Induction of tissue- and stressor-specific kinomic responses in chickens exposed to hot and cold stresses.

Induction of tissue- and stressor-specific kinomic responses in chickens exposed to hot and cold stresses Scott Napper,∗,†, 1 Samira Dadgar,§ Ryan J. Arsenault,¶ Brett Trost,‡ Erin Scruten,∗ Anthony Kusalik,‡ and Phyllis Shand§ ∗

Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E3, Canada; † Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada; ‡ Department of Computer Science, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada; § Department of Food and Bioproduct Science, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada; and ¶ United States Department of Agriculture, Agricultural Research Service, SPARC, College Station, TX 77845 USA

Key words: kinome, peptide array, thermal stress, chickens 2015 Poultry Science 00:1–13 http://dx.doi.org/10.3382/ps/pev046

INTRODUCTION

ture stress may eventually lead to the breeding of poultry with increased thermal tolerance. Laying hens and broilers have optimal performance within a temperature range of 18–22◦ C (Lin et al., 2006). At higher temperatures birds may experience heat stress and heat burden, which are associated with increased mortality, decreased feed consumption, and reduced weight gain (Cooper and Washburn, 1998; Quinteiro-Filho et al., 2010). Long-term and acute heat stresses also have significant consequences on meat quality. Preslaughter heat stress has been reported to accelerate the rate and extent of rigor mortis development, postmortem glycolysis, and postmortem metabolism and biochemical changes in the muscle (Sams 1999). Exposure of chickens to heat stress before slaughter also results in breast meat with lower ultimate pH (pHu) (Sandercock et al., 1999), reduced water-binding capacity (WBC) (Petracci et al., 2001; Dadgar et al., 2010), and reduced tenderness (Petracci

Transportation of animals is an essential, but stressful, component of the poultry processing industry. The temperature fluctuations to which the birds may be exposed during transport are significant from the perspectives of animal welfare and meat quality. Specifically, hot and cold stresses diminish survival rates and compromise the quality of breast and thigh meat (Lin et al., 2006; Petracci et al., 2001; Zhang et al., 2012). A greater understanding of the cellular responses of these muscles to such stresses may enable more effective management strategies. Additionally, evaluation of phosphorylationassociated biomarkers of poultry exposed to tempera C 2015 Poultry Science Association Inc. Received July 24, 2014. Accepted December 10, 2014. 1 Corresponding author: [email protected]

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tion of AMPK, a key regulatory enzyme of metabolism. In thigh, cold stress induced responses suggestive of the occurrence of tissue damage, including activation of innate immune signaling pathways and tissue repair pathways (TGF-β ). In contrast, heat stress in thigh activated pathways associated with protein and fat metabolism through adipocytokine and mammalian target of rapamycin (mTOR) signaling. Defining the responses of these tissues to these stresses through conventional markers of pH, glycolytic potential, and meat quality offered a similar conclusion of the tissue- and stressor-specific responses, validating the kinome results. Collectively, the results of this study highlight the unique cellular responses of breast and thigh tissues to heat and cold stresses and may offer insight into the unique susceptibilities, as well as functional consequences, of these tissues to thermal stress.

ABSTRACT Defining cellular responses at the level of global cellular kinase (kinome) activity is a powerful approach to deciphering complex biology and identifying biomarkers. Here we report on the development of a chicken-specific peptide array and its application to characterizing kinome responses within the breast (pectoralis major) and thigh (iliotibialis) muscles of poultry subject to temperature stress to mimic conditions experienced by birds during commercial transport. Breast and thigh muscles exhibited unique kinome profiles, highlighting the distinct nature of these tissues. Against these distinct backgrounds, tissue- and temperature-specific kinome responses were observed. In breast, both cold and hot stresses activated calciumdependent metabolic adaptations. Also within breast, but specific to cold stress, was the activation of ErbB signaling as well as dynamic patterns of phosphoryla-

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surrogate kinase substrates (Zhu et al., 2000). These peptides are highly amenable to array technologies that can be used to monitor global cellular kinase activity (the kinome) (Arsenault et al., 2011). Historically, peptide kinome arrays were created based on phosphorylation events characterized for a particular species and applied for investigations of that same species. Because the majority of the information that is available within the phosphorylation site databases relates to humans and mice, there was a barrier to the application of this technology to other species. Because sites of phosphorylation are often conserved across species, we hypothesized, and demonstrated, that it was possible to predict the sequence contexts of phosphorylation events in proteins of other species based on genomic information (Jalal et al., 2009). The process of generating species-specific peptide arrays has been automated in the form of DAPPLE, a software program that enables the prediction of phosphorylation sites in species of interest (Trost et al., 2013a). Furthermore, a software platform developed by our group customized for the analysis of kinome data has been equally important in expanding the value of peptide arrays for kinome analysis by enabling more effective and accurate extraction of biological information (Li et al., 2012; Trost et al., 2013b). With a maintained priority on livestock species, but with an interest in applying species-specific peptide arrays across greater evolutionary distances, we developed a chicken-specific array. An initial priority of these arrays was to characterize signaling responses of the breast and thigh muscles of poultry exposed to temperatures that mimic the stresses birds often face during transport. In addition to defining the signaling responses of these tissues to temperature stress, conventional markers, such as pH, glycolytic potential, and other meat quality measurements, were also conducted. Collectively, this investigation provides a valuable tool for the characterization of cellular responses in poultry and offers insight into the cellular responses of chickens to thermal stress.

