cascades that affect uremia and exposure to glucocorticoids [7,45,46]. Glutamine muscle growth (Table 4). The differentially expressed proteins synthetase catalyzes de novo glutamine synthesis from list includes two isoforms of growth hormone receptors, GHR1 glutamate and ammonia maintaining glutamine homeostasis and GHR2. Growth hormone, GH, is the major peptide under conditions of increased glutamine demand by other hormone stimulating somatic growth in fish. The GH/IGF-I tissues for gluconeogenesis or as a result of its limited supply axis is a critical mediator of skeletal muscle growth and [47]. Muscle atrophy induces glutamine efflux, thereby deplet- adaptation [52]. Growth hormone action involves binding to ing muscle glutamine stores [48]. These effects suggest that membrane receptors in many tissues including skeletal glutamine synthetase may be a suitable marker for monitor- muscle [53]. Nutritional status had a major influence on the ing muscle catabolism during vitellogenesis and spawning in animal's somatotrophic axis, and regulation at the level GH rainbow trout. Further studies are needed to determine the receptors is significant [54]. Therefore, the reduced abundance effect of glutamine feed supplementation on prevention of of GHR is consistent with insufficient calories available for fish muscle atrophy during vitellogenesis. 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. Protein name Symbol Fold Change No. unique peptides qValue GO:ID GO aspect Gene ontology/function NCBI acc#

Inhibitor of nuclear factor kappa B alpha NFKBIA 1.3 1 0.005 GO:0042345 P Regulation of NF-kappaB ref|NP_001117840 778 (2010) 73 PROTEOMICS OF JOURNAL Interleukin-1 receptor type 1 IL-1R1 −1.5 1 0.005 GO:0004872 F Receptor activity ref|NP_001117832 Interleukin-1 receptor type II IL-1RII −2.4 1 0.003 GO:0004872 F Receptor activity ref|NP_001138892 Interleukin-20 receptor alpha IL20Ra 1.2 1 0.001 GO:0004872 F Receptor activity ref|NP_001118088 MHC class I heavy chain ONMY-UA-B13 −1.8 1 0.003 GO:0006955 P Immune response gb|AAB62228 Nonclassical MHC class I antigen ONMY-LCA −1.9 1 0.002 GO:0006955 P Immune response gb|ABI21845 MHC class I antigen ONMY-UBA 1.4 1 0.001 GO:0006955 P Immune response gb|AAK84490 Complement component C6 C6 1.2 1 0.002 GO:0006955 P Immune response ref|NP_001118093 Complement factor B/C2-B BFC2-B −1.6 1 0.003 GO:0006955 P Immune response ref|NP_001117673 Complement component C8 beta C8B 1.3 1 0.017 GO:0006955 P Immune response ref|NP_001118079 Complement component C3-4 C3-4 1.8 1 0.007 GO:0006955 P Immune response gb|AAG40610 Complement component C8 alpha chain C8A −1.6 1 0.001 GO:0006955 P Immune response ref|NP_001118096 Immunoglobulin mu heavy chain IGH-6 −1.3 1 0.047 Immune response gb|ABR15659 LBP (LPS binding protein)/BPI (bactericidal/ LBP/BPI-1 −1.3 1 0.045 GO:0006953 P Acute-phase response ref|NP_001118057 permeability-increasing protein)-1 Liver-expressed antimicrobial peptide 2B LEAP2B −2.0 1 0.002 GO:0042742 P Defense response to bacterium ref|NP_001117937 − Chemokine receptor-like protein 1 CMKLR1 2.0 1 0.006 GO:0004930 F G-protein coupled receptor ref|NP_001117878 – T-cell receptor V-alpha5 chain TCR V-ALPHA5 −2.0 1 0.001 GO:0004872 F Receptor activity gb|AAA98475 789 T-cell receptor beta chain NITR1 1.2 1 0.019 GO:0004872 F Receptor activity emb|CAD57366 Myxovirus resistance 2 RBTMX3 −1.9 1 0.001 GO:0005525 F GTP binding ref|NP_001117162 Precerebellin-like protein CBLNL 1.4 1 0.001 Acute phase response ref|NP_001117737 Tapasin-related TPSNR 1.3 1 0.004 GO:0019885 P Antigen processing ref|NP_001118026 Table 4 – Differential expression of signal transduction/transcription regulation proteins. GO aspects are C, cellular component; P, biological process and F, molecular function. Protein name Symbol Fold No. unique qValue GO:ID GO Gene ontology/function NCBI acc# Change peptides aspect

