Freeze-responsive regulation of MEF2 proteins and downstream gene networks in muscles of the wood frog, Rana sylvatica

Journal of Thermal Biology 67 (2017) 1–8

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Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio

Freeze-responsive regulation of MEF2 proteins and downstream gene networks in muscles of the wood frog, Rana sylvatica Oscar A. Aguilar1, Hanane Hadj-Moussa, Kenneth B. Storey

MARK

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Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6

A R T I C L E I N F O

A B S T R A C T

Keywords: Freeze tolerance Myocyte enhancer factor 2 Heart Skeletal muscle Transcriptional regulation

The wood frog survives frigid North American winters by retreating into a state of suspended animation characterized by the freezing of up to 65% of total body water as extracellular ice and displaying no heartbeat, breathing, brain activity, or movement. Physiological and biochemical adaptations are in place to facilitate global metabolic depression and protect against the consequences of whole body freezing. This study examined the myocyte enhancer factor 2 (MEF2) transcription factor family, proteins responsible for coordinating selective gene expression of a myriad of cellular functions from muscle development and remodelling to various stress responses. Immunoblotting, subcellular localization, and RT-PCR were used to analyze the regulation of MEF2A and MEF2C transcription factors and selected downstream targets under their control at transcriptional, translational, and post-translational levels in skeletal and cardiac muscles from control, frozen and thawed frogs. Both MEF2A/C proteins were freeze-responsive in skeletal muscle, displaying increases of 1.7–2 fold for phosphorylated MEF2AThr312 and MEF2CThr300 during freezing with an enrichment of nuclear phosphorylated MEF2 proteins (by 1.7–2.1 fold) observed as early as 4 h post-freezing. Despite the reduced response of total and phosphorylated MEF2A/C protein levels observed in cardiac muscle, the MEF2 downstream gene targets (glucose transporter-4, calreticulin, and creatine kinase brain and muscle isozymes) displayed similar increases in transcript levels (1.7–4.8 fold) after 24 h freezing in both muscle types. This study describes a novel freezeresponsive function for MEF2 transcription factors and further elaborates our understanding of the molecular mechanisms underlying natural freeze tolerance. This novel freeze-responsive regulation suggests a role for MEF2s and downstream genes in cryoprotectant glucose distribution, calcium homeostasis, and maintenance of energy reserves vital for successful freeze tolerance.

1. Introduction Many North American animals are faced with environmental challenges in winter including food scarcity, short photoperiods, and most importantly, subzero temperatures. Enduring this winter landscape requires a myriad of behavioural, physiological, and molecular adaptations, and while some animals are able to escape the cold by migration, most employ energy-saving strategies to survive. Winter hardiness strategies including hibernation, freeze-avoidance, and freeze tolerance rely on the depression of metabolic rate to allow organisms to survive for prolonged periods using only internal fuel reserves (Storey, 2015). In particular, freeze tolerance is one of the more extreme overwintering strategies and has been documented in species including various soil microfauna, selected intertidal marine invertebrates, many insects and various ectothermic vertebrates including turtles, snakes, salamanders, and frogs (Storey, 1990; Storey and Storey, 2017). Freeze

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tolerance is the ability to survive long-term freezing of a very high proportion of total body water as extracellular ice at subzero temperatures experienced naturally in winter hibernacula coupled with the ability to endure the various consequences of freezing including anoxia/ischemia, cell dehydration and shrinkage, elevated osmolality, and the cessation of vital processes (Storey and Storey, 2017). Wood frogs, Rana sylvatica (also known as Lithobates sylvaticus) have developed multiple mechanisms to aid freezing survival and can endure 65–70% of total body water as extracellular ice and, in their most northern locations in Alaska, survive temperatures as low as −18 °C for months at a time (Larson et al., 2014). Wood frog natural habitats stretch from the Southern Appalachians to above the Arctic Circle, making them the most northerly-distributed amphibian in North America (Lee‐Yaw et al., 2008). When in a frozen state of suspended animation, these frogs display no measurable brain activity, no heartbeat, no breathing and cease all skeletal muscle movement (Layne et al.,

Corresponding author. E-mail address: [email protected] (K.B. Storey). Present address: Department of Immunology, University of Toronto and Sunnybrook Research Institute, 2075 Bayview Avenue (S206B), Toronto, Ontario, Canada M4N 3M5.

http://dx.doi.org/10.1016/j.jtherbio.2017.04.007 Received 16 January 2017; Received in revised form 24 March 2017; Accepted 18 April 2017 Available online 19 April 2017 0306-4565/ © 2017 Elsevier Ltd. All rights reserved.