MATERIALS AND METHODS Design and Development of Chicken-Specific Peptide Arrays The chicken-specific peptide array was developed through a bioinformatics approach developed by our group termed DAPPLE (Trost et al., 2013a). DAPPLE predicts phosphorylation sites in species of interest based on homology to known phosphorylation sites in other species. Using DAPPLE, known phosphorylation sites from the PhosphoSitePlus database (Hornbeck et al., 2011) were used as BLAST queries against the chicken proteome. For each known phosphorylation site, DAPPLE outputs the best match in the chicken proteome, the number of sequence


et al., 2001; Northcutt et al., 1994; Holm and Fletcher, 1997). These responses result in undesirable changes in meat characteristics similar to the pale, soft, and exudative (PSE) condition (Northcutt et al., 1994; McKee and Sams, 1997; Sandercock et al., 2001). Heat-stressinduced PSE meat also compromises further processing because the impaired protein functionality leads to poor water-holding capacity, cook yield, and textural properties. Exposure of poultry to a cold environment imposes acute demands on energy metabolism and increases glycogenolysis, which may result in meat with dark, firm, dry (DFD) characteristics (Dadgar et al., 2010; Dadgar et al., 2012). For example, up to 8% of broilers that experienced cold conditions during transport exhibited DFD breast meat (Dadgar et al., 2010). The value was even higher in thigh meat, which is more sensitive to transportation stresses. Inconsistencies in the color and eating quality of PSE and DFD meat can decrease consumer acceptance of the product. Short-term heat or cold stress during transport can also lead to bird mortality, which has both welfare and economic implications. There have been extensive investigations to define the consequences of temperature stress on poultry mortality, morbidity, and meat quality (SchwartzkopfGenswein et al., 2012; Mitchell and Kettlewell, 1998). However, there have been limited investigations of global cellular responses to thermal stress. As such, information on the cellular mechanism of poultry temperature tolerance is limited, and there is an absence of reliable, high-throughput biomarkers to guide breeding efforts toward thermotolerant birds. This, in our opinion, reflects the challenges associated with deciphering complex biology, in particular within the context of a mixed genetic population, which is exacerbated by the limited availability of research tools for livestock species. Our group has overcome similar challenges in other livestock species through the development and application of species-specific peptide arrays (Arsenault et al., 2012). Reversible protein phosphorylation, as catalyzed by protein kinases, is frequently the defining event for initiating cellular responses and phenotypes (Arsenault et al., 2011). Investigations of cellular responses at the level of phosphorylation-mediated signal transduction are advantaged in that these posttranslational modifications are often directly responsible for the changes in protein function that result in a particular phenotype. As such, investigations of changes in phosphorylationmediated signal transduction have the potential to offer clearer insight into complex biology than can be achieved with traditional genomic or proteomic approaches (Arsenault et al., 2011). The specificity of many kinases is determined by the residues immediately surrounding the phosphorylation site (Kreegipuu et al., 1998). This offers the opportunity to employ short peptides (approximately 15 amino acids) representing these phosphorylation targets as

KINOME RESPONSES OF POULTRY TO TEMPERATURE STRESS

Thermal Stress of Chickens Thirty male broiler chickens (Ross 308 at 5 weeks of age) (n = 10/group) were exposed to temperature stress for 3 h using a previously described thermal treatment chamber (Dadgar et al., 2010; Dadgar et al., 2012). Birds were taken off feed 7 h prior to the start of the trial but had access to water until insertion of iButtons, which was 2–2.5 h prior to the start of the trial. Birds were wing banded, weighed, and orally dosed with miniature temperature loggers (iButton Thermocron DS1922L, Maxim Integrated Products, San Jose, CA) into the proventriculus to measure the core body temperature (CBT). The loggers recorded temperature once each minute and were retrieved from the euthanized birds at the end of the trial. Temperature stress was defined as approximately +35◦ C for heat stress, −15◦ C for cold stress, and +20◦ C for control birds. These temperatures were achieved within +/−2◦ C. All animal experiments were performed with the approval of the University of Saskatchewan Animal Care Committee. The temperature at each bird location was also monitored as previously described (Dadgar et al., 2010; Dadgar et al., 2012). Temperature was interpolated for the center position of each grid space that a bird occu-

pied using mapping software (Version 10, Tecplot Inc, Bellevue, WA). The calculated temperature is referred to as the experienced temperature and represents an estimate of the air temperature surrounding each bird. After the 3-h exposure period, the drawer was removed from the chamber. Birds were weighed and blood samples were collected within 10 min into heparinized syringes via brachial puncture. The samples were stored on ice until being analyzed. Blood glucose levels were measured using a glucose kit (i-STAT 1 Handheld Clinical Analyzer, Heska Inc., Loveland, CO) with a reportable range of 1.1 to 38.9 mmol/L for the glucose cartridges and resolution of 1. Birds were then sacrificed in a simulated commercial abattoir as previously described (Dadgar et al., 2012) and iButtons were retrieved. Samples were collected immediately upon sacrifice from the pectoralis major muscle of the breast and iliotibialis muscle of the thigh and were immediately placed in liquid nitrogen and then stored at −70◦ C until further analysis.

Meat Quality Assessment Quality parameters, including instrumental color L∗ (100 = white, 0 = black), a∗ (redness), b∗ (yellowness), initial and ultimate pH (pHi , pHu ), water binding capacity (WBC), processing cook yield (PCY), glycogen concentration, and glycolytic potential (GP), were measured as previously described (Dadgar et al., 2010; Dadgar et al., 2012). Meat quality assessments and the kinome analysis were performed on 5 randomly selected birds from each temperature group (cold, control, and hot). Data were subjected to ANOVA using the generalized linear model (GLM) procedure of SAS (SAS Institute, Cary, NC), and results were reported as leastsquares means with their standard deviation (SD). Differences among means were evaluated using the Least Significant Difference test option of SAS. Unless stated otherwise, the means were considered different at P < 0.05.

