Differential expression between normal and pale, soft, and exudative turkey meat

Y. Malila ,* R. J. Tempelman ,† K. R. B. Sporer ,*† C. W. Ernst ,† S. G. Velleman ,‡ K. M. Reed ,§ and G. M. Strasburg * 1

* Department of Food Science and Human Nutrition, and † Department of Animal Science, Michigan State University, East Lansing 48824; ‡ Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster 44691; and § Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul 55108

ABSTRACT In response to high consumer demand, quantitative real-time PCR. Selection of differentially turkeys have been intensively selected for rapid growth expressed for pathway analysis was performed us- rate and breast muscle mass and conformation. The suc- ing a combination of fold change (FC) ranking (FC < cess in breeding selection has coincided with an increas- −1.66, FC >1.66) and false discovery rate (<0.35) as ing incidence of pale, soft, and exudative (PSE) meat criteria. The pathway was highlighted defect, especially in response to heat stress. We hypoth- as the top canonical pathway associated with differen- esized that the underlying mechanism responsible for tial between normal and PSE turkey. the development of PSE meat arises from differences Dramatic downregulation of fast-twitch heavy in expression of several critical genes. The objective of chain coupled with upregulation of slow-twitch myosin this study was to determine differential gene expres- and C suggested a switch of skeletal muscle sion between normal and PSE turkey meat using a 6K isoforms, which may alter muscle fiber arrangement turkey skeletal muscle long oligonucleotide microarray. and formation of -myosin complexes. Changes in Breast meat samples were collected from Randombred expression of genes in the actin signaling Control Line 2 turkeys at 22 wk of age, and classi- pathway also suggest altered structures of actin fila- fied as normal or PSE primarily based on marinade ments that may affect cell motility as well as strength uptake (high = normal, low = PSE). Total RNA was and flexibility of muscle cells. Substantial downregula- isolated from meat samples with the highest (normal, tion of pyruvate dehydrogenase kinase, isozyme 4 was n = 6) and the lowest (PSE, n = 6) marinade uptake. observed in PSE samples, suggesting altered regulation Microarray data confirmation was conducted using of the aerobic metabolic pathway in the birds that de- veloped PSE meat defect. Key words: pale soft and exudative , microarray , global gene expression , turkey 2013 Poultry Science 92 :1621–1633 http://dx.doi.org/10.3382/ps.2012-02778

INTRODUCTION the prevalence of a significant quality defect known as pale, soft, and exudative (PSE) meat (Dransfield and Consumer demand for inexpensive food with low Sosnicki, 1999; Owens et al., 2000). fat and high content has led to a tremendous Pale, soft, and exudative meat was originally identi- growth of the poultry industry over the past several fied in pork with flaccid texture and unusually light decades. Turkeys have become an attractive protein color. Processed meat products made from either pork source because of their larger portion of lean meat com- or poultry PSE meat are often of inferior quality (Ab- pared with chickens. To meet increasing demand for erle et al., 2001) and lower customer acceptability (Fer- turkey meat, birds have been intensively selected for nandez et al., 2002). This has been attributed to an ex- rapid growth rate and breast muscle mass accretion tensive denaturation of in PSE meat, resulting and conformation (Barbut et al., 2008). However, the in a loss of protein functionalities including solubility, success of breeding has coincided with an increase in water-holding capacity, and binding properties. Over- all, the PSE meat defect substantially lowers process- ing yields and causes significant economic loss to the © 2013 Poultry Science Association Inc. poultry industry (Owens et al., 2009). Received September 14, 2012. Accepted February 4, 2013. In pigs, it is generally accepted that development of 1 Corresponding author: [email protected] PSE pork is associated with a rapid rate of postmortem