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genes whose protein products have crucial supporting roles in muscle function such as glucose transporter 4 (glut4), creatine kinase isozymes (ckb, ckm), and calreticulin (calr) (Amacher et al., 1993; Thai et al., 1998; Shen et al., 2002; Lynch et al., 2006). Glut-4 is the insulindependent glucose transporter essential for the efficient uptake of glucose into muscle and several other organs and is of high importance for wood frogs in order to rapidly distribute glucose cryoprotectant (made in liver) to all other organs during the early minutes/hours when frogs are freezing (Storey and Storey, 1992; King et al., 1993). Efficient management of ATP levels and energy homeostasis is a main function of creatine kinase that catalyzes a reversible reaction to synthesize phosphocreatine pools when ATP is plentiful or facilitate rapid ATP production from phosphocreatine when energy demands are high (Wallimann et al., 1992). Creatine kinase is prominent in muscle (both skeletal and cardiac) and brain where MM-CK and BB-CK dimers dominate. These are encoded by the muscle type (ckm) and brain type (ckb) genes, respectively, both being responsive to MEF2 activation (Amacher et al., 1993; Shen et al., 2002). Calreticulin is a protein present in the lumen of the endoplasmic reticulum where it acts as a Ca2+-binding chaperone, crucial for both the maintenance of Ca2+ homeostasis and regulation of the unfolded protein response (Michalak et al., 1999; Lynch et al., 2006). Calreticulin also plays a key role in coordinating protein quality control processes (Ellgaard and Helenius, 2003). Recent years have seen advances in our understanding of the molecular underpinnings of freeze tolerance such as the elucidation of cell cycle regulation, transcriptional signalling, and microRNA controls (Zhang and Storey, 2012; Aguilar et al., 2016; Bansal et al., 2016). Given the extensive role that MEF2 proteins play in regulating muscle metabolism, we hypothesized that MEF2 transcription factors are actively involved in facilitating the muscle pro-survival pathways necessary to adapt/protect muscles during wood frog freezing. The present study used immunoblotting, subcellular localization, and RTPCR to investigate the stress-activated responses and role of MEF2 transcription factors in wood frog freezing survival. We demonstrate that MEF2 transcription factors are activated in skeletal muscle of frozen wood frogs, showing enhanced levels of active phosphorylated MEF2 protein, MEF2A and MEF2C translocation to the nuclei of skeletal muscle during freeing, and enhanced transcription of downstream MEF2 targets (glut4, calr, ckb, ckm) in both skeletal and cardiac muscle. Thus, we reveal that MEF2A and MEF2C are freeze-responsive transcription factors in wood frog muscles. Understanding the mechanisms that vertebrates employ to survive the various stresses that accompany freezing can have important biomedical implications, especially with respect to organ cryopreservation and low oxygen tolerance that are well developed in the wood frog but poor in mammals.