Peptide Arrays For kinome analysis, 40 mg of each muscle tissue sample were homogenized by a hand-held Qiagen TissueRuptor (Valencia, CA) in 100 uL lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl,1 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3 VO4 , 1 mM NaF, 1 μg/mL leupeptin, 1 g/mL aprotinin, and 1 mM phenylmethylsulphonyl fluoride (all products from Sigma Aldrich, St. Louis, MO, unless indicated]. Following homogenization, the peptide array protocol was carried out as described earlier (Jalal et al., 2009) with alterations (Arsenault et al., 2012).


differences between the known site and its best match in the chicken proteome, the position of each phosphorylation site in its corresponding protein, functional descriptions of the proteins, and peer-reviewed papers relating to the phosphorylation site. DAPPLE predicted approximately 43,000 phosphorylation events within the chicken proteome. A total of 299 peptides were manually selected from this panel of 43,000 predicted chicken phosphorylation events. Selection was based on confidence in the phosphorylation event as well as interest in the corresponding biology. Confidence in the phosphorylation event was evaluated by the following criteria: (a) degree of sequence conservation between predicted and known protein, (b) degree of sequence conservation between predicted and known phosphopeptides, and (c) similar positioning of phosphorylation site within the proteins. Peptides were selected on the basis of biological interest with priority given to ensuring representation of critical, welldocumented phosphorylation events associated with a broad spectrum of signaling pathways as well as to placing emphasis on pathways and processes implicated in metabolism and stress adaptations. The rationale was to generate an array with a global perspective on signaling events in order to facilitate novel discovery while still enabling targeted consideration of events likely associated with responses to temperature stress. This chicken-specific peptide array design was previously used to investigate the metabolic consequences of host–pathogen interactions in a chicken model of Salmonella infection (Arsenault et al., 2013).

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Data Analysis Data Sets The data sets contain the signal intensities associated with each of the 299 peptides on the array for the animals under the different treatment conditions. For each animal and each treatment, there are 1 intra-array replicates. All data processing and pathway analysis were done as per Li et al. (2012). Briefly, the kinome data were analyzed by subtracting the background intensity from the foreground intensity, normalizing to bring all of the arrays onto the same scale, and then performing clustering and pathway analysis (Li et al., 2012).

RESULTS

DAPPLE predicted approximately 43,000 phosphorylation events within the chicken proteome. There was a modest degree of phosphorylation site conservation between chickens and the other organisms represented in the PhosphoSitePlus database [Table 1]. Approximately 10% of the predicted phosphorylation sites were exactly conserved over a peptide of 15 amino acids (7 residues flanking each side of the phosphoacceptor site). This limited degree of conservation highlights the importance of developing species-specific arrays as opposed to simply transferring commercially available arrays across species. A total of 299 peptides were manually selected from this panel of 43,000 predicted chicken phosphorylation events. The complete peptide list can be found in Supplementary Table S1. Peptide synthesis, array spotting, and quality control checks were performed as a commercial service of JPT Peptide Technologies GmbH (Berlin, Germany). Representative images of the quality assurance printing of the chicken Table 1. Conservation between known phosphorylation sites from PhosphoSitePlus database and chicken proteome. Sequence differences

All chicken peptides (%)

Peptides on array (%)

0 1 2 3 4 5 6 7+ No homology

10.63 7.65 6.21 5.00 4.41 3.60 2.56 1.56 58.41

95 0 0 0 0 0 0 0 5

The first column indicates the number of sequence differences between a known phosphorylation site from the PhosphoSitePlus database and its best match in the chicken proteome. The second column indicates the percentage of known phosphorylation sites with the indicated number of sequence differences. The “no homology” row indicates known phosphorylation sites for which either there was no match in the chicken proteome or the E-value of the match between the full protein corresponding to the known phosphorylation site and the full protein corresponding to the site’s best match in the chicken proteome was greater than 1e-5. The third column is the same as the second, except for only the peptides ultimately included on the array.

array, as well as a representative data image from this study, are presented in Figure 1.

Thermal Stress of Poultry Poultry (n = 10) were exposed to temperatures consistent with the thermal stresses that birds typically experience during transport; the average heat stress applied was 35.0±0.7◦ C, and the corresponding cold stress was −15.0±1.1◦ C. Control animals were maintained at 20±0.7◦ C. The experienced temperature, core body temperature (averages over 3-h exposure), and blood glucose (10 min after exposure) of chickens exposed to cold and hot simulated transport are presented in Table 2. The magnitude and duration of the heat stress matched that reported in similar investigations (Sandercock et al., 1999; Debut et al., 2005; Geraert et al., 1996). Compared to the changes observed for cold stress, heat stress had minimal impact on core body temperature and blood glucose levels (Table 2). Similarly, and to be discussed later, the changes that were observed in response to heat stress at the levels of meat quality indicators, as well as biochemical and kinomic characterizations, were also consistent with more moderate responses to heat rather than cold stress. Table 2. Experienced temperature, core body temperature (averages over 3-h exposure), and blood glucose (10 min after exposure) of chickens exposed to cold- and hot-simulated transport (mean +/−SD, n = 5/group). Groups Cold-stressed Control Heat-stressed P value

Experienced temperature (◦ C)

Core body temperature (◦ C)

Blood glucose (mmol/L)

−13.5±1.1c 22.2±0.7b 36.0±0.7a P < 0.0001

34.4±1.6b 40.6±0.2a 42.0±0.6a P < 0.0001

9.1±1.7b 12.2±0.5a 12.1±0.7a P < 0.003

The P value is the result of an ANOVA test. Means within a column with different superscript letters are significantly different.