1621 1622 Malila et al. anaerobic glycolysis, resulting in high carcass tempera- turkeys from the Randombred Control Line 2 (RBC2), ture and rapid decrease in pH, leading to protein dena- a line representative of the commercial turkey of the turation. The accelerated postmortem glycolysis is asso- late 1960s and maintained without selection pressure ciated with abnormal Ca2+ homeostasis in muscle cells. at The Ohio Agricultural Research and Development A Ca2+ leak via sarcoplasmic reticulum (SR) calcium Center of The Ohio State University were used in this release channel proteins (ryanodine receptors, RYR) study (Nestor et al., 1967; Nestor, 1977). The birds were in skeletal muscle cells, results from a single point mu- raised at the Michigan State University (MSU) Poul- tation in RYR1 that changes the amino acid sequence try farm (Chiang et al., 2008). All methods were ap- from arginine at position 615 to cysteine (Mickelson et proved by the Institutional Animal Care and Use Com- al., 1988; Fujii et al., 1991; Otsu et al., 1994). mittee (AUF#: 06/05–081–00). Turkeys at 22 wk of In contrast, a hypersensitivity of the SR calcium re- age were slaughtered using standard industry practices lease channels between different turkey lines has been in the MSU Meat Laboratory. Breast muscle samples suggested (Wang et al., 1999), but no has yet from one side of each bird were collected immediately been observed (Chiang et al., 2004). We hypothesized postbleed, snap-frozen in liquid nitrogen, and stored at that some turkeys may be PSE-susceptible because of −80°C for later isolation of total RNA. Breast muscle differences in the abundance of key proteins involved samples from the opposite side were processed under in regulation of intracellular [Ca2+]. Recently, we de- commercial conditions. Breast muscle was classified af- termined relative mRNA abundance of 4 major genes ter 24 h as normal, primarily based on high marinade involved in Ca2+ homeostasis in skeletal muscle cells uptake, and secondarily on low cook loss. Conversely, between normal and PSE turkey breast meat (Sporer PSE samples were grouped by low marinade uptake et al., 2012). With the onset of heat stress, the PSE and high cook loss (Sporer et al., 2012). Six samples for meat showed a significant delay in the upregulation of each extreme of normal and PSE characteristics (n = RYR isoforms αRYR and βRYR, and of , 6) were used for microarray experiments. the high-capacity, low-affinity Ca2+ binding protein lo- cated in the lumen of the SR. Transcript abundance of the sarco/endoplasmic reticulum Ca2+-ATPase 1 RNA Isolation (SERCA1) remained unchanged. This previous study Total RNA was isolated from breast meat samples us- of Sporer et al. (2012) suggests a complex manifesta- ing Ambion TRI Reagent Solution (Applied Biosystems tion of changes in gene expression associated with de- Inc., Foster City, CA), and subsequently purified with velopment of PSE in turkey. It is possible that differ- the Qiagen RNeasy Mini spin column (Qiagen Inc., Va- ential expression of unidentified genes other than SR 2+ lencia, CA) according to the manufacturer’s instruc- Ca regulators may be revealed in PSE turkey. In ad- tions. Quantity of total RNA was measured using a dition, comparison of gene expression between normal Nanodrop ND-1000 spectrophotometer (Thermo Fisher and PSE turkey meat from birds not subjected to heat Scientific, Waltham, MA). Integrity of total RNA was stress was not examined in the study of Sporer et al. confirmed using an Agilent 2100 Bioanalyzer (Santa (2012). This information is important to advance our Clara, CA). Samples with an RNA integrity number fundamental comprehension at the transcriptional level equal to or exceeding 8.0 (RNA integrity number = 10 regarding the development of PSE turkey meat. is the best) were used for microarray and quantitative A turkey skeletal muscle long oligonucleotide (TSK- real-time PCR (qPCR). MLO) microarray was constructed with the initial pur- pose of screening the skeletal muscle transcriptome for candidate genes critical for growth and development. Microarray Experimental Design The platform has been subsequently validated and used for studies of domestic turkey muscle biology (Sporer et The 6K TSKMLO microarrays were used for tran- al., 2011a,b; Nierobisz et al., 2012). Utilization of the scriptome analysis of normal and PSE meat samples. TSKMLO platform enables a simultaneous investiga- Details of the array design are available at the National tion of the expression of thousands of skeletal muscle Center for Biotechnology Information’s Gene Expres- genes. The objective of the current study was to in- sion Omnibus (NCBI GEO) with the platform acces- vestigate differential expression in the turkey skeletal sion GPL9788. Gene expression between normal and muscle transcriptome between normal and PSE meat PSE turkey skeletal muscle samples was directly com- using the TSKMLO microarray. pared. Six biological replicates were run for each meat quality level (n = 6). Dye swapping was performed to minimize dye bias; i.e., in 3 arrays, the normal samples MATERIALS AND METHODS were labeled with Cy3 fluorescent dye (GE Healthcare, Experimental Birds and Sample Collection Piscataway, NJ), whereas PSE samples were labeled with Cy5; for the other 3 arrays, the dye assignments Breast meat samples used in this study were ob- were reversed. A total of 6 arrays were used in this tained from the study of Chiang et al. (2008). Briefly, study. GENE EXPRESSION IN PALE, SOFT, AND EXUDATIVE TURKEY 1623 RNA Amplification and Microarray Operon Inc. (Huntsville, AL). The confirmation proto- Hybridizations col was as described in Sporer et al. (2011a). Briefly, 5 μg of total RNA from the same samples used for mi- Amplification of RNA samples, microarray prepara- croarray was reverse transcribed to cDNA using Super- tion and microarray hybridization were performed as script III (Invitrogen). The cDNA was purified using described elsewhere (Sporer et al., 2011a). Briefly, 2 ethanol precipitation and quantified with a Nanodrop μg of total RNA was incubated with T7 Oligo(dT)18 ND-1000 spectrophotometer. Reactions included 10 ng as primer and reverse-transcribed into cDNA followed of cDNA, 300 nM primer mix, and POWER SYBR by in vitro for amplified RNA (aRNA) Green Master Mix (Applied Biosystems) and were run using an Amino Allyl MessageAmp II aRNA kit (Am- in an ABI Prism 7900 Sequence Detection System (Ap- bion Inc., Austin, TX) according to the manufacturer’s plied Biosystems). Threshold cycle was analyzed using instructions. For dye labeling, 10 μg of aRNA samples Sequence Detection Systems Software version 2.3 (Ap- were dye-coupled with Cy3 or Cy5 fluorescent dye at plied Biosystems). β-Actin was used as an endogenous room temperature in the dark for 1 h. Dye-labeled control gene (Sporer et al., 2012). Relative expression aRNA was purified; then, 5 μg of each sample was frag- of genes of interest in PSE samples relative to normal mented into 60 to 200 nucleotide fragments using RNA samples was calculated using 2−ΔΔCT method (Livak Fragmentation Reagents (Ambion Inc.) at 70°C for 15 and Schmittgen, 2001). Student’s t-test was performed min. The fragmented Cy3-labeled aRNA was equally to evaluate significant difference (P < 0.05) in gene mixed with its Cy5-labeled counterpart. Hybridization expression between PSE and normal samples. of microarray slides was performed in a GeneTac Hy- bridization Station (Genomic Solutions, Ann Arbor, MI) for 18 h. Arrays were washed with a series of wash Pathway Analysis solutions and dried by centrifugation. Afterward, the To identify biological functions of the annotated arrays were scanned using a Molecular Devices Ge- genes, pathway analysis was performed using Ingenu- nePix 4000B scanner (Molecular Devices, Sunnyvale, ity Pathway Analysis (IPA; Ingenuity Systems, http:// CA), and image analysis was performed using Molecu- www.ingenuity.com). Two criteria, FC and FDR, were lar Devices GenePix Pro 6.0 software. Array spot inten- used to select the genes for pathway analysis. Genes sities were exported as GenePix Results (GPR) files for with FC <−1.66 or FC >1.66 and FDR <0.35 were up- statistical analysis. loaded to the IPA. Canonical pathways associated with focus genes were generated from the software. Microarray Statistical Analysis and Gene Annotation RESULTS Dye intensity bias was normalized using “normexp” background correction method based on Ritchie et al. Differential Expression of Genes Between (2007). Normalized data were described as log2 fluores- Normal and PSE Turkey Skeletal Muscle cent intensities ratio (Cy5/Cy3) or M-value, and sta- tistically analyzed with a linear model using LIMMA Differential expression of 49 transcripts between PSE (Smyth, 2005). Specific hybridization was confirmed by and normal turkey skeletal breast muscle was identified monitoring fluorescence intensities of negative control (FDR <0.1). Seventeen transcripts were downregulated and mismatched oligonucleotides (Sporer et al., 2011a). and 32 transcripts were upregulated (Table 2). Among The microarray data were submitted to the NCBI GEO those transcripts, 2 corresponded to oligos annotated as with GEO accession number GSE36660. The oligos α sarcoglycan (SGCA), 3 oligos annotated as myosin were annotated using NCBI BLASTn (http://www. heavy chain isoform 2 (MYH2), 2 oligos annotated as ncbi.nlm.nih.gov). nebulin (NEB), 4 oligos annotated as myosin heavy chain isoform 1 (MYH1), 2 oligos annotated as (TTN), and 1 unknown oligo (Table 3). The top 10 Confirmation of Expression Patterns down- and upregulated genes (based on FC only) are Thirteen genes were selected for further analysis by shown in Table 4. Downregulated genes refer to the qPCR to confirm the microarray gene expression re- genes expressed lower in PSE relative to normal sam- sults. Of 13 genes, 12 genes were chosen based on their ples. Conversely, upregulated genes are those expressed large fold changes (FC, gene expression in PSE relative higher in PSE compared with normal samples. to normal sample) obtained from the microarray study. In addition, (PFN) was selected as it showed Confirmation of Microarray Results statistical significance in expression between normal by qPCR and PSE sample (estimated false discovery rates, FDR < 0.1). Confirmation of microarray analysis was performed Primers (Table 1) were designed using Primer Express by conducting qPCR analysis of 13 selected genes 3.0 software (Applied Biosystems) and synthesized by (Figure 1). The majority of the selected genes showed 1624 Malila et al. Table 1. Primer information for genes chosen for confirmation of expression using quantitative real-time PCR

Amplification Amplification length temperature Gene symbol Primer1 (bp) (°C) ACTB F: GTCCACCTTCCAGCAGATGTG 71 79 (β-actin) R: CAATGGAGGGTCCGGATTC ANKRD1 F: CGCCGATGCATGATGCT 56 81 R: AATCAAAAGCCGGACCATTTT ATP1B4 F: TACCCTGGAAACGGCACATT 70 80 R: TGTAGTTGACGTGCGTGAGCTT CA3 F: CAACCTGATGGTGTGGCTGTT 62 79 R: TCTGGTTTGGGAGTTTTTCCA CCDC135 F: ATTACCTGGCACCTTTTCTTATTCA 67 79 R: CGGAGGGCCTGCCTTTT COL6A1 F: TTCCATTGGTGCTCTTGCTATG 79 78 R: TTTGGGATGATGGCGATACC CST3 F: GTGATCTCCAGAGCTGCGAAT 67 80 R: ACAAAGGTGCATGTGGTATACTTAGC IGFN1 F: TCCCTGGTGATCTTTAGTGTTTCC 71 79 R: CCTGAATCATTGGTGGCTTCA MYH4 F: AGCAGGCATTCACCCAACA 104 82 R: GTCATGACGAGCAGACTGCAA MYOM1 F: TGAGCCAACTCCACAAGACAAA 100 76 R: ATCAAATGCTTGCCCAGAAAGA PDK4 F: ATGAATGTCTGTAATAGTGCTTGCAA 90 74 R: CATGTCTTCATTGTATGTTCTGCATATAC PFN2 F: CGGTCTTTCTGCCAGATCACT 65 82 R: CGTCCAAACAGCGGCTTT RGS2 F: AGGCTCCCAAGGAGATAAACATT 64 79 R: TCCTGGAGGTTCTGTGCTATCA TNNT3 F: CCCGTGCCTCAGTGATAACTAAA 68 78 R: AGAAGAAAAGCAGCAGCAATAGC 1F = forward; R = reverse. statistical significance in expression between normal sion regarding development of PSE turkey also altered and PSE turkey (P < 0.05) with a similar direction as pathways related to development of muscle and muscle observed in microarray analysis. Five genes including contraction. regulator of G-protein signaling 2 (RGS2), immuno- globulin-like and fibronectin type 3 domain contain- DISCUSSION ing 1 (IGFN1), cystatin C (CST3), type 3 (TNNT3), and myomesin 1 (MYOM1), did not Development of PSE in poultry poses one of the show statistically significant differential expression in greatest challenges to the meat processing industry the qPCR experiments. It should be noted that, among (Anthony, 1998; Petracci and Cavani, 2012; Samuel et those 5 genes, the FDR from the microarray study for al., 2012). The prevalence of PSE turkey meat can be RGS2, CST3, and TNNT3 exceeded 0.35. as high as 50% in commercial plants depending on the flock, time of the year, and other factors such as trans- Functional and Pathway Analysis Using IPA portation (Barbut, 1998; Woelfel et al., 2002). How- ever, even a small amount of PSE turkey entering the To perform pathway analysis, 2 parameters were processing line can have significant negative effects on used for gene selection from the microarray results. The quality of the final meat products. primary criterion was FC <−1.66 or FC >1.66. The second criterion was FDR <0.35. Using these criteria, expression data from 174 transcripts were uploaded to Table 2. Number of differentially expressed transcripts between IPA. According to the IPA knowledge base, 86 genes pale, soft, and exudative and normal turkey skeletal muscle at were recognized and mapped into canonical pathways different false discovery rate (FDR) significance levels (well-characterized cellular signaling pathways). Ca- Number of transcripts nonical pathways associated with development of PSE turkey are shown in Table 5. The calcium signaling FDR Downregulated Upregulated Total pathway (Figure 2) was the first pathway suggested by 0.05 10 19 29 the IPA and supports an association between develop- 0.10 17 32 49 2+ 0.15 32 43 75 ment of PSE meat and abnormal Ca homeostasis. 0.20 47 57 104 Ras homology family member A (RhoA) signaling and 0.25 74 71 145 actin cytoskeleton signaling (Figure 3) were also iden- 0.30 101 97 198 tified. As suggested by IPA, changes in gene expres- 0.35 131 113 244 GENE EXPRESSION IN PALE, SOFT, AND EXUDATIVE TURKEY 1625 Table 3. Differentially expressed transcripts between pale, soft, and exudative (PSE) and normal turkey skeletal muscle (false dis- covery rate < 0.1)