1989). Yet when temperatures rise weeks or months later, they are able to thaw unscathed and resume normal life (Storey, 1999). To allow for a slow rate of ice formation, freezing is instigated by an initial ice nucleation event at temperatures near the freezing point of body fluids, just below −0.5 °C. Freezing at this temperature minimizes the instantaneous ice surge (instant conversion of water to ice) that occurs if animals supercool extensively before freezing and provides frogs with sufficient time to adjust their metabolism and synthesize and distribute cryoprotectant as the freezing front moves through the body (Storey, 1997). Wood frogs rely on the synthesis of the colligative cryoprotectant glucose as their main line of defence to limit the amount of water converted to extracellular ice, minimize cell volume reduction, and help stabilize macromolecules (Storey, 1997). Freeze tolerance and other hypometabolic states are characterized by a global suppression of most metabolic functions and an overall reduction in ATP turnover with a reprioritization of energy use towards basic survival requirements. This is accomplished via multiple controls at transcriptional, translational and posttranslational levels (Storey and Storey, 2013). Against a background of global suppression of transcription and translation, a variety of genes are upregulated during freezing to elevate protective proteins over freeze-thaw cycles and enhance prosurvival pathways (Cai and Storey, 1997; McNally et al., 2002; Storey, 2004). However, the transcriptional regulation of these genes in response to freezing remains largely elusive. Of interest is the role of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors. These are emerging as essential stress-responsive elements capable of converting extracellular signals into specific nuclear responses to elicit the expression of genes that are important in developmental processes, differentiation, myogenesis, survival, apoptosis, and other cellular processes (Potthoff and Olson, 2007). MEF2 transcription factors are evolutionarily conserved proteins of the MADS-box family that consist of four members in vertebrates (MEF2A, MEF2B, MEF2C, MEF2D) (Black and Olson, 1998). Initially described as factors regulating muscle development and remodelling (Gossett et al., 1989), MEF2 proteins are now recognized as being ubiquitously expressed and involved in development, cardiac stress, osmotic stress, and immune responses (Ornatsky and McDermott, 1996; McKinsey et al., 2002; Savignac et al., 2007; Barry and Townsend, 2010). The conserved MEF2 transcription factor DNA binding motifs are present in the control regions of numerous muscle-specific genes, making MEF2 proteins essential regulators of structural proteins, contractile proteins, and overall muscle metabolism. They contain key threonine residues in their transactivation domain that become activated upon phosphorylation, allowing them to activate gene expression that adjusts muscle molecular attributes (Black and Olson, 1998). In addition, MEF2 proteins can form complexes with other proteins such as histone deacetylases (HDAC), SMAD, and specificity protein 1 (Sp1), and in this way modulate MEF2 activity to impose either positive and negative controls on muscle metabolism (Grayson et al., 1998; Quinn, 2001; Potthoff and Olson, 2007; Ginnan et al., 2012). During freezing, wood frog tissues experience cell stresses including anoxia, ischemia, hyperglycaemia, osmotic stress, and can experience oxidative stress when oxygen is reintroduced upon thawing (Storey, 1990). These stresses present different functional challenges for skeletal and cardiac muscles, both of which undergo periods of inactivity that can leave them vulnerable to atrophy, a process that results in decreased muscle mass and strength (Bassel-Duby and Olson, 2006). Indeed, studies have shown that wood frog skeletal muscles exhibit substantial (> 30%) decreases in muscle mass during winter conditioning and that despite this proteolytic response, they are still able to partake in strenuous mating upon spring emergence (Costanzo et al., 2015). Skeletal muscles must be able to adapt to changing metabolic demands and resources by altering their metabolic capacity and muscle fibre composition. Wood frog hearts must also be able to endure months in a cryopreserved state and then rapidly regain complete functionality upon thawing. MEF2 transcription factors regulate the expression of

2. Materials and methods 2.1. Animals Male wood frogs (5–7 g body mass) were collected by net from melt water breeding ponds near Oxford Mills, Ontario, Canada in April over several nights (< 1 week) when frogs were chorusing/breeding. Animals were washed in a tetracycline bath and acclimated at 5 °C in plastic containers lined with damp sphagnum moss for two weeks prior to experimentation. Control frogs were randomly sampled directly from this condition. For freezing experiments, frogs were placed in closed plastic containers lined with damp paper towels, and put in an incubator set at −3 °C. A 45 min cooling period was allowed during which the body temperature of the frogs cooled below −0.5 °C, at which point ice nucleation was triggered due to skin contact with ice crystals formed on the paper towels. Following the initial 45 min, incubator temperature was raised to −2.5 °C and the length of freezing was timed from this point, frogs were then randomly sampled after undergoing 4 or 24 h freezing exposures. Frogs randomly assigned to 2

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dissolved in TBST, followed by overnight incubations with the appropriate primary antibodies (1:1000 v-v dilution in TBST) at 4 °C. Primary α-MEF2A and α-phospho-MEF2AThr312 antibodies were purchased from GenScript whereas α-MEF2C and α-phospho-MEF2CThr300 were purchased from Santa Cruz Biotechnology. For both skeletal and cardiac muscle, the MEF2A and MEF2C antibodies detected single bands at approximately 50 kDa and 43 kDa, respectively, in accordance with the expected size of MEF2 transcription factors. This was consistent with immunoblots using antibodies against phosphorylated MEF2AThr312 (pMEF2A) and phosphorylated MEF2CThr300 (p-MEF2C) sites. After probing with primary antibody, membranes were washed in TBST and incubated with anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) (Cell Signalling Technology; 1:8000 v-v dilution in TBST), except for MEF2C that was probed with anti-goat HRP-linked secondary antibody (BioShop, 1:8000v/v dilution in TBST), for 1.5 h at room temperature. Membranes were washed in TBST and bands were visualized with enhanced chemiluminescence (Millipore) on a ChemiGenius Bio-Imaging System (Syngene, Frederick, MD). To visualize total protein, membranes were then stained with Coomassie blue (0.25% w-v Coomassie brilliant blue, 7.5% v-v acetic acid, and 50% v-v methanol).