Development of a Chicken-Specific Peptide Array

Figure 1. Printing and validation of chicken-specific peptide array. (A) The arrays were printed by a commercial partner (JPT Technologies). For each array each spot is printed in triplicate within each block. Each block is then printed in triplicate for 3 technical repeats of each peptide. This image, taken as a quality control step in array production, illustrates the consistency and reproducibility of peptide spotting. (B) An image of a data scan of a representative array that was used for the analysis of a chicken breast sample. All of the arrays in this work were of comparable quality with respect to the clarity and consistency of peptide phosphorylation. A clear and consistent pattern of extents of peptide phosphorylation is apparent across the three printed blocks.


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Cluster Analysis

Kinome data were processed through the Platform for Integrated, Intelligent Kinome Analysis (PIIKA), an in-house peptide array data processing pipeline (Li et al., 2012). Within each array there was a high degree of technical reproducibility; specifically, an average of 97% of the peptides demonstrated a consistent pattern of phosphorylation across the technical replicates (P < 0.01). This high degree of technical reproducibility was consistent for the different tissues and stress conditions (lowest percentage = 96%, highest = 99%). Further, the extent of variability across the kinome data sets of the different animals was consistent with that observed for other outbred species (Trost et al., 2013c).

Kinome data were subjected to hierarchical clustering analysis to identify global kinomic patterns across the different tissues and treatments. Within the heatmaps there was an absolute segregation of the samples on the basis of tissue type (Figure 2). This indicates that, despite the overall similarities of breast and thigh muscle, these tissues exhibit unique profiles of signal transduction activity. This also provides confidence in the ability of the arrays to detect and discriminate biological differences between these closely related tissue types. The existence of distinct tissue-specific kinomic profiles was confirmed with three-dimensional principal component analysis (PCA), which again showed clear and distinct separation of the samples on the basis of tissue type (Figure 3).

Figure 2. Cluster analysis of kinome data sets of thigh and breast samples of temperature-stressed birds. Kinome data sets were subjected to hierarchical clustering analysis. The distance metric used was 1 – Pearson correlation, and the linkage method used was McQuitty (McQuitty, 1966). The animal codes are indicated under the heat map. The first letter of each code indicates the treatment condition (C cold, N normal, H Hot), the number is the individual bird number, and the last letter is the tissue of origin (T thigh, B breast). Each column represents the kinome activity of individual muscle biopsy.

Figure 3. Principle component analysis of thigh and breast samples of temperature-stressed birds. The first three principal components from PCA are shown. Separation of the samples on the basis of tissue of origin (pyramids for thigh, spheres for breast) is clearly observed.


Technical Reproducibility of Kinome Data

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responses (Figure 4). This allows for a representative kinomic response irrespective of potential variation between stressed individuals.

Metabolic Responses of Poultry to Thermal Stress

Having demonstrated tissue-specific kinome profiles, we sought to determine whether there was a consistent kinomic response within each tissue to each treatment. Within the tissue-specific subclusters, considerable variability occurred, which was largely independent of the treatment condition. In particular, within each of the breast and thigh subclusters, the data sets corresponding to the controls and heat-stressed conditions appeared randomly interspersed. This suggests minimal kinomic response within either of these tissues in response to the heat stress. This is consistent with a minimal impact of heat stress on core temperature, pH, blood glucose, and other physiological indicators. In an effort to minimize the contributions of individual bird-to-bird variation, and to identify responses that are consistent and conserved across animals, the data sets from each of the treatment conditions were averaged. Clustering of the averaged data sets confirmed preservation of the tissue-specific nature of the

Dynamic Phosphorylation of AMPK in Response to Thermal Stress AMPK is the principal energy sensor of most eukaryotic cells (Proszkowiec-Weglarz and Richards, 2007). In all species, AMPK functions as a heterotrimer, consisting of a catalytic α-subunit as well as regulatory β - and γ -subunits (Hardie et al., 2012). Given the pivotal role of this enzyme in sensing metabolic stress and regulating a plethora of metabolic adaptations, we wanted to have extensive representation of the phosphorylation events that have been described for AMPK. The chicken-specific peptide array contained 9 peptides that represented unique phosphorylation events of different isoforms of the α, β , and γ subunits. A complete description of the changes in phosphorylation of each of these peptides in each of the tissue types in response to each thermal stress is presented in Table 5. The greatest consequences of AMPK dynamic phosphorylation were observed in the breast muscle in

Table 3. Biochemical properties [pH, glycolytic potential (GP)], meat color, and water binding capacity of breast and thigh meat from chickens exposed to cold- and hot-simulated transport (mean +/−SD, n = 5 per group). Group

pHi (5 min)

pHu (30 h)

GP–0 min (umol/g)

GP–30 h (umol/g)

L∗ (lightness)

WBC (%)

Cold-stressed Control Heat-stressed P value

6.8±0.1 6.6±0.2 6.6±0.2 P = 0.12

6.4±0.13a 6.1±0.08b 6.0±0.05b P = 0.0003

Breast 63.8±7.9b 103.1±8.7a 111.9±11.3a P < 0.0001

85.9±13.6 104.0±8.8 101.2±12.6 P = 0.11

41.6±2.2b 48.1±1.8a 48.3±0.7a P< 0.0001

38.6±3.5 31.7±5.1 33.9±2.8 P = 0.08

14.5±6.2b 45.1±7.3a 50.7±6.2a P < 0.0001

40.5±2.1b 50.4±1.6a 52.1±1.1a P < 0.0001

83.9±5.9a 35.1±17.5b 40.3±8.3b P < 0.0001

Thigh Cold-stressed Control Heat-stressed P value

a

6.9±0.07 6.4±0.15b 6.5±0.14b P = 0.0002

a

7.2±0.04 6.2±0.12b 6.2±0.04b P < 0.0001

b

11.4±3.3 76.5±12.3a 64.5±8.3a P < 0.0001

The P value is the result of an ANOVA test. Means within a column and muscle type with different superscript letters are significantly different.