GenBank accession Gene symbol Gene name Fold change DQ993255 MHC Major histocompatibility complex, B −14.6 NM_001044651 ATP1B4 Adenosine triphosphatase (ATPase), (Na+)/K+ transporting, β 4 −12.0 polypeptide NM_204228.1 MYH21 Myosin, heavy chain 2, skeletal muscle −7.1 XM_419317.3 RIN2 Ras and Rab interactor 2 −4.2 XM_003209725 CCDC135 Coiled-coil domain containing 135 −3.6 NM_001024577 LINGO1 Leucine rich repeat and Ig domain containing 1 −2.7 XM_429975 STBD1 Starch binding domain 1 −2.4 NM_001242311 ATL2 Atlastin Guanosine triphosphatase 2 −2.3 XR_118375 ACSF3 Acyl-CoA synthetase family member 3 −2.3 XM_003203019 USP9X Ubiquitin specific peptidase 9, X-linked −2.1 NM_001006494 PSMC6 Proteasome (prosome, macropain) 26S subunit, ATPase, 6 −2.0 XM_423397 SELENBP1 Selenium binding protein 1 −2.0 NM_001079760 PFN2 Profilin 2 −2.0 NM_001006260 EIF2S3 Eukaryotic translation initiation factor 2, subunit 3 gamma −1.9 XM_003207146 HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2/B1 −1.9 XM_003209920 VPS4 Vacuolar protein sorting-associated protein 4A −1.8 XM_003213364 AQP3 Aquaporin-3 1.8 NM_205177 CTSD Cathepsin D 1.9 XM_003643520 SGCA2 Alpha sarcoglycan 1.9 NM_001030899 THRAP3 Thyroid hormone receptor associated protein 3 2.0 XM_003643520 SGCA2 Alpha sarcoglycan 2.4 AJ419877 18S rRNA gene 2.4 XR_118187 PHKA1 Phosphorylase b kinase regulatory subunit α, skeletal muscle isoform 2.5 XM_003203043 RPS6KA3 Ribosomal protein S6 kinase, 90 kDa, polypeptide 3 2.5 XM_003207155 TAX1BP1 Human T-cell leukemia virus type I (Tax 1)-binding protein 1 2.6 NM_205096 HDLBP High density lipoprotein binding protein (vigilin) 2.8 XM_415578 MYH21 Myosin, heavy chain 2, skeletal muscle, adult 2.8 BC067379 Negative cofactor 2, alpha 3.2 AB024330 NEB3 Nebulin 3.3 NM_001013396 MYH14 Myosin heavy chain isoform 1, skeletal muscle, adult 3.7 XM_003204036 ACTA1 Actin, α skeletal muscle 3.7 NM_001012945 DNAJA1 Heat shock protein 40 kDa homolog, subfamily A, member 1 3.8 XM_001236426 HDGF Hepatoma-derived growth factor 3.8 XM_415578 MYH21 Myosin, heavy chain 2, skeletal muscle 3.9 NM_204289 HSP90B1 Heat shock protein 90 kDa β, member 1 3.9 XM_420181 PRPS1 Phosphoribosyl pyrophosphate synthetase 1 3.9 NM_001031489 HBE1 Hemoglobin subunit β 4.1 XR_118264 TTN5 Titin 4.2 NM_001013396 MYH14 Myosin heavy chain isoform 1, skeletal muscle, adult 4.2 DQ018757 Clone AY006 28S ribosomal RNA gene, partial sequence 4.9 NM_001013396 MYH14 Myosin heavy chain isoform 1, skeletal muscle, adult 5.0 AB024330 NEB3 Nebulin 5.8 NM_204959 MYOM1 Myomesin 1 7.4 FM165415 28S rRNA gene, clone GgLSU-1 7.4 XR_118264 TTN5 Titin 8.0 NM_001013396 MYH14 Myosin heavy chain isoform 1 8.1 XM_418319 CA3 Carbonic anhydrase 3 14.5 EF153719 Mitochondrion, complete genome 18.9 Unknown −2.1 1Different oligos on the microarray but were annotated as myosin heavy chain isoform 2. 2Different oligos on the microarray but were annotated as sarcoglycan. 3Different oligos on the microarray but were annotated as nebulin. 4Different oligos on the microarray but were annotated as myosin heavy chain isoform 1. 5Different oligos on the microarray but were annotated as titin.

Several investigators have attempted to define PSE of carcass color for on-line sorting of PSE from normal meat by using objective measurements to establish a meat is problematic for the meat industry. cut-off value that would separate PSE meat from nor- The overall goal of this study is to gain a greater mal (Barbut, 1998; Garcia et al., 2010; Eadmusik et mechanistic understanding of the development of PSE al., 2011). One of the indicators frequently studied is in turkey so that new strategies can be developed to carcass color because this characteristic can be mea- identify PSE-susceptible animals in the breeding popu- sured rapidly and is amendable to use on the process- lation and thus reduce the number of PSE birds enter- ing line. However, this indicator is weakly correlated ing the processing line. Turkeys from the RBC2 line, with percent marinade uptake (Chiang et al., 2008) and developed without selection pressure, were chosen for water-holding capacity (Samuel et al., 2012). Thus, use this study. Analysis of normal and PSE breast samples 1626 Malila et al. Table 4. Top 10 downregulated and top 10 upregulated genes [based on fold change (FC) alone] in pale, soft, and exudative relative to normal turkey skeletal muscle revealed by the turkey skeletal muscle long oligonucleotide microarray