the thawed recovery group were also frozen for 24 h and then transferred back to 5 °C and sampled after 8 h of thawing. Frogs were euthanized by pithing, hearts and mixed hind leg thigh skeletal muscles were immediately excised, flash frozen in liquid nitrogen, and then stored at −80 °C until use. All animal procedures including protocols for the care, experimentation, and euthanasia of the animals were approved by the Carleton University Animal Care Committee (Protocol # 13683) and followed the guidelines set by the Canadian Council on Animal Care. 2.2. Protein extractions Total soluble protein extracts were prepared from skeletal muscle and heart tissue of control, 24 h frozen, and 8 h thawed wood frogs. Samples (500 mg) of frozen tissue were homogenized with a Polytron PT10 homogenizer (Kinematica) in 1 ml of homogenizing buffer [20 mM HEPES, 0.1 mM EDTA, 1 mM Na3VO4, 200 mM NaCl, 10 mM NaF, 10 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Homogenates were centrifuged for 15 min at 10,000 rpm and 4 °C, and protein-containing supernatants were collected. Nuclear extracts were prepared using modified versions of protocols previously described (Dignam et al., 1983; Mamady and Storey, 2006). Briefly, skeletal muscle samples were homogenized in buffer [10 mM HEPES, 10 mM KCl, 10 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM PMSF, pH 7.9] using a Dounce homogenizer. These lysates were centrifuged for 10 min at 8000g and 4 °C to collect cytoplasmic fractions (supernatant). The nuclear extracts were isolated by resuspending pellets in 150 μl of nuclear extraction buffer [10 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% v-v glycerol, and 1.5 μl PMSF, pH 7.9], with slow mixing on a rocking platform for 1 h at 4 °C. Samples were then centrifuged at 10,000 rpm for 10 min at 4 °C and the supernatant containing the nuclear fraction was collected. Protein concentrations of both total soluble protein extracts and cytoplasmic/nuclear extracts were quantified using the BioRad assay, concentrations were standardized, and aliquots were mixed 1:1 with 2× Laemmli buffer (100 mM Tris-HCl, 4% w-v SDS, 20% v-v glycerol, 0.2 w-v bromophenol, 10% v-v β-mercaptoethanol, pH 6.8), and boiled for 5 min. Samples were stored at −20 °C until use. Integrity of nuclear extracts was confirmed by immunoblotting the cytoplasmic and nuclear fractions for histone H3 (Cell Signalling Technologies), a nuclear marker.

2.4. RNA isolation Total RNA was prepared from ~50 to 100 mg tissue samples. Tissue was homogenized in Trizol reagent using a Polytron PT1200 homogenizer and following manufacturer's protocol (ThermoFisher). Total RNA was quantified by measuring absorbance at 260 nm on a GeneQuant Pro Spectrophotometer (Pharmacia), while maintaining A260/280 ratio > 1.7. RNA integrity was verified by the presence of sharp bands for 28S and 18S ribosomal RNA on a 1% native agarose gel stained with 0.01% ethidium bromide (EtBr) and run at 130 V for 25 min. Samples were frozen at −20 °C until use. 2.5. cDNA synthesis and PCR amplification First strand synthesis was performed using 5 µg of total RNA with oligo-dT primers and SuperScript II reverse transcription system, following manufacturer's instructions (Thermo Fisher). The resulting cDNA was used as a template for PCR amplification of all genes of interest including the reference gene α-tubulin that was used for standardization. Primers were designed from the consensus sequences derived from alignments of sequences from several vertebrate species using MegAlign (DNASTAR) and Primer Designer (Scientific and Educational Software). Primers were synthesized by Sigma Genosys and are listed in Table 1. PCR reactions were performed on a Mastercycler Gradient Thermal Cycler (Eppendorf) in a total volume of 25 μl by mixing 5 μl of cDNA dilution with 1.25 μl of gene specific primer mixture (0.5 μM forward and 0.5 μM reverse primers), 15 μl of DEPC-treated water, 0.5 μl of 10× PCR buffer, 1.75 μl 50 mM MgCl2, 0.5 μl 10 mM dNTPs, and 1 μl of Taq polymerase. The optimized PCR protocol consisted of an initial denaturation step of 2 min at 95 °C, followed by 37–40 cycles of 95 °C for 45 s, optimal annealing temperature for 45 s, and 72 °C for 45 s. This was followed by a final step of