Figure 4. Differentially modified peptides between different tissues and treatment conditions. Kinome data sets were subjected to hierarchical clustering analysis. The distance metric used was 1 – Pearson correlation, and the linkage method used was McQuitty (McQuitty, 1966).

The biochemical responses of the birds to thermal stress were evaluated through a number of traditional indicators, including pH, glycolytic potential, meat color, and water binding. No significant effect of heat stress was observed on meat quality attributes of either breast or thigh (Table 3). In contrast, cold stress caused a significant increase in pHu and water binding capacity, as well as a significantly darker color and lower glycolytic potential of both breast pectoralis major and thigh iliotibialis muscles. Further, a more dramatic effect of cold stress prior to slaughter was observed on total glucose reserves for thigh compared to breast muscle [Table 3].

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KINOME RESPONSES OF POULTRY TO TEMPERATURE STRESS Table 4. Pathway analysis of responses of breast muscle to hot and cold stresses of poultry. Cold Stress (Breast) Upregulated

Pathway

↑↓

P

Metabolism

Ca-calmodulin-dependent protein kinase activation Regulation of pgc-1a CREB phosphorylation through activation of CaMKII Carbohydrate digestion and absorption LPA receptor-mediated events Ras activation upon Ca2+ influx through NMDA receptor Trafficking of AMPA receptors Unblocking of NMDA receptor, glutamate binding, and activation Nectin adhesion pathway ErbB signaling pathway Bioactive peptide-induced signaling pathway Vascular smooth muscle contraction Heat Stress (Breast) Pathway CREB phosphorylation through activation of CaMKII Ca-calmodulin-dependent protein kinase activation Calcium signaling pathway Glycolysis and gluconeogenesis Regulation of pgc-1a Ras activation upon Ca2+ influx through NMDA receptor Trafficking of AMPA receptors Unblocking of NMDA receptor, glutamate binding, and activation Bioactive peptide-induced signaling pathway None

4 4 3 3 3 3 3 3 3 15 5 7

4 4 3 3 3 3 3 3 3 9 4 7

0.02 0.02 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.04 0.06 0.02

3 3 5 5 3 3 3 3 3

↑↓ 3 3 4 4 3 3 3 3 3

p 0.04 0.04 0.05 0.05 0.04 0.04 0.04 0.04 0.04

NMDA Structural Signaling Downregulated Upregulated

Process Metabolism

NMDA Signaling Downregulated

InnateDB is a publicly available pathway analysis tool (Lynn et al., 2008). Pathways are assigned a probability value (P) based on the number of peptides present for a particular pathway. Output also includes the number of the uploaded peptides associated with a particular pathway as well as the subset of those that are differentially phosphorylated. For our investigation, fold change cutoffs were set at a confidence of difference between treatment and control equal to a P value of 0.1. The number of peptides on the array is indicated by the symbol , while ↑↓ indicates the number of those peptides with increased or decreased phosphorylation under the experimental conditions.

response to cold stress, where 5 of the 9 peptides displayed significant changes in their phosphorylation state in response to cold stress. This was consistent with the other results from 2 key perspectives. First, that these patterns occurred specifically in response to cold stress is consistent with the theme throughout many of the experiments that cold stress was the more taxing of the thermal challenges. Further, that these changes were observed in the breast rather than the thigh is consistent with the pathways implicated for each tissue in that the responses to cold stress had a significantly different metabolic component in the breast than the thigh.

Pathway Responses of Breast to Thermal Stress InnateDb pathway overrepresentation analysis was performed on the data sets of the representative animals to identify cellular responses initiated within breast and thigh muscles of poultry following exposure to hot and cold stress (Lynn et al., 2008). Within the breast samples, a number of pathways were activated for both the cold- and heat-stressed birds. Specifically, the patterns of peptide phosphorylation were consistent with activation of calcium influx and subsequent activation of processes associated with carbohydrate metabolism (Table 4). This included activation of the calcium signaling pathway, increased phosphorylation of cAMP response element-binding protein (CREB) through CAMKII,

and activation of calmodulin-dependent kinase. Collectively, the differential phosphorylation of peptides relating to metabolic control in response to heat and cold stress suggests that this is a more general stress or, perhaps, a temperature-stress response rather than specific adaptations to either hot or cold stress in this tissue. There was strong evidence for activation of ErbB signaling in breast following cold stress (Table 4). A large number of peptides (15) on the array related to this signaling pathway, and many of these (9) were differentially phosphorylated in the cold stress condition. Accordingly, there is high confidence (P < 0.04) for activation of this pathway specifically in breast in response to cold stress.

Pathway Responses of Thigh to Thermal Stress The responses within thigh muscle to cold stress were distinct from those within breast. Within thigh, there was strong evidence for upregulation of a number of pathways, many of which share a common denominator of their involvement in innate immune responses (Table 6). While activation of the innate immune pathways, such as the Toll-like receptors (TLRs), is classically associated with recognition of microbial molecules, these systems are also activated by a number of endogenous molecules, including heat shock proteins. Activation of innate immune responses, such as TLR signaling, in response to temperature (or other) stress can reflect


Process

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DISCUSSION Livestock researchers often face complex biological problems while being disadvantaged by an absence of cutting edge research technologies. Because a disproportionate amount of research is devoted to humans and mice, so too are the available research tools. Unfortunately, the species specificity of many of these tools limits their application to other species. For example, while there is an ongoing trend within human medicine to monitor and influence cellular responses at the level of phosphorylation-mediated signal transduction, the species specificity of the research tools prohibits the application of this perspective to livestock. To address this issue, our group developed a protocol for the creation of species-appropriate peptide arrays for kinome analysis, including for chicken.