Gene symbol Gene name FC FDR1 Downregulated gene MYH4 Myosin, heavy chain 4, skeletal muscle −26.2 0.11 PDK4 Pyruvate dehydrogenase kinase, isozyme 4 −25.9 0.33 MB Myoglobin −13.8 0.25 ATP1B4 Adenosine triphosphatase, Na+/K+ transporting, β 4 polypeptide −12.0 0.04 ANKRD1 repeat domain 1 (cardiac muscle) −6.8 0.15 RSG2 Regulator of G-protein signaling 2, 24 kDa −4.7 0.41 IGFN1 Immunoglobulin-like and fibronectin type 3 domain containing 1 −4.2 0.14 TNNT3 Troponin T type 3 (skeletal, fast) −3.8 0.41 CCDC135 Coiled-coil domain containing 135 −3.6 0.04 DNAJA4 Heat shock protein 40 kDa homolog, subfamily A, member 4 −3.0 0.23 Upregulated gene CA3 Carbonic anhydrase III, muscle specific 14.6 0.06 MYH12 Myosin, heavy chain 1, skeletal muscle, adult 8.1 0.01 TTN Titin 8.0 0.08 MYOM1 Myomesin 1 7.4 0.02 TNNC1 type 1 (skeletal muscle, slow) 7.0 0.34 NEB Nebulin 5.8 0.04 COL6A1 Collagen, type VI, α 1 5.7 0.25 CST3 Cystatin C 4.3 0.37 MYH12 Myosin, heavy chain 1, skeletal muscle, adult 4.2 <0.01 HBB Hemoglobin, β 4.1 0.02 1FDR = false discovery rate. 2Different oligos on the microarray but were annotated as myosin heavy chain isoform 1. from this line will serve as a basis for comparison with ing pathway analysis. Using these expanded criteria, growth-selected turkey lines in future studies. changes in gene expression for several of the selected In previous studies, delayed upregulation of Ca2+ genes, including MYH4 and PDK4, were confirmed by regulatory proteins was observed in PSE turkey sam- qPCR (P < 0.05). ples upon heat stress treatment (Sporer et al., 2012). Pathway analysis revealed that several cellular sig- The results suggested a greater complexity of the de- naling pathways are associated with the development velopment of the PSE meat defect than a single gene of PSE turkey meat, with numerous genes associated mutation. It is possible that additional genes may be with more than one pathway. For example, MYH4 was involved that have not yet been identified. Unlike the mapped into calcium signaling, actin cytoskeleton sig- study of individual gene expression, the microarray naling, and protein kinase A signaling pathways. This technique enables simultaneous analysis of thousands result indicates interactions among molecular pathways of genes with the capability of revealing differential ex- associated with development of this meat defect. The pression of unidentified genes and interaction among potential roles of the calcium signaling, RhoA signal- genes. ing, and actin cytoskeleton signaling pathways in devel- In this study, the 6K TSKMLO microarray was used opment of PSE meat are discussed below. to identify relative transcript abundance between nor- mal and PSE turkey meat samples. Considering the top Calcium Signaling Pathway 10 down- and upregulated genes, several genes displayed large FC but were not significantly different based on The calcium signaling pathway (Figure 2) was the statistical criteria (FDR <0.1); examples include myo- top pathway highlighted by the IPA. This pathway is sin heavy chain isoform 4 (MYH4, FC = −26.2, FDR of particular interest because several previous studies = 0.11) and pyruvate dehydrogenase kinase, isozyme 4 have implicated abnormal Ca2+ homeostasis in the de- (PDK4, FC = −25.9, FDR = 0.33). velopment of PSE meat. In pigs, it is widely accepted The MicroArray Quality Control project suggested that development of PSE pork is associated with a sin- a rationale for gene selection (Shi et al., 2008). After gle point mutation in RYR1, which results in abnormal completing statistical analysis of numerous platforms, Ca2+ homeostasis in skeletal muscle of MH-susceptible this group found that selection of genes based on a com- pigs (Mickelson et al., 1988; Fujii et al., 1991; Otsu et bination FC ranking and a less stringent P threshold al., 1994). The rate of Ca2+ release from SR in stress- improves reproducibility and specificity of microarray susceptible pigs is about 2 times greater than that of analyses. The more stringent the P cutoff used for gene normal pigs (Mickelson and Louis, 1996). The high lev- selection, the less reproducible the list of differentially el of sarcoplasmic Ca2+ postmortem activates muscle expressed genes (Shi et al., 2008). Based on this proj- hypermetabolism and accelerates pH decline. With the ect, a FC cutoff (FC <−1.66 or FC >1.66) with a less combination of high acidity and high carcass tempera- stringent FDR (FDR <0.35) was used in the present ture in the initial postmortem phase, proteins undergo study to identify genes for more confidently perform- denaturation, causing the PSE meat defect. GENE EXPRESSION IN PALE, SOFT, AND EXUDATIVE TURKEY 1627

Figure 1. Confirmation of gene expression analyzed by microarray using quantitative real-time PCR (qPCR). Results are presented as rela- tive expression or fold change for gene expression in pale, soft, and exudative (PSE) relative to normal samples. Bars below the origin indicate lower expression (downregulation) of the gene in PSE samples; bars above the origin indicate higher expression (upregulation) in PSE samples. Statistical significance indicates change in expression between PSE and normal samples within each technique (†FDR <0.1 for microarray, *P < 0.05 for qPCR).

Previously, Sporer et al. (2012) found a delay in of ATP2A1 results in a reduction of SERCA1 activ- upregulation of αRYR and βRYR expression in PSE ity and is associated with an exercise-induced impair- turkey compared with normal samples when the birds ment of skeletal muscle relaxation and severe cramps in underwent heat stress. However, in the current study, humans (Odermatt et al., 2000) and cattle (Sacchetto there was no evidence of differential gene expression of et al., 2009). In this study, lower ATP2A1 expression RYR, either α- or β- isoform, between normal and PSE was found in PSE samples (FC = −2.0). This may im- turkey meat when the birds were not undergoing heat ply an overload of intracellular Ca2+ causing a severe stress. This discrepancy may be due to the fact that the muscle contraction in the susceptible birds. It should fold-change differences observed by qPCR were modest be noted that expression of the gene encoding SERCA1 and thus not determined to be significant by the mi- in PSE turkey remained unchanged with the onset of croarray method. These results agree with the study of heat stress (Sporer et al., 2012); however, differential Oda et al. (2009) who observed an unchanged expres- expression of this gene between normal and PSE turkey sion of broiler αRYR, although they found decreased without heat stress treatment was not determined in expression of βRYR. This may be due to the biological the previous study. differences among species. Pathway analysis clustered genes encoding myofi- Apart from RYR, the intracellular [Ca2+] is also regu- brillar proteins within the calcium signaling pathway. lated by the SERCA, the SR-Ca2+ pump. The SERCA Expression of MYH4 encoding myosin heavy chain isoform 1 is expressed exclusively in fast-twitch skeletal (MHC) isoform IIb (Tonge et al., 2010) dramatically muscle and is encoded by the ATPase, Ca2+-transport- decreased in PSE samples (FC = −26.1). In contrast, ing gene (ATP2A1; Lytton et al., 1992). Mutation α actin (ACTA1) was upregulated (FC = 3.7). Myo- 1628 Malila et al. Table 5. Top canonical pathways associated with development of pale, soft, and exudative turkey