2.3. Immunoblotting SDS-PAGE was run using equal amounts of total protein (10–30 µg for different targets) or 20 µg of nuclear extracted proteins. Samples were run on 10–12% polyacrylamide gels and prepared with 5% upper stacking gels. Proteins were separated by electrophoresis in SDS-PAGE running buffer (25 mM Tris-base, 190 mM glycine, 0.1% w/v SDS) at 180 V for 45 min with a Mini Protean III system (BioRad). Proteins were then electroblotted by wet transfer onto a 0.45 µm polyvinylidene difluoride membrane in a pre-chilled transfer solution (25 mM, Tris pH 8.5, 192 mM glycine, and 20% v/v methanol) at 4 °C for 1.5 h at 160 mA. Membranes were washed in TBST (20 mM Tris base, 140 mM NaCl, 0.05% v-v Tween-20) and blocked for 30 min in 5% w-v milk

Table 1 Forward and reverse primers used for analysis of gene expression by RT-PCR; α-tubulin was used as the reference gene. Gene

Forward primer

Reverse primer

Mef2a Mef2c Glut 4 Calr Ckb Ckm α-tubulin

5′-AGG CTC TGA CAG AAG GAA CA-3′ 5′-GGT TTC CAT TCC GGT GAG CA-3′ 5′-GAS ATG AAG GAG GAG AAG AG-3′ 5′-GAY AAC AGC AAG GTK GAR TC-3′ 5′-GAC AGR CAY GGY GGC TAC AA-3′ 5′-GGA CTG GCC GYA GCA TYA AG-3′ 5′-AAG GAA GAT GCT GCC AAT AA-3′

5′-CAC AGA GAT GTC CGA TGA AC-3′ 5′-TCC TTT GAT TCA CAG AGG GC-3′ 5′-GAA GTT GGM GGT CCA GTT GG-3′ 5′-TTC TTG CGW TCC TCY TCA TC-3′ 5′-GTC TTR TTG TCA TTG TGC CA-3′ 5′-AGT GTC MAC ACC ACC TGT GC-3′ 5′-GGT CAC ATT TCA CCA TCT G-3′

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tubulin was experimentally determined to be a suitable reference gene for this study based on its stable expression in R. sylvatica heart and skeletal muscle under control and frozen conditions. Data are derived from independent protein or RNA extracts with n=3–5 biological replicates, each containing tissue from two or more animals. This was necessary since frogs are small and organ samples from several animals were required to meet sample weight requirements for different extraction processes. Immunoblot data were graphed and statistically analyzed with GraphPad Prism software. A one-way ANOVA was done and where significant differences were detected between groups, a posthoc Bonferroni test was applied (GraphPad Software). PCR data were analyzed using two-tailed Student's t-tests. Graphs show mean values ± SEM and p-value thresholds for significance are; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 1. Analysis of MEF2 transcription factor protein levels in wood frog skeletal muscle over a freeze-thaw cycle using immunoblotting. Upper panel displays the histogram showing protein levels, relative to control, of MEF2A and MEF2C total protein and phosphorylated MEF2AThr312 and MEF2CThr300 under control, 24 h frozen and 8 h thawed conditions; data are means ± SEM of 4 independent biological replicates, except for pMEF2AThr312 (n=3). Data were analyzed using a one-way ANOVA with a Bonferroni post hoc test, *p < 0.05, **p < 0.01. Bottom panel shows representative immunoblots from each group.