These arrays were applied to understand cellular changes associated with events involved in the transport of poultry, specifically in describing patterns of signal transduction resulting from hot and cold stresses. Within the experimental model of exposing poultry to hot and cold stresses that are consistent with those that occur during shipping, we investigated changes in kinome profiles within breast and thigh as well as a number of traditional indicators of thermal stress, including core temperature, muscle pH, and glycolytic potential. We also investigated indicators of meat quality, including color and water holding capacity. To our knowledge, this is the first description of cellular responses of poultry to production stress at the level of signal transduction. It was encouraging that a number of significant trends within the responses were measured from each of the three perspectives (biological, meat quality, and kinomic). In this investigation, the heat stress challenge had minimal impact on the birds, a finding that was apparent across the biological, meat-quality, and kinome experiments. In particular, the kinome profiles associated with the heat-stressed animals did not display unique profiles for either breast or thigh, which is consistent with the absence of a measurable phenotype for these tissues. Others have used similar thermal temperatures (32 to 36◦ C) and duration of exposure (1 to 6 h) and found marked differences in meat pH and color and in blood parameters such as levels of circulating creatine kinase and other enzymes (Tang et al., 2013). However, in contrast to the present study, none simulated transport conditions with significant air movement that may have moderated a hyperthermic effect. There was congruence between the readouts at the levels of biological indicators, meat quality, and kinome profiles of the breast and thigh samples in response to cold stress. Further, the magnitude of change in pH and other parameters in thigh meat due to cold exposure was greater than that observed for breast meat, which is in agreement with previously reported data by our group (Dadgar et al., 2013). Specifically, in this investigation, a more dramatic effect of cold stress prior to slaughter was observed on total glucose reserves for thigh compared to breast muscle (Table 3). This might be due to an initial lower level of glycogen reserve in thigh than breast muscle. Thigh (iliotibialis) muscle, with approximately 35% fast-twitch red (Type IIR) fibers, has lower glycogen content compared to the breast pectoralis major muscle, which is homogeneously composed of over 99% fast white fibers (Type IIW) (Sakakibara et al., 2000). It has been shown that glycogen depletion happens selectively based on muscle fiber type and the type of stress (Lacourt and Tarrant, 1985). Earlier studies reported greater effects of transport stress (Warriss et al., 1993; Debut et al., 2003), crating duration (Kannan et al., 1997), and feed withdrawal (Warriss et al., 1993) on thigh as compared to breast meat, which, in contrast, was more sensitive to physical activity on the shackle prior to slaughter.


damage of the tissues by the stress, leading to the release of endogenous molecules that can serve as TLR ligands (Arslan et al., 2011). From this perspective, activation of the TLR system in response to cold stress is suggestive of tissue damage. This hypothesis, and the observation that this response was not observed in the more thermal-tolerant breast, is consistent with accumulation of and response to tissue damage in thigh as a result of cold stress. This tissue damage may be a result of significant oxidative stress placed on the tissue. GP results showed a significant drop in GP in coldstressed thigh muscle (Table 3). This is indicative of a thermogenic response most likely facilitated by significant oxidative metabolism. The tissue damage due to the combination of shivering thermogenesis and rapid oxidative metabolism may be the cause of the proposed tissue damage. Within the thigh muscle, in response to both hot and cold stress, there was evidence for activation of TGF beta signaling [Table 6]. This response, based on the number of peptides relating to the pathway that are differentially phosphorylated, was much more pronounced in response to cold stress than heat stress because it involved differential phosphorylation of 10 rather than 4 peptides. This was again consistent with the hypothesis that the cold stress imposed a more demanding stress challenge. TGF beta is involved in the regulation of healing and is upregulated during the regeneration of skeletal muscle following injury; also, it is believed to be responsible for an acute inflammatory response to muscle damage (Serrano and Mu˜ noz-C´anoves, 2010). Collectively, the activation of innate immune and tissue repair pathways, as well as the absence of cytoprotective ErbB signaling, was consistent with previous findings that thigh meat quality is more sensitive than breast to temperature stress (Dadgar et al., 2010; Dadgar et al., 2012). Also within thigh, the response to the heat stress involved activation of adipocytokine and mammalian target of rapamycin (mTOR) signaling pathways (Table 6).


evidence for a significant energy expenditure occurring in these tissues. [Tables 4 and 5]. While the responses to heat stress were not as definitive as for cold stress, nevertheless, within thigh the response to heat stress involves activation of two primary pathways, including adipocytokine and mTOR signaling (Table 6). Signaling through the mTOR pathway promotes anabolic processes in response to growth factors, nutrients (amino acids and glucose), and stress (Wullschleger et al., 2006). A central role of the mTOR system is in the regulation of protein synthesis (Wang and Proud, 2006). Activation of mTOR in heat-stressed thigh is of significance because the heat stress of poultry has significant consequences for protein metabolism, including decreased body protein content, protein gain, and the ratio of protein retained to protein intake in broilers (Geraert et al., 1996). It has been suggested that this indicates that heat stress changes protein metabolism, decreases protein synthesis, and increases the catabolic rate (Geraert et al., 1996). Others have observed a similar trend in which elevated temperatures decreased muscle protein synthesis and accelerated protein breakdown (Yunianto et al., 1997; Temim et al., 1999). Heat stress lowers the ribosomal capacity for protein synthesis through decreased ribosomal gene transcription (Zhang et al., 2012; Jacob 1995; Temim et al., 2000). Activation of the mTOR system in the thigh of heat-stressed birds is likely indicative of cellular countermeasures to maintain protein synthesis in the face of faltering protein synthesis capabilities. Within the breast samples, there were a number of pathways that were activated for the cold- and heat-stressed birds. Specifically, the patterns of peptide phosphorylation were consistent with activation of calcium influx and subsequent activation of processes associated with carbohydrate metabolism. Others have also reported on altered calcium metabolism in chickens in response to heat stress and speculated on its role in the regulation of metabolic processes (Debut et al., 2005). This included quantification of a number of markers associated with metabolic and, in particular, glycolytic activity. The increased carbohydrate metabolism observed in breast in response to temperature stress may have significance for overall meat quality. For example, after slaughter there is a disruption of the nutrient and oxygen supply to muscle cells. The subsequent anaerobic metabolism of glycogen and glucose results in the accumulation of lactic acid (Poso and Puolanne, 2005), which may explain the increase in GP at 0 min in heat-stressed breast muscle compared to control. Additionally, the activation of the gluconeogenesis pathway in breast muscle following heat stress may also explain this increase in GP compared to control at 0 min and subsequent drop at 30 h in heat-stressed tissue (Table 3). Recent studies have focused on AMPK as a key enzyme in postmortem metabolism (Du et al., 2005; Shen et al., 2006; Shen et al., 2007; Sibut et al., 2008). Since no difference was observed in energy reserve between