Canonical pathway Gene symbol FC1 FDR2 1. Calcium signaling MYH4 −26.1 0.11 ATP2A1 −2.0 0.25 TNNI2 2.9 0.16 ACTA1 3.7 0.08 TNNC1 7.0 0.34 2. Ras homology family member A signaling MYLK2 −2.1 0.28 PFN2 −2.0 0.02 IGF1 2.0 0.28 ACTA1 3.7 0.08 3. Hypoxia signaling in the cardiovascular system P4HB 1.8 0.28 UBE2D2 2.0 0.17 HSP90B1 3.9 0.08 4. Caveolar-mediated endocytosis signaling FLNC −2.0 0.25 ITGA7 −1.7 0.25 ACTA1 3.7 0.08 5. Protein ubiquitination pathway USP9X −2.1 0.09 PSMC6 −2.0 0.02 UBE2D2 2.0 0.17 DNAJA1 3.8 0.02 HSP90B1 3.9 0.08 6. Nuclear factor-erythroid 2-related factor 2- mediated DNAJA4 −2.9 0.23 oxidative stress response AOX1 −1.9 0.25 DNAJA1 3.8 0.02 ACTA1 3.7 0.08 7. Integrin signaling MYLK2 −2.1 0.28 ITGA7 −1.9 0.25 CAPN3 2.1 0.18 ACTA1 3.7 0.08 8. Actin cytoskeleton signaling MYH4 −26.2 0.11 MYLK2 −2.1 0.28 PFN2 −2.0 0.02 ACTA1 3.7 0.08 1FC = fold change. 2FDR = false discovery rate. sin and actin are major contractile proteins in skeletal Upregulation of slow-muscle troponin C (TNNC1, muscle, which constitute approximately 45 and 20% FC = 7.0) corresponds to a shift of myosin isoforms, of myofibrillar proteins, respectively (Aberle et al., supporting the hypothesis of the fast-to-slow muscle 2001). By regulating myofibrillar assembly, changes in type conversion. Biological properties of slow-muscle expression of genes encoding myosin and actin may af- troponin C, including Ca2+ binding, the Ca2+-bound fect myofibril accumulation and stability (Wells et al., conformation, and interaction with differ 1996) and potentially lead to irregular organization of from that of the fast muscle isoform (Sia et al., 1997). muscle fibers in PSE meat as previously found in PSE In addition, fast-twitch troponin I (TNNI2, FC = 2.9) pork (Laville et al., 2005; Obi et al., 2010) and PSE was upregulated in PSE samples. The differential ex- chicken (Wilhelm et al., 2010). Interestingly, whereas pression of the regulatory proteins in turkey skeletal MYH4 (which encodes fast-twitch glycolytic MHCIIb) muscle may result in change in the ratio of regulatory was downregulated, MYH2 (which encodes fast-twitch proteins after protein translation. Together, changes in oxidative glycolytic MHCIIa) and MYH1 (intermedi- expression of myofibrillar proteins can directly and in- ate between type IIa and IIb MHCIIx) showed upregu- directly alter interactions among the proteins and their lation. Because turkey breast muscle mainly consists response to Ca2+ flux, with the net effect being altered of fast-twitch glycolytic muscle fibers (Rosser et al., meat quality. 1996), we hypothesized that the change in transcript abundance of MYH gene family members in the PSE RhoA Signaling and Actin Cytoskeleton turkey may cause fast-to-slow muscle transformation Signaling Pathway that is associated with various functional changes at the muscle cell level as well as cytosol-regulating Ca2+ In this study, an alteration of pathways involved in dynamics (Kaprielian et al., 1991; Jakubiec-Puka et al., regulation of arrangement of actin and actomyosin in 1999; Pette and Vrbová, 1999; Pette and Staron, 2001; PSE turkey meat was identified. These pathways in- Tonge et al., 2010). However, previous reports on the clude RhoA signaling and actin cytoskeleton signaling relative proportion of myosin heavy-chain isoforms in (Figure 3). RhoA is a subtype of the Ras superfamily, PSE meat have not been consistent (Ryu and Kim, a low-molecular-weight phosphoprotein family of GT- 2006; Franck et al., 2007; Golding-Myers et al., 2010). Pases (McClung et al., 2004) that links extracellular GENE EXPRESSION IN PALE, SOFT, AND EXUDATIVE TURKEY 1629

Figure 2. Schematic diagram of the calcium signaling pathway associated with development of pale, soft, and exudative (PSE) turkey meat. The pathway, suggested by the Ingenuity Pathway Analysis, showed direct interaction among differentially expressed genes associated with regulation of Ca2+ concentration between normal and PSE turkey skeletal muscle. FC means fold change or gene expression in PSE relative to normal samples. Negative fold change indicates downregulation of gene in PSE samples, whereas a positive number indicates upregulation in PSE samples. SERCA = sarco/endoplasmic reticulum Ca2+-ATPase; CASQ = calsequestrin; TRDN = triadin; ASPH = aspartate beta-hydroxylase; RYR = . growth signals or intracellular stimuli to the assembly accommodate changes in cell shape during muscle con- and organization of the actin cytoskeleton (Schmidt traction. The cytoskeleton is also intimately involved and Hall, 1998). in other cellular functions including cell division and Insulin-like growth hormone 1 (IGF-1) was mapped transmembrane signaling (Schmidt and Hall, 1998). by IPA into the RhoA signaling pathway. This protein, Organization of the actin cytoskeleton is determined structurally similar to insulin, acts via either autocrine by the turnover of actin filaments and actin-binding or paracrine mechanisms (McMurtry et al., 1997) and proteins, which are modulated by either internal or regulates tissue growth and development in various ver- environmental signals (Sheterline and Sparrow, 1994). tebrates (Jones and Clemmons, 1995), including tur- One of the important actin-binding proteins is profilin keys (Bacon et al., 1993; Richards et al., 2005). Infusion encoded by PFN2. Profilin catalyzes ATP/ADP ex- of IGF-1 into chicken has been shown to affect protein change of actin monomers and inhibits the hydrolysis of synthesis (Conlon and Kita, 2002) and mediate pro- ATP bound to monomeric actin, thereby maintaining tein degradation (Czerwinski et al., 1998; Tomas et al., concentration of readily polymerizing ATP-bound actin 1998). Upregulation of IGF-1 in PSE turkey (FC = 2.0) (Sohn and Goldschmidt-Clermont, 1994; dos Remedios may imply changes in protein turnover, including actin, et al., 2003). The protein also promotes formation of thus affecting downstream actin cytoskeleton function actin filaments by transporting the monomer to the in the defective meat. growing end of the filament (dos Remedios et al., 2003). The actin cytoskeleton is a polymer of actin mono- In this study, transcript abundance of PFN2 was lower mers assembled together via condensation reaction in PSE samples (FC = −2.0), which may affect the (Schmidt and Hall, 1998). The primary function of the concentration of ATP-bound actin monomers and alter actin cytoskeleton in skeletal muscle is to tether struc- arrangement of actin filaments. tural components and maintain overall structural order Actin cytoskeleton can be formed along myosin fila- of those components inside the cell, but in contrast to ments and generate force in the appropriate direction myofibrillar actin, the cytoskeleton is not directly in- based on polarity of actin filaments. Myosin-based mo- volved in muscle contraction (Stromer, 1998). The ac- tility of actin cytoskeleton accounts for muscle contrac- tin cytoskeleton contributes strength and flexibility to tion (when it interacts with muscle myosin) or cellular 1630 Malila et al.