Growth hormone receptor isoform 1 GHR1 −1.5 1 0.002 GO:0004872 F Receptor activity ref|NP_001118007 Growth hormone receptor isoform 2 GHR2 −1.5 2 0.043 GO:0004872 F Receptor activity ref|NP_001118203 IGF binding protein 4 IGFBP4 1.3 1 0.017 GO:0005520 F Insulin-like growth factor binding ref|NP_001133058

IGF binding protein 5 IGFBP5 −1.3 1 0.002 GO:0001558 P Regulation of cell growth ref|NP_001117121 778 (2010) 73 PROTEOMICS OF JOURNAL TMyogenin TMYOGENIN −1.4 1 0.005 GO:0003677 F DNA binding ref|NP_001118199 Pit-1 POU1F1 −1.2 1 0.031 GO:0003677 F DNA binding ref|NP_001118118 Follistatin FST −1.6 1 0.001 GO:0005515 F Protein binding ref|NP_001153960 Follistatin-like 3 glycoprotein FSTL3 −1.8 1 0.002 GO:0030514 F Inhibition of BMP signaling ref|NP_001153959 Id2 protein ID2 1.2 1 0.006 GO:0005634 C Nucleus ref|NP_001118195 MAPK /ERK kinase TMEK 1.3 1 0.001 GO:0000166 F Nucleotide binding ref|NP_001117896 Signal transducer and activator of transcription 1 alpha STAT3 1.3 1 0.006 GO:0003700 F Transcription factor activity ref|NP_001117126 Transcription factor jun-B JUNb 1.3 1 0.003 GO:0003677 F DNA binding ref|NP_001133373 Estrogen receptor beta 1 ERB1 −2.2 1 0.005 GO:0006355 ] F Regulation of transcription ref|NP_001118225 Estrogen receptor beta 2 ERB2 −1.9 1 0.001 GO:0006355 ] F Regulation of transcription ref|NP_001118042 Beta2-adrenergic receptor ADRB2 −1.8 1 0.001 GO:0004871 F Signal transducer activity ref|NP_001117912 Rab protein RAB24 −1.2 1 0.046 GO:0007264 P Small GTPase mediated signaling ref|NP_001117845 Regulator of G-protein signalling 18 RGS18 −1.4 1 0.017 GO:0004871 F Signal transducer activity ref|NP_001118112 Corticotropin-releasing factor receptor type 1 CRHR1 −1.3 1 0.001 GO:0004871 F Signal transducer activity gb|AAT38872 Macrophage colony stimulating factor receptor-like M-CSFR 1.2 1 0.001 GO:0004672 F Protein kinase activity ref|NP_001118211 Granulocyte colony stimulating factor receptor CSF3R 1.3 2 0.007 GO:0004872 F Receptor activity ref|NP_001117874 –

Mineralocorticoid receptor form B MRB 1.3 1 0.001 GO:0004872 F Receptor activity ref|NP_001118212 789 Toll-like leucine-rich repeat TLR5 1.4 1 0.009 GO:0004872 F Receptor activity ref|NP_001117163 cyclin B2 CCNB2 −1.9 1 0.002 GO:0007049 P Cell cycle ref|NP_001118131 homeobox protein HoxA4an HOXA4AII 1.2 1 0.000 GO:0003677 F DNA binding ref|NP_001133041 Homeobox protein Hox-D4a HOXD4AI 1.3 1 0.021 GO:0003677 F DNA binding ref|NP_001134844 Multidrug resistance associated protein 2 MRP2 −1.6 3 0.000 GO:0000166 F Nucleotide binding ref|NP_001118127 14 kDa transmembrane protein I14K 3.2 1 0.008 GO:0009607 P Response to biotic stimulus ref|NP_001136191 Synaptonemal complex protein 3 SCP3 1.3 1 0.036 GO:0007049 Cell cycle ref|NP_001117979 785 786