Moreover, leg muscles are involved in maintaining balance in a moving vehicle during transport or simulated transport due to vibration, and finally the fibers responsible for shivering are mostly the slow-contracting type; therefore, animals exposed to severe cold prior to slaughter may have less glycogen in these fibers (Lupandin and Poleshchuk, 1979). All these observations were indicative of differences between muscle types, perhaps owing to different fiber types and different responses of muscles to cold and heat exposure prior to slaughter. Therefore, GP could predict postmortem meat quality of thigh oxidative red muscle that has a greater proportion of fast red fiber than breast glycolytic muscle that is mainly composed of fast white fibers [Table 3]. Critically, kinome analysis demonstrated the activation of tissue-specific signaling patterns that were consistent with, and offer insight into, changes that are observed in terms of meat quality. For example, within breast, cold stress appeared to activate responses that enable this tissue to better withstand cold challenge through activation of metabolic responses and cytoprotective mechanisms. There was strong evidence for the activation of ErbB signaling in breast following cold stress [Table 4]. A common response to stress is the release of epidermal growth factor (EGF), which activates ErbB1 signaling through the EGF receptor (Yarden and Sliwkowski, 2001). Interestingly, there have been several reports highlighting the cytoprotective action of ErbB1 signaling in a number of animal models (Hoffmann et al., 1997; Ishikawa et al., 1994; Pillai et al., 1999). EGF signaling has been shown to be involved in muscle proliferation (Harper and Buttery, 2001), and ErbB signaling is directly associated with muscle repair and muscle cell survival (Golding et al., 2007). Activation of this pathway under the more stressing condition (as evaluated by changes in core body temperature and other physiological indicators of response) is consistent with a cytoprotective role. Activation of the ErbB1 pathway was not observed in thigh muscle in response to cold stress. Rather, in thigh the pattern of pathway activation suggests the occurrence of tissue damage, which would again support a cytoprotective function of this signaling in breast. It has been reported that thigh muscle is more sensitive to cold and produces a more rapid and prolonged shivering thermogenesis in chicken exposed to cold than breast muscle (Aulie and Toien, 1988). This may explain the more significant drop in GP during cold stress in the thigh muscle compared to the breast (Table 3). Given sufficient exposure to cold temperatures, both thigh and breast muscle initiate shivering thermogenesis (Aulie and Toien, 1988), and this energetic expenditure would explain the drop in GP of both muscle types following cold stress. In addition, the pathway analysis of both breast and thigh muscle following cold stress showed an activation of the carbohydrate digestion and absorption pathways in these tissues, providing further

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NAPPER ET AL. Table 5. Patterns of phosphorylation of AMP-activated protein kinase.

Table 6. Pathway analysis of responses of thigh muscle to hot and cold stresses of poultry. Process Upregulated

Metabolic Innate immunity Signaling Cell cycle

Downregulated

Cold stress (thigh) Pathway Carbohydrate digestion and absorption IL-1 signaling (through JNK cascade) Toll-like receptor signaling pathway JNK cascade TGF beta receptor Cell cycle Aurora A signaling P53 signaling pathway FOXM1 transcription factor network None

↑↓

P

3 5 7 3 17 3 3 3 3

3 4 5 3 10 3 3 3 3

0.07 0.09 0.10 0.07 0.08 0.07 0.07 0.07 0.07

4 4 4 3

↑↓ 4 4 4 3

p 0.06 0.06 0.06 0.07

Heat stress (thigh) Upregulated

Process Metabolism Signaling

Downregulated

Pathways Adipocytokine signaling pathway MTOR signaling pathway TGF beta receptor FGF signaling pathway

For details see caption of Table 4.

normal and DFD breast meat of the control birds, it was hypothesized that a lack of AMPK activity might be responsible for the cessation of glycolysis and the resultant high pHu of the breast meat. However, a preliminary study by our group found no difference in phosphoAMPK, which is an indication of its activity (Dadgar et al., 2010). Studies on the effect of AMPK activity and pHu are contradictory. Specifically, Du (Du et al., 2005) and Shen (Shen et al., 2006; Shen et al., 2007) reported that a higher activity of AMPK correlated with a lower ultimate pH in mice and pigs. However, in a study by Sibut et al. on broiler chickens, a higher activity of AMPK was correlated with a higher ultimate pH, which was not explained by the authors (Sibut et al., 2008). Our results show a general decrease in the phosphorylation state of AMPK in breast tissue following cold stress. We did not see significant changes in the AMPK phosphorylation status of breast tissue following heat stress or in thigh tissue following either temperature stress (Table 5). We observed no significant change in the pH

of breast tissue under either stress condition, though thigh showed an increase in pH following cold stress (Table 3). This lack of correlation between pH and AMPK activity, coupled with the contradictory information on the relationship between pH level and AMPK activity, indicates that there is no significant correlation between pH and AMPK activity in chicken due to temperature stress. The broad decrease in phosphorylation of AMPK observed in cold-stressed breast tissue would correlate to an activation of anabolic responses. One of the anabolic processes influenced by the dephosphorylation of AMPK is lipogenesis (Proszkowiec-Weglarz and Richards, 2007). It has been reported that in brown adipose tissue during cold-induced thermogenesis fatty acid synthesis and oxidation occur simultaneously (Yu et al., 2002); though chickens do not have brown adipose tissue, a similar fatty acid driven thermogenesis may be occurring. From our GP results, it appears that more GP is retained in breast muscle than thigh muscle following cold stress [Table 3]. This may be due to


The various isoforms and specific sites of phosphorylation are indicated. The fold change (FC) is the extent of increased or decreased phosphorylation relative to the control condition. The P values of increased (↑) or decreased (↓) phosphorylation of the peptide under the treatment conditions are provided. Changes in phosphorylation at P < 0.1 are shaded.


Competing Interests The authors have no competing interests to declare.

Authors’ Contributions PS and SD were responsible for the development and performance of thermal stress experiments. SN and RA were responsible for the development and application of the chicken-specific peptide arrays. TK and BT were responsible for bioinformatics analysis. ES was responsible for designing and performing the kinome array experiments. All authors contributed to the preparation of the manuscript.

SUPPLEMENTARY DATA Supplementary data is available at PSA Journal online.

ACKNOWLEDGMENTS The study was supported by funds provided by the Natural Science and Engineering Research Coun-

cil, Agriculture and Agri-Food Canada, Saskatchewan Chicken Industry Development Fund, Alberta Farm Animal Care, Chicken Farmers of Saskatchewan, Alberta Chicken Producers, Canadian Poultry Research Council, Poultry Industry Council, and Sofina Foods Inc. (Lilydale). Special thanks to T. Crowe, M. Strawford, and the staff of the Poultry Research Center at the University of Saskatchewan for their support and participation in data collection.

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the difference in a more rapid and sustained shivering thermogenesis in thigh muscle and a nonshivering fattyacid-based thermogenesis in breast muscle. The deactivation of AMPK and potential lipogenesis and nonshivering thermogenesis may be one of these responses in breast tissue. In contrast, thigh tissue, in response to cold stress, displayed an absence of these metabolic and cytoprotective signaling events and instead (and likely as a consequence) showed activation of pathways that are consistent with tissue damage and repair. In addition, cold-stressed thigh muscle showed a significant drop in GP, possibly owing to a large energy expenditure resulting from shivering thermogenesis. In poultry the quality of meat products reflects the interaction between the genotype and environmental conditions. In particular, stress emerges as an important environmental determinant of meat quality (Debut et al., 2003; Berri, 2000). This offers the opportunity to reduce stress through better management practices and by breeding for birds with increased capabilities to manage particular stresses. There is an emerging body of research that considers the ability of various genotypes to manage a variety of stresses (Le Bihan-Duval et al., 2003), including exposure to heat stress (Debut et al., 2005). This introduces an additional layer of complexity onto breeding efforts, which are typically dictated by priorities such as growth rate and breast yield. In addition to the obvious short-term advantages of these characteristics to industry and consumers, these phenotypes are also readily quantified. The establishment of breeding programs for thermal or other stress tolerance will likely depend on the identification of more complex biomarkers. The ability to characterize the cellular responses of poultry to stress, as demonstrated here, may represent a valuable contribution toward achieving this objective.

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Effects of constant and cyclic heat stress on muscle metabolism and meat quality of broiler breast fillet and thigh meat. Poult. Sci. 91:2931–2937. Zhu, H., J. F. Klemic, S. Chang, P. Bertone, A. Casamayor, K. G. Klemic, D. Smith, M. Gerstein, M. A. Reed, and M. Snyder. 2000. Analysis of yeast protein kinases using protein chips. Nature Genet. 26:283–289.


Symmetry in cold-to-hot and hot-to-cold valuation gaps.

Metabolomic analysis of wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses: unraveling metabolic responses.

Hot flushes and cold turkey.

Thermal responses of unclothed men exposed to both cold temperatures and high altitudes.

Shivering and nonshivering thermogenic responses of cold-exposed rats to hypothalamic warming.

Geophysics. Inside Earth runs hot and cold.

Blue inhalers: blowing hot and cold.

Resistance of cold-exposed rats to aflatoxin.

Tissue-specific antioxidant responses in pale chub (Zacco platypus) exposed to copper and benzo[a]pyrene.

Differential regulation of microRNAs in response to osmotic, salt and cold stresses in wheat.

Rat lung response to PM2.5 exposure under different cold stresses.

Glutathione and tissue amino acid responses to light-exposed parenteral nutrients.

Normalization for Relative Quantification of mRNA and microRNA in Soybean Exposed to Various Abiotic Stresses.

Physiological and leukocyte subset responses to exercise and cold exposure in cold-acclimatized skaters.

The hot and cold issues of laser angioplasty.

Murder or not? Cold temperature makes criminals appear to be cold-blooded and warm temperature to be hot-headed.

Acute exposure to cold air and metabolic responses to exercise.

Plant responses to environmental stresses-from gene to biotechnology.

restraint stress.

Thermoregulatory responses of young and older men to cold exposure.

"Cold training" affects rat liver responses to continuous cold exposure.

Responses of the hands and feet to cold exposure.

Finger and Toe Temperature Responses to Cold After Freezing Cold Injury in Elite Alpinists.

Responses of Populus trichocarpa galactinol synthase genes to abiotic stresses.