3. Results 3.1. Analysis of MEF2 protein levels in response to freezing We analyzed protein relative abundance using immunoblotting with anti-MEF2 antibodies that cross-reacted with wood frog heart and skeletal muscle tissues. In skeletal muscle, MEF2A levels remained constant during freezing and showed a 1.34 ± 0.08 fold (p < 0.01) increase in the thawed condition (Fig. 1). Levels of phosphorylated MEF2AThr312 displayed a 1.98 ± 0.23 fold (p < 0.05) and 2.18 ± 0.26 fold (p < 0.01) increase over controls after 24 h frozen or 8 h thawed, respectively, highlighting the activation of MEF2A in the freeze-thaw cycle of skeletal muscles (Fig. 1). Similar to total MEF2A levels, total MEF2C levels also did not change during freezing, whereas phosphoMEF2CThr300 showed a 2.00 ± 0.23 fold (p < 0.01) increase during freezing (Fig. 1). There were no changes in total MEF2A protein levels in cardiac tissues, however there was a 1.41 ± 0.10 fold (p < 0.05) increase in phosphorylated MEF2AThr312 after 8 h of thawing (Fig. 2). Similarly, no changes in MEF2C or phosphorylated MEF2CThr300 levels were observed in heart tissues (Fig. 2). This demonstrates that although there was generally no change in total protein abundance of MEF2A and MEF2C, both proteins appeared to be activated in skeletal muscle in response to freezing, whereas only MEF2A was phosphorylated during thawing in heart.

72 °C for 4 min. Optimal annealing temperatures were 65 °C for mef2a, mef2c, ckm, and ckb; 60 °C for calr and glut-4; 53 °C for α-tubulin. Aliquots (12 μl) of PCR products were mixed with 1 μl of DNA loading dye (0.25% w-v xylene cyanol FF, 30% v-v glycerol in ddH2O) and run on 1% agarose gels, stained with EtBr and visualized on ChemiGenius Bio-Imaging System (Syngene). PCR reactions were tested over a tenfold cDNA dilution series and quantification of band intensities was performed on the most dilute cDNA samples to ensure signal saturation was not reached. PCR amplicons were gel purified, sequenced at the Ottawa Hospital Research Institute (Ottawa, ON), and validated using BLAST (http://blast.ncbi.nlm.nih.gov/). The confirmed sequences were deposited into GenBank under the following accession numbers: mef2a, KU737543; mef2c, KU737544; glut4, KU737542; calr, KU737541; ckb, KU737540; ckm, KU737545. 2.6. Bioinformatics analysis Wood frog partial cDNA sequences were analyzed using ClustalW on MegAlign (DNASTAR Lasergene 8). The R. sylvatica mef2a (KU737543) sequence was aligned and compared to Xenopus laevis (NM_001101746. 1), Gallus gallus (NM_204864.3), Mus musculus (NM_001033713.2), and Homo sapiens (NM_001319206.1) and R. sylvatica mef2c (KU737544) was compared to X. laevis (NM_001092414.1), G. gallus (XM_ 015280562.1), M. musculus (NM_001170537.1), and H. sapiens (NM_ 002397.4). Wood frog glut-4 sequence (KU737542) was compared to X. laevis (NM_001092138.1), M. musculus (NM_009204.2), and H. sapiens (NM_001042.2), and the calr sequence (KU737541) was compared to X. laevis (NM_001087296.1), G. gallus (XM_015300202.1), M. musculus (NM_007591.3), and H. sapiens (NM_004343.3). Wood frog ckb sequence (KU737540) was compared to X. laevis (NM_001086894.1), G. gallus (NM_205310.1), M. musculus (NM_021273.4), and H. sapiens (NM_001823.4) and the ckm sequence (KU737545) was compared to X. laevis (NM_001086604.1), G. gallus (NM_205507.1), M. musculus (NM_007710.2), and H. sapiens (NM_001824.4).

3.2. Activated MEF2 in nuclei of skeletal muscle during freezing Nuclear MEF2 protein levels were measured in skeletal muscles during freezing and similar to total protein extracts (Fig. 1), skeletal muscle nuclear extracts revealed constant levels of MEF2A during

2.7. Data analysis and statistics Densitometric analysis was performed with Gene Tools software (Syngene) on bands visualized by the ChemiGenius BioImaging System. Immunoblot chemiluminescent band intensity in each lane was normalized against a group of Coomassie-stained protein bands in the same lane to correct for any minor variations in sample loading. For RT-PCR, band intensities were normalized against the corresponding intensity of the α-tubulin band amplified from the same cDNA sample. Alpha-

Fig. 2. Analysis of MEF2 transcription factor protein levels in wood frog heart over a freeze-thaw cycle using immunoblotting. Upper panel displays the histogram showing protein levels, relative to control, of MEF2A and MEF2C total protein and phosphorylated MEF2AThr312 and MEF2CThr300 under control, 24 h frozen and 8 h thawed conditions; data are means ± SEM of 4 independent biological replicates. Data were analyzed using a oneway ANOVA with a Bonferroni post hoc test, *p < 0.05. Bottom panel shows representative immunoblots from each group.