Table 5 – Differential expression of miscellaneous function proteins. GO aspects are C, cellular component; P, biological process and F, molecular function. Protein name Symbol Fold No. unique qValue GO:ID GO Gene ontology/function NCBI acc# Change peptides aspect ORA FPOEMC 3(00 778 (2010) 73 PROTEOMICS OF JOURNAL Cytochrome P450 21-hydroxylase CYP21 −1.9 1 0.002 GO:0004497 F Monooxygenase activity gb|ABX10835 Cytochrome b-245, beta polypeptide CYBB −1.5 1 0.000 GO:0005506 F Iron ion binding ref|NP_001138891 Cytochrome P450 2K5 CYP2K5 −1.3 1 0.003 GO:0004497 F Monooxygenase activity ref|NP_001118214 Aryl hydrocarbon receptor 2 delta AHR2D −1.3 1 0.003 GO:0003677 F DNA binding ref|NP_001117015 11-beta-hydroxylase CYP11B 1.3 1 0.007 GO:0004497 F Monooxygenase activity ref|NP_001117736 Fatty acid binding protein H-FABP FABP3 1.7 6 0.000 GO:0005215 F Transporter activity ref|NP_001118185 Delta 6-desaturase FD6D −1.3 35 0.000 GO:0005506 F Iron ion binding ref|NP_001117759 Vitellogenin VTG1 −1.9 2 0.007 GO:0005319 F Lipid transporter activity dbj|BAH10127 Zona radiata structural protein ZP2.3 −2.3 1 0.002 GO:0006869 P Lipid transport ref|NP_001118072 Rh30-like3 RH30 1.3 1 0.002 GO:0016021 C Integral to membrane ref|NP_001118135 Bile salt export pump ABCB11 1.2 3 0.006 GO:0005215 F Transport activity ref|NP_001118128 Potassium channel TSK3 LOC100135972 −1.3 1 0.045 GO:0005216 F Ion channel activity ref|NP_001117784 Inwardly-rectifying channel, subfamily J, 12 KIR2.2 −2.3 2 0.004 GO:0005216 F Ion channel activity gb|ABE02699 Voltage-gated sodium channel alpha type IV SCN4AA −1.5 4 0.000 GO:0001518 C Voltage-gated sodium channel complex ref|NP_001118204 Neuronal-type voltage-gated calcium channel Cav2 CACNA1B −1.3 3 0.006 GO:0005216 F Ion channel activity ref|NP_001118101 Potassium voltage-gated channel subfamily H2 KCNH2 1.2 1 0.010 GO:0000155 F Two-component sensor activity ref|NP_001118148

Calcium channel, voltagedependent, L type, alpha 1D CACNA1D 1.3 1 0.004 GO:0005216 F Ion channel activity ref|NP_001117800 – 789 Anion exchanger LOC100136955 1.2 1 0.023 GO:0005215 F Transporter activity ref|NP_001118213 Transport-associated protein TAP1 1.3 1 0.003 GO:0000166 F Nucleotide binding ref|NP_001117145 Transferrin TRF −1.2 2 0.006 GO:0005576 C Extracellular region ref|NP_001118024 Terminal deoxynucleotidyl transferase DNTT 1.3 1 0.001 GO:0006260 F DNA replication ref|NP_001118178 Hypothetical protein LOC100136074 LOC100136074 −1.7 2 0.004 GO:0005576 C Extracellular region ref|NP_001117853 JOURNAL OF PROTEOMICS 73 (2010) 778– 789 787 reproduction. Actions of GH are mediated via stimulation of dance of the estrogen receptors on the cell appears to permit IGF-1 synthesis in the liver for systemic release and in skeletal catabolism of macromolecules in muscle while maintaining a muscle to elicit a local effect [55].IGFbindingproteins(IFGBPs) high level of plasma estrogen. Three proteins of the G-protein play an important role in regulating the availability of IGF 1 and signaling mechanism, beta2-adrenergic receptor (ADRB2), Rab thus, its action. Skeletal muscle produces four isotypes of the protein (RAB24) and regulator of G-protein signaling 18 (RGS18) IGFBP family; IGFBP-3 through 6 [56]. Relative abundances of were reduced in atrophying muscle. Binding of catecholamine these IGFBPs can modulate IGF-1 bioavailability. IGFBP-4 to the Beta2-adrenergic receptor increases muscle accretion inhibits IGF-1 [57]; whereas IGFBP-5 can either activate [58] or through inhibition of protein catabolism, thereby moderating inhibit IGF-1 actions [59]. In our results, IGFBP5 was down- fish muscle atrophy [72]. regulated and IGFBP4 was up-regulated in atrophying muscle (Table 4). These changes suggest inhibition of IGF actions that is 3.6. Miscellaneous functions possibly coordinated with down-regulation of GHR1&2 in atrophying muscle. Evidence exits supporting a relationship In addition to the aforementioned protein clusters, differential between declining GH and IGF-1 levels and the age-related expression of several proteins involved in a broad array of decline in human muscle mass [55]. Consequently, changes in functions was observed (Table 5). A well organized down- GHRs and IGFBPs suggest an important role of the somato- regulated expression of five members of the cytochrome P450 trophic axis in regulating the vitellogenesis-associated muscle superfamily of enzymes was noted. The cytochrome P450 atrophy, perhaps through inhibiting protein synthesis [60]. proteins are monooxygenases that catalyze many reactions Degenerating muscle also had under-expressed abundances of involved in xenobiotics metabolism and synthesis of choles- myogenin, a muscle-specific transcription factor that can terol, steroids and other lipids. Further study is necessary to induce myogenesis. This was coordinated with up-regulated characterize the role(s) of monooxygenases in fish muscle expression of Id2 protein that block transcription and induces degeneration. degradation of myogenin [61] (Table 4). Myogenin is IGF-1- sensitive; reduced expression has been observed in mammalian loss of muscle mass [62,63]. A change in myogenin expression is 4. Conclusion consistent with the signal transduction cascade role of GH/IGF in mediating vitellogenesis-associated muscle atrophy. Outcomes of this study indicate that changes in protein Atrophying muscle of spawning fish exhibited differential expression are generally consisted with corresponding expression of other important arrays of transcription factors changes at the transcriptional level; atrophying muscle (Table 4). Abundance of two follistatin proteins, FST and tends to have reduced enzymes of anaerobic respiration and FSTL3, was reduced in degenerating muscle. Follistatin is a protein biosynthesis. Other changes including the inflamma- potent positive regulator of muscle growth, and it binds and tory/immune response and signal transduction/ transcription inhibits several negative regulators of muscle growth, includ- regulation appear to represent changes that are regulated at ing myostatin and activin [64]. In addition, STAT3, JunB and post transcriptional level. Proteomic and transcriptomic data MAPK/ERK kinase were more abundant in degenerating allow a rational interpretation of the adaptive changes of muscle. In IL-6-induced, skeletal muscle atrophy, IL-6 induced muscle metabolism to support the fish reproductive cycle, in activation, via phosphorylation, of STAT3 [52]. In atrogin- general, and oocyte growth and maturation, specifically. 1induced muscle atrophy, atrogin-1 increased phosphoryla- Muscle degradation is induced primarily as a consequence of tion of JNK (STAT3 activator) and c-Jun through a mechanism imbalanced protein turnover described by decreased protein that involved degradation of MAPK phosphatase. Together, synthesis and increased protein degradation and resulting these changes suggest a shift in the balance of transcription from the caloric demands of the rainbow trout reproductive factors to favor a more catabolic state in muscle. Jun was cycle. Rainbow trout are ectothermic animals that rapidly use down-regulated in our microarray expression studies of proteins as oxidative substrates [16]. Trout can acclimate to a muscle atrophy [7] suggesting posttranscriptional regulation considerable range of environmental stressors. During sexual of Jun in trout muscle atrophy. JunB and STAT3 have been maturation and reproductive cycle, females orchestrate me- identified as hub proteins in the general muscle atrophy tabolism to support the dominant process of oocyte develop- network [65]. Additional studies are warranted to characterize ment. Specific muscle regulatory mechanisms are involved roles of these transcription factors, and thus potential as including the GH/IGF-I axis and the muscle regulatory proteins markers of muscle growth in fish. myogenin and follistatin. 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 Acknowledgements report that estrogen therapy can reduce contraction-induced and disuse muscle damage, but other studies indicated no This project was supported by National Research Initiative effect [66–68]. Fish, throughout gonadal maturation and Competitive Grant No.2007-35205-17914 from the USDA Co- spawning, require large quantities of lipids and proteins to operative State Research, Education, and Extension Service; be drawn from body stores for oocyte development [69].In and USDA-ARS Cooperative Agreement No. 58-1930-5-537. teleosts, estrogen increases during gonadal maturation and is It is published with the approval of the Director of the West the principal ovarian steroid responsible for the hepatic yolk Virginia Agriculture and Forestry Experiment Station as precursor production [70,71]. Therefore, a decreased abun- scientific paper No. 3053. 788 JOURNAL OF PROTEOMICS 73 (2010) 778– 789

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