Figure 3. Schematic diagram of Ras homology family member A (RhoA) and actin cytoskeleton signaling pathways associated with develop- ment of pale, soft, and exudative (PSE) turkey meat. The pathway, suggested by Ingenuity Pathway Analysis, shows direct interaction among differentially expressed genes associated with polymerization of actin filament and formation of actomyosin complex between normal and PSE turkey skeletal muscle. FC means fold change or gene expression in PSE relative to normal samples. Negative fold change indicates downregu- lation of gene in PSE samples, whereas a positive number indicates upregulation in PSE samples. ACTN = alpha ; DIAPH = lactate dehydrogenase-B; IGF1 = insulin-like growth factor hormone I; IGF1R = insulin-like growth factor hormone I receptor; LARG = Rho guanine nucleotide exchange factor; MLCP = kinase phosphatase; MYL = myosin light chain; MYLK = myosin light chain kinase; PFN = profilin; PI4P5K = phosphatidylinositol-4-phosphate 5-kinase; PIP = phosphatidylinositol 4-monophosphate; PIP2 = 1-phosphatidyl-1D-myo- inositol 4,5-bisphosphate; ROCK = Rho-associated, coiled-coil containing protein kinase 1. morphogenic movement (when it interacts with non- et al. (2005), knockout of the MYLK2 gene decreased muscle myosin; Sheterline and Sparrow, 1994). Thus, RLC phosphorylation in mouse skeletal muscle. Thus, altered assembly of actomyosin may affect cell motility. change in expression of MYLK2 (FC = −2.1) in PSE In this study, changes in expression of the gene en- meat suggests an alteration of stability of myosin mol- coding myosin light chain kinase (MYLK) may alter ecules and the actomyosin complex. actomyosin assembly. The MYLK is a Ca2+/calmod- ulin-dependent enzyme (Park et al., 2011) that phos- Postmortem Oxidative Metabolism phorylates the myosin regulatory light chain (RLC). When phosphorylated, the RLC mediates Ca2+ sen- and PDK4 sitivity in and promotes movement of The PDK4 gene was substantially downregulated in myosin cross bridges away from the thick filament sur- PSE meat (FC = −25.9). This gene was not mapped by face toward actin filaments (Stull et al., 2011) and af- IPA into a specific canonical pathway. However, consid- fects skeletal muscle twitch potentiation (Manning and ering its function, PDK4 may be one of the key play- Stull, 1982). The MYLK enzyme encoded by the gene ers regarding development of PSE turkey meat. This MYLK2 is expressed predominantly in fast-twitch skel- gene encodes the PDK4 enzyme, one of the regulators etal muscle fibers (Zhi et al., 2005). In the study of Zhi of oxidative metabolism in the mitochondria. The en- GENE EXPRESSION IN PALE, SOFT, AND EXUDATIVE TURKEY 1631 zyme inhibits conversion of pyruvate into acetyl CoA In a study on differential expression in chickens dif- by phosphorylating pyruvate dehydrogenase (Wynn et fering in glycogen content, Sibut et al. (2011) observed al., 2008). The dramatic downregulation of the PDK4 downregulation of PDK4 in high-glycogen chicken skel- gene suggests altered oxidative metabolism in PSE etal muscle compared with low-glycogen samples. They turkey meat. It can be hypothesized that, for normal did not find differences in direct measures of meat meat, a small amount of oxygen is still present in the quality such as drip loss, but the high-glycogen meat muscle cells at the early stage of postmortem muscle, showed lower ultimate pH and lighter color (Sibut et enabling glucose to undergo oxidative metabolism. al., 2011), determinants often used as PSE indicators in When all of the oxygen is consumed, the metabolic previous studies (Oda et al., 2009; Ziober et al., 2010). pathway switches to anaerobic metabolism to generate However, although it is of interest that differential ATP for cellular activities. Lactic acid is produced and PDK4 expression was observed in the study of Sibut accumulated, resulting in a pH drop. However, due to et al. (2011), their results are not directly comparable drastically lowered expression of the PDK4 gene, the with our study. They classified chicken muscle samples initial conversion of pyruvate to acetyl CoA and rate based on glycogen content to determine the molecular of oxygen consumption may be greater in PSE meat. mechanisms involved in variation of meat quality. In The rate of metabolic switch from aerobic to anaerobic the current study, samples were first classified as PSE may be faster, resulting in a rapid pH drop that causes or normal and then analyzed for differential gene ex- protein denaturation in the defective turkey meat. This pression. hypothesis is supported by a lower pH at 15 min post- In conclusion, this study is the first evidence of mortem in PSE turkey (Chiang et al., 2008). global differential gene expression between normal and Eadmusik et al. (2011) used 20-min postmortem pH PSE turkey. Pathway analysis shows several molecu- to classify turkey breast samples as fast-glycolysing or lar signaling pathways associated with development of normal-glycolysing muscle. Samples from the rapid- the turkey meat defect including the calcium signal- glycolysing group had reduced solubility, which is an ing pathway that supports an abnormality of Ca2+ ho- indicator of protein denaturation, but the ultimate pH meostasis in susceptible birds. The results also suggest was not significantly different from that of samples skeletal muscle fast-to-slow isoform switch, which may with a normal rate of glycolysis. Their results support reflect different molecular properties and disorganiza- our hypothesis that although the magnitude of ultimate tion of muscle fibers in PSE turkey. Altered stability of pH may influence meat quality as previously indicated nonmuscle actin polymer and actomyosin assembly in (Barbut, 1993; Fernandez et al., 2002), a rapid rate of PSE turkey is implicated due to differential expression postmortem pH decline, due to decreased expression of of genes in the RhoA signaling and actin cytoskeleton PDK4, combined with high early-postmortem carcass signaling pathway. Clearly, development of this turkey temperature, may have a greater impact on protein de- meat defect is complex and associated with interac- naturation associated with development of PSE turkey. tions of more than one pathway. Here, evidence was Differential expression of PDK4 has been observed found of dramatically lower expression of the PDK4 in previous studies on meat quality. Lan et al. (2009) gene in PSE turkey meat, which may alter oxidative reported reduced mRNA abundance of PDK4 in mus- metabolism and be associated with an unusually high cle from commercial lean-bred Yorkshire pigs compared rate of postmortem metabolism in PSE turkey. Studies with a Chinese traditional breed. The authors claimed are in progress to quantify protein levels and enzymatic that the traditional breed has superior meat quality activity in PSE and normal muscles, and to investigate compared with the Yorkshire breed, but they did not the mechanisms regulating expression of PDK4 to bet- directly measure meat quality indices between the 2 ter understand the role of this protein in turkey meat breeds. They suggested that PDK4 expression is cor- quality. related with fiber type (i.e., increased levels of PDK4 would be associated with a higher percentage of slow- twitch fibers). In our study, we observed upregulation ACKNOWLEDGMENTS of several genes in PSE turkey that would suggest a shift in expression from fast-twitch muscle proteins to The authors gratefully acknowledge the MSU Meat slow-twitch muscle proteins (Tables 3 and 4). Slow- Laboratory Team (East Lansing, MI) for their assis- twitch muscle generally has a greater degree of oxida- tance in harvesting of birds and sample collection, and tive metabolism (Donoghue et al., 2007). Our results Hui-Ren Zhou (MSU, East Lansing, MI) for her techni- are further consistent with this transition in that the cal assistance during the RNA isolation process. This mitochondrial genome was upregulated in the defective project was supported by National Research Initia- meat (Table 3). We would expect to see increased ex- tive Grant no. 2005-35503-16348 and Grant no. 2005- pression of PDK4 in association with increased expres- 35604-15628 from the USDA National Institute of Food sion of oxidative-metabolism genes. However, the fact and Agriculture (Washington, DC). Yuwares Malila is that PDK4 was downregulated in PSE muscle suggests grateful for the financial support from the Royal Thai that in this subset of birds, there is impaired regulation Government Scholarship, Ministry of Science and Tech- of expression of this gene. nology (Bangkok, Thailand). 1632 Malila et al. REFERENCES Jones, J. I., and D. R. Clemmons. 1995. Insulin-like growth fac- tors and their binding proteins: Biological actions. Endocr. Rev. 16:3–34. Aberle, E. D., J. C. Forrest, D. E. Gerrard, and E. W. Mills. 2001. Kaprielian, Z., E. Bandman, and D. M. Fambrough. 1991. Expres- Structure and composition of animal tissues. Pages 2–43 in Prin- sion of Ca2+-ATPase isoforms in denervated, regenerating, and ciples of Meat Science. 4th ed. Kendall/Hunt Publishing Com- dystrophic chicken skeletal-muscle. Dev. Biol. 144:199–211. pany, Dubuque, IA. Lan, J., M. G. Lei, Y. B. Zhang, J. H. Wang, X. T. Feng, D. Q. Xu, Anthony, N. B. 1998. A review of genetic practices in poultry: Ef- J. F. Gui, and Y. Z. Xiong. 2009. Characterization of the porcine forts to improve meat quality. J. Muscle Foods 9:25–33. differentially expressed PDK4 gene and association with meat Bacon, W. L., K. E. Nestor, D. A. Emmerson, R. Vasilatos-Younken, quality. Mol. Biol. Rep. 36:2003–2010. and D. W. Long. 1993. Circulating IGF-I in plasma of grow- Laville, E., T. Sayd, V. Sante-Lhoutellier, M. Morzel, R. Labas, M. ing male and female turkeys of medium and heavy weight lines. Franck, C. Chambon, and G. Monin. 2005. Characterisation of Domest. Anim. Endocrinol. 10:267–277. PSE zones in semimembranosus pig muscle. Meat Sci. 70:167– Barbut, S. 1993. Color measurements for evaluating the pale soft 172. exudative (PSE) occurrence in turkey meat. Food Res. Int. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene 26:39–43. expression data using real-time quantitative PCR and the 2(T) Barbut, S. 1998. Estimating the magnitude of the PSE problem in (-Delta Delta C) method. Methods 25:402–408. poultry. J. Muscle Foods 9:35–49. Lytton, J., M. Westlin, S. E. Burk, G. E. Shull, and D. H. MacLen- Barbut, S., A. A. Sosnicki, S. M. Lonergan, T. Knapp, D. C. Cio- nan. 1992. Functional comparisons between isoforms of the sar- banu, L. J. Gatcliffe, E. Huff-Lonergan, and E. W. Wilson. 2008. coplasmic or endoplasmic-reticulum family of calcium pumps. J. Progress in reducing the pale, soft and exudative (PSE) problem Biol. Chem. 267:14483–14489. in pork and poultry meat. Meat Sci. 79:46–63. Manning, D. R., and J. T. Stull. 1982. Myosin light chain phosphor- Chiang, W., C. P. Allison, J. E. Linz, and G. M. Strasburg. 2004. ylation-dephosphorylation in mammalian skeletal-muscle. Am. Identification of two alpha RYR alleles and characterization of J. Physiol. 242:C234–C241. alpha RYR transcript variants in turkey skeletal muscle. Gene McClung, J. M., R. W. Thompson, L. L. Lowe, and J. A. Carson. 330:177–184. 2004. RhoA expression during recovery from skeletal muscle dis- Chiang, W., A. Booren, and G. Strasburg. 2008. The effect of heat use. J. Appl. Physiol. 96:1341–1348. stress on thyroid hormone response and meat quality in turkeys McMurtry, J. P., G. L. Francis, and Z. Upton. 1997. Insulin-like of two genetic lines. Meat Sci. 80:615–622. growth factors in poultry. Domest. Anim. Endocrinol. 14:199– Conlon, M. A., and K. Kita. 2002. Muscle protein synthesis rate is 229. altered in response to a single injection of insulin-like growth fac- Mickelson, J. R., K. M. Johnson, L. A. Litterer, and C. F. Louis. tor-I in seven-day-old Leghorn chicks. Poult. Sci. 81:1543–1547. 1988. Abnormal ryanodine receptor properties correlate with al- Czerwinski, S. M., J. M. Cate, G. Francis, F. Tomas, D. M. Brocht, tered calcium release of sarcoplasmic-reticulum from malignant and J. P. McMurtry. 1998. The effect of insulin-like growth fac- hyperthermia susceptible pigs. Biophys. J. 53:A336–A336. tor-I (IGF-I) on protein turnover in the meat-type chicken (Gal- Mickelson, J. R., and C. F. Louis. 1996. Malignant hyperthermia: lus domesticus). Comp. Biochem. Physiol. C Pharmacol. Toxicol. Excitation-contraction coupling, Ca2+ release channel, and cell Endocrinol. 119:75–80. Ca2+ regulation defects. Physiol. Rev. 76:537–592. Donoghue, P., P. Doran, K. Wynne, K. Pedersen, M. J. Dunn, and Nestor, K. E. 1977. The use of a paired mating system for the K. Ohlendieck. 2007. Proteomic profiling of chronic low-frequen- maintenance of experimental populations of turkeys. Poult. Sci. cy stimulated fast muscle. Proteomics 7:3417–3430. 56:60–65. dos Remedios, C. G., D. Chhabra, M. Kekic, I. V. Dedova, M. Nestor, K. E., M. G. McCartney, and W. R. Harvey. 1967. Genetics Tsubakihara, D. A. Berry, and N. J. Nosworthy. 2003. Actin of growth and reproduction in the turkey. 1. Genetic and nonge- binding proteins: Regulation of cytoskeletal . netic variation in body weight and body measurements. Poult. Physiol. Rev. 83:433–473. Sci. 46:1374–1384. Dransfield, E., and A. A. Sosnicki. 1999. Relationship between mus- Nierobisz, L. S., K. R. B. Sporer, G. M. Strasburg, K. M. Reed, S. G. cle growth and poultry meat quality. Poult. Sci. 78:743–746. Velleman, C. M. Ashwell, J. V. Felts, and P. E. Mozdziak. 2012. Eadmusik, S., C. Molette, X. Fernandez, and H. Remignon. 2011. Differential expression of genes characterizing myofibre pheno- Are one early muscle pH and one early temperature measurement type. Anim. Genet. 43:298–308. sufficient to detect PSE breast meat in turkeys? Br. Poult. Sci. Obi, T., M. Matsumoto, K. Miyazaki, K. Kitsutaka, M. Tamaki, K. 52:177–188. Takase, A. Miyamoto, T. Oka, Y. Kawamoto, and T. Nakada. Fernandez, X., V. Sante, E. Baeza, E. Lebihan-Duval, C. Berri, H. 2010. Skeletal ryanodine receptor 1-heterozygous PSE (pale, soft Remignon, R. Babile, G. Le Pottier, and T. Astruc. 2002. Effects and exudative) meat contains a higher concentration of myoglo- of the rate of muscle post mortem pH fall on the technological bin than genetically normal PSE meat in pigs. Asian-australas. quality of turkey meat. Br. Poult. Sci. 43:245–252. J. Anim. Sci. 23:1244-1249. Franck, M., P. Figwer, C. Godfraind, M. T. Poirel, A. Khazzaha, Oda, S. H. I., A. L. Nepomuceno, M. C. Ledur, M. C. N. de Oliveira, and M. M. Ruchoux. 2007. Could the pale, soft, and exudative S. R. R. Marin, E. I. Ida, and M. Shimokomaki. 2009. Quantita- condition be explained by distinctive histological characteristics? tive differential expression of alpha and beta ryanodine receptor J. Anim. Sci. 85:746–753. genes in PSE (pale, soft, exudative) meat from two chicken lines: Fujii, J., K. Otsu, F. Zorzato, S. Deleon, V. K. Khanna, J. E. Weiler, Broiler and layer. Brazilian Archives of Biology and Technology P. J. O’Brien, and D. H. MacLennan. 1991. Identification of a 52:1519–1525. mutation in porcine ryanodine receptor associated with malig- Odermatt, A., K. Barton, V. K. Khanna, J. Mathieu, D. Escolar, T. nant hyperthermia. Science 253:448–451. Kuntzer, G. Karpati, and D. H. MacLennan. 2000. The mutation Garcia, R. G., L. W. de Freitas, A. W. Schwingel, R. M. Farias, of Pro(789) to Leu reduces the activity of the fast-twitch skeletal F. R. Caldara, A. M. A. Gabriel, J. D. Graciano, C. M. Komi- muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) yama, and I. Paz. 2010. Incidence and physical properties of PSE and is associated with Brody disease. Hum. Genet. 106:482–491. chicken meat in a commercial processing plant. Braz. J. Poult. Otsu, K., K. Nishida, Y. Kimura, T. Kuzuya, M. Hori, T. Kamada, Sci. 12:233–237. and M. Tada. 1994. The point mutation Arg615 -> Cys in the Golding-Myers, J. D., C. D. Showers, P. J. Shand, and B. W. C. Ca2+ release channel of skeletal sarcoplasmic reticulum is re- Rosser. 2010. Muscle fiber type and the occurrence of pale, soft sponsible for hypersensitivity to caffeine and halothane in malig- exudative pork. J. Muscle Foods 21:484–498. nant hyperthermia. J. Biol. Chem. 269:9413–9415. Jakubiec-Puka, A., I. Ciechomska, J. Morga, and A. Matusiak. 1999. Owens, C. M., C. Z. Alvarado, and A. R. Sams. 2009. Research Contents of myosin heavy chains in denervated slow and fast rat developments in pale, soft, and exudative turkey meat in North leg muscles. Comp. Biochem. Physiol. B 122:355–362. America. Poult. Sci. 88:1513–1517. GENE EXPRESSION IN PALE, SOFT, AND EXUDATIVE TURKEY 1633

Owens, C. M., E. M. Hirschler, S. R. McKee, R. Martinez-Dawson, Smyth, G. K. 2005. Limma: Linear models for microarray data. and A. R. Sams. 2000. The characterization and incidence of Pages 397–420 in Bioinformatics and Computational Biology So- pale, soft, exudative turkey meat in a commercial plant. Poult. lutions using R and Bioconductor. R. Gentleman, V. Carey, S. Sci. 79:553–558. Dudoit, R. Irizarry, and W. Huber, ed. Springer, New York, NY. Park, I., C. Han, S. Jin, B. Lee, H. Choi, J. T. Kwon, D. Kim, J. Sohn, R. H., and P. J. Goldschmidt-Clermont. 1994. Profilin—At Kim, E. Lifirsu, W. J. Park, Z. Y. Park, D. H. Kim, and C. Cho. the crossroads of signal-transduction and the actin cytoskeleton. 2011. Myosin regulatory light chains are required to maintain Bioessays 16:465–472. the stability of myosin II and cellular integrity. Biochem. J. Sporer, K. R. B., W. Chiang, R. J. Tempelman, C. W. Ernst, K. M. 434:171–180. Reed, S. G. Velleman, and G. M. Strasburg. 2011a. Characteriza- Petracci, M., and C. Cavani. 2012. Muscle growth and poultry meat tion of a 6K oligonucleotide turkey skeletal muscle microarray. quality issues. Nutrients 4:1–12. Anim. Genet. 42:75–82. Pette, D., and R. S. Staron. 2001. Transitions of muscle fiber pheno- Sporer, K. R. B., R. J. Tempelman, C. W. Ernst, K. M. Reed, typic profiles. Histochem. Cell Biol. 115:359–372. S. G. Velleman, and G. M. Strasburg. 2011b. Transcriptional Pette, D., and G. Vrbová. 1999. What does chronic electrical stimula- profiling identifies differentially expressed genes in developing tion teach us about muscle plasticity? Muscle Nerve 22:666–677. turkey skeletal muscle. BMC Genomics 12:143. http://dx.doi. Richards, M. P., S. M. Poch, and J. P. McMurtry. 2005. Expression org/10.1186/1471-2164-12-143. of insulin-like growth factor system genes in liver and brain tis- Sporer, K. R. B., H. R. Zhou, J. E. Linz, A. M. Booren, and G. sue during embryonic and post-hatch development of the turkey. M. Strasburg. 2012. Differential expression of calcium-regulating Comp. Biochem. Physiol. A Mol. Integr. Physiol. 141:76–86. genes in heat-stressed turkey breast muscle is associated with Ritchie, M. E., J. Silver, A. Oshlack, M. Holmes, D. Diyagama, A. meat quality. Poult. Sci. 91:1418–1424. Holloway, and G. K. Smyth. 2007. A comparison of background Stromer, M. H. 1998. The cytoskeleton in skeletal, cardiac and correction methods for two-colour microarrays. Bioinformatics smooth muscle cells. Histol. Histopathol. 13:283–291. 23:2700–2707. Stull, J. T., K. E. Kamm, and R. Vandenboom. 2011. Myosin light Rosser, B. W. C., M. Wick, D. M. Waldbillig, and E. Bandman. chain kinase and the role of myosin light chain phosphorylation 1996. Heterogeneity of myosin heavy-chain expression in fast in skeletal muscle. Arch. Biochem. Biophys. 510:120–128. twitch fiber types of mature avian pectoralis muscle. Biochem. Tomas, F. M., R. A. Pym, J. P. McMurtry, and G. L. Francis. 1998. Cell Biol. 74:715–728. Insulin-like Growth Factor (IGF)-I but not IGF-II promotes lean Ryu, Y. C., and B. C. Kim. 2006. Comparison of histochemical growth and feed efficiency in broiler chickens. Gen. Comp. En- characteristics in various pork groups categorized by postmortem docrinol. 110:262–275. metabolic rate and pork quality. J. Anim. Sci. 84:894–901. Tonge, D. P., S. W. Jones, R. G. Bardsley, and T. Parr. 2010. Char- Sacchetto, R., S. Testoni, A. Gentile, E. Damiani, M. Rossi, R. acterisation of the sarcomeric myosin heavy chain multigene fam- Liguori, C. Droegemueller, and F. Mascarello. 2009. A defective ily in the laboratory guinea pig. BMC Mol. Biol. 11:52. http:// SERCA1 protein is responsible for congenital pseudomyotonia in dx.doi.org/10.1186/1471-2199-11-52. Chianina cattle. Am. J. Pathol. 174:565–573. Wang, L. J., T. M. Byrem, J. Zarosley, A. M. Booren, and G. M. Samuel, D. D., L. Billard, D. Pringle, and L. Wicker. 2012. Influence Strasburg. 1999. Skeletal muscle calcium channel ryanodine bind- of growth rate on occurrences of pale muscle in broilers. J. Sci. ing activity in genetically unimproved and commercial turkey Food Agric. 92:78–83. populations. Poult. Sci. 78:792–797. Schmidt, A., and M. N. Hall. 1998. Signaling to the actin cytoskel- Wells, L., K. A. Edwards, and S. I. Bernstein. 1996. Myosin heavy eton. Annu. Rev. Cell Biol. 14:305–338. chain isoforms regulate muscle function but not myofibril assem- Sheterline, P., and J. C. Sparrow. 1994. Actin. Protein Profile 1:1– bly. EMBO J. 15:4454–4459. 121. Wilhelm, A. E., M. B. Maganhini, F. J. Hernandez-Blazquez, E. I. Shi, L. M., W. D. Jones, R. V. Jensen, S. C. Harris, R. G. Perkins, F. Ida, and M. Shimokomaki. 2010. Protease activity and the ul- M. Goodsaid, L. Guo, L. J. Croner, C. Boysen, H. Fang, F. Qian, trastructure of broiler chicken PSE (pale, soft, exudative) meat. S. Amur, W. J. Bao, C. C. Barbacioru, V. Bertholet, X. M. Cao, Food Chem. 119:1201–1204. T. M. Chu, P. J. Collins, X. H. Fan, F. W. Frueh, J. C. Fuscoe, Woelfel, R. L., C. M. Owens, E. M. Hirschler, R. Martinez-Dawson, X. Guo, J. Han, D. Herman, H. X. Hong, E. S. Kawasaki, Q. Z. and A. R. Sams. 2002. The characterization and incidence of Li, Y. L. Luo, Y. Q. Ma, N. Mei, R. L. Peterson, R. K. Puri, R. pale, soft, and exudative broiler meat in a commercial processing Shippy, Z. Q. Su, Y. A. Sun, H. M. Sun, B. Thorn, Y. Turpaz, plant. Poult. Sci. 81:579–584. C. Wang, S. J. Wang, J. A. Warrington, J. C. Willey, J. Wu, Wynn, R. M., M. Kato, J. L. Chuang, S. C. Tso, J. Li, and D. T. Q. Xie, L. Zhang, S. Zhong, R. D. Wolfinger, and W. D. Tong. Chuang. 2008. Pyruvate dehydrogenase kinase-4 structures reveal 2008. The balance of reproducibility, sensitivity, and specificity of a metastable open conformation fostering robust core-free basal lists of differentially expressed genes in microarray studies. BMC activity. J. Biol. Chem. 283:25305–25315. Bioinformatics 9(Suppl. 9):S10. http://dx.doi.org/10.1186/1471- Zhi, G., J. W. Ryder, J. Huang, P. G. Ding, Y. Chen, Y. M. Zhao, 2105-9-s9-s10. K. E. Kamm, and J. T. Stull. 2005. Myosin light chain kinase Sia, S. K., M. X. Li, L. Spyracopoulos, S. M. Gagne, W. Liu, J. A. and myosin phosphorylation effect frequency-dependent poten- Putkey, and B. D. Sykes. 1997. Structure of cardiac muscle tropo- tiation of skeletal muscle contraction. Proc. Natl. Acad. Sci. nin C unexpectedly reveals a closed regulatory domain. J. Biol. USA 102:17519–17524. Chem. 272:18216–18221. Ziober, I. L., F. G. Paiao, D. F. Marchi, L. L. Coutinho, E. Bin- Sibut, V., C. Hennequet-Antier, E. Le Bihan-Duval, S. Marthey, neck, A. L. Nepomuceno, and M. Shimokomaki. 2010. Heat and M. J. Duclos, and C. Berri. 2011. Identification of differentially chemical stress modulate the expression of the alpha-RYR gene expressed genes in chickens differing in muscle glycogen con- in broiler chickens. Genet. Mol. Res. 9:1258–1266. tent and meat quality. BMC Genomics 12:112. http://dx.doi. org/10.1186/1471-2164-12-112.