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Fig. 5. Cloning and analysis of mef2 genes and their downstream gene targets in wood frog heart during freezing. Histogram displays standardized relative mRNA transcript levels of mef2a, mef2c and downstream glut4, calr, ckb, and ckm genes under control and 24 h frozen conditions. Transcript levels were internally controlled using α-tubulin as the reference gene. Data are means ± SEM of 4–5 independent biological replicates and were analyzed using a two-tailed Student's t-test, *p < 0.05, **p < 0.01.

Fig. 3. Analysis of nuclear distribution of MEF2 transcription factors in wood frog skeletal muscle during freezing using immunoblotting. Upper panel displays the histogram showing nuclear protein levels, relative to control, of MEF2A and MEF2C total protein and phosphorylated MEF2AThr312 and MEF2CThr300 under control, 4 h frozen and 24 h frozen conditions; data are means ± SEM of 4 independent biological replicates. Data were analyzed using a one-way ANOVA with a Bonferroni post hoc test, *p < 0.05. Bottom panel shows representative immunoblots from each group.

1.97 ± 0.24 fold (p < 0.05) increase in mef2a transcripts and a 1.74 ± 0.05 fold (p < 0.01) downregulation in mef2c (Fig. 5), skeletal muscle MEF2 transcripts remained stably expressed (Fig. 4). 3.4. Transcriptional activation of MEF2 downstream target genes

freezing (Fig. 3). However, a significant increase in phosphorylated MEF2AThr312 was observed as early as 4 h frozen (2.12 ± 0.22 fold; p < 0.05) and persisted after 24 h frozen (1.52 ± 0.06 fold; p < 0.05) (Fig. 3). Similarly, although MEF2C demonstrated no protein level changes in total protein extracts of skeletal muscle (Fig. 1), nuclear fractions showed increases in phosphorylated MEF2CThr300 after 4 h (1.70 ± 0.07 fold; p < 0.05) and 24 h (2.12 ± 0.23 fold; p < 0.05) of freezing (Fig. 3). Collectively, these skeletal muscle results demonstrate that although total MEF2A and MEF2C protein levels showed no change during freezing, a large population of MEF2 proteins appears to be phosphorylated and localized to the nucleus in response to freezing.

To investigate transcriptional activity of MEF2A/C in muscles, the mRNA levels of four MEF2 gene targets were measured using RT-PCR; glucose transporter 4 (glut4), calreticulin (calr), creatine kinase brain isozyme (ckb), and creatine kinase muscle isozyme (ckm). The glut4 PCR product [GenBank accession # KU737542] was a 475 bp fragment that encoded 158 amino acids and corresponded to nucleotides 800–1300 bp of the X. laevis coding sequence and shared 80% sequence identity with X. laevis glut4 (Supp. Fig. 1C). The calr amplicon [GenBank accession # KU737541] was 476 bp in length, which encoded 158 amino acids and aligned to nucleotide positions 630–1100 relative to other vertebrate calreticulin genes and had 74.4–80% identity upon comparison to other vertebrate sequences (Supp. Fig. 1D). The ckb primer amplified a product [GenBank accession # KU737540] of 597 bp (198 amino acids) corresponding to nucleotides 150–750 of reference vertebrate sequences and shared a sequence conservation of 83% with X. laevis (Supp. Fig. 1E). The wood frog ckm product [GenBank accession # KU737545] was 534 bp encoding 177 amino acids; the partial cDNA sequence corresponded to nucleotide positions 450–980 of the reference vertebrate sequences and shared 85% identity with X. laevis (Supp. Fig. 1F). Glut4, calr, ckb, and ckm all had increased transcript levels in both skeletal and heart muscle after 24 h of freeze exposure (Figs. 4 and 5). Glut4 transcripts were increased in skeletal muscle by 1.71 ± 0.24 fold (p < 0.05) and heart by 2.48 ± 0.24 fold (p < 0.05) during freezing (Figs. 4 and 5). Calreticulin transcripts were also increased during freezing by 2.35 ± 0.39 fold (p < 0.05) and 4.85 ± 0.80 fold (p < 0.01) fold in skeletal and heart muscle, respectively (Figs. 4 and 5). Similarly, both creatine kinase brain and muscle isozyme transcripts were significantly upregulated in 24 h frozen muscle by 1.56 ± 0.21 fold and 1.88 ± 0.34 fold, respectively (both p < 0.05) and in heart tissue by 1.68 ± 0.21 fold (p < 0.05) and 2.78 ± 0.40 fold; (p < 0.01) (Figs. 4 and 5). Collectively, these data reveal that transcript levels of the selected MEF2 downstream targets examined were upregulated in response to freezing.

3.3. cDNA cloning and transcript expression of mef2 genes The PCR products of mef2 genes were sequenced to confirm the identity of DNA amplicons and subsequently compared to mef2 coding sequences of other vertebrates. The confirmed mef2a/c sequences were deposited into GenBank under the following accession numbers: mef2a, KU737543 and mef2c, KU737544. The mef2a product was 434 bp and encoded 140 amino acids, which corresponded to nucleotides 400–800 of the X. laevis coding sequence and demonstrated 79% identity with X. laevis (Supp. Fig. 1A). The R. sylvatica mef2c amplicon was 512 bp and encoded 170 amino acids, which corresponded to nucleotides 370–900 of the X. laevis coding sequence, and revealed an 88% identity to X. laevis (Supp. Fig. 1B). Skeletal muscle showed no changes in mef2a or mef2c mRNA levels (Fig. 4), correlating with measurements of protein levels (Fig. 1). Together, these results show that wood frog mef2a and mef2c genes have conserved coding sequences and while they were found to exhibit differential expression in heart during freezing with a

4. Discussion Wood frog organs must adjust to low temperature, dehydration, ischemia and encasement in extracellular ice when they are frozen. During this period of prolonged inactivity muscles may become vulnerable to disuse atrophy potentially requiring molecular adaptations to safely transition into the frozen state and recover after thawing. MEF2 proteins are key muscle-specific transcription factors responsible for muscle differentiation, remodelling, and various aspects of basic cell survival (Bassel-Duby and Olson, 2006). The present study investigated

Fig. 4. Cloning and analysis of mef2 genes and their downstream gene targets in wood frog skeletal muscle during freezing. Histogram displays standardized relative mRNA transcript levels of mef2a, mef2c and downstream glut4, calr, ckb, and ckm genes under control and 24 h frozen conditions. Transcript levels were internally controlled using αtubulin as the reference gene. Data are means ± SEM of 5 independent biological replicates and were analyzed using a two-tailed Student's t-test, *p < 0.05, **p < 0.01.

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thawing in skeletal muscle but p-MEF2C Thr300 rose only during freezing (Fig. 1). Since active phosphorylated forms of MEF2 proteins must translocate to the nucleus to activate transcription of muscle-specific genes (Black and Olson, 1998), we also examined the population of MEF2A/C and p-MEF2A/C in skeletal muscle nuclei. During freezing, levels of p-MEF2A and p-MEF2C increased significantly in skeletal muscle nuclei and p-MEF2C increased even more upon thawing (Fig. 3). This increase in MEF2 phosphorylation corresponds with previously measured increases in the protein abundance and activity of selected MAPK components during wood frog freezing (Greenway and Storey, 2000a). Hence, these results suggest a general trend of MEF2 activation in response to freezing and thawing in skeletal muscle, potentially in response to the selected members of the MAPK signal transduction cascade. While heart mRNA transcript levels of mef2a were significantly elevated during freezing, mef2c levels were significantly downregulated (Fig. 5). This suggests that, unlike skeletal muscle, MEF2A and MEF2C are differentially regulated at the gene expression level in cardiac muscle. The protein levels of total MEF2A, MEF2C and p-MEF2C remained constant over the freeze-thaw cycle, with only p-MEF2A showing a significant increase in relative protein levels after 8 h thawed (Fig. 2). The reduced MEF2 protein response measured in frozen hearts may be due to the much higher concentrations of glucose cryoprotectant sequestered in hearts, as compared to skeletal muscles (Storey and Storey, 2017), suggesting that cardiac tissues may be less prone to freezing damage. Our findings also show a significant upregulation of pMEF2A relative to control levels during thawing (Fig. 2); this thawspecific upregulation was also observed for skeletal muscle p-MEF2A and MEF2A (Fig. 1). The upregulation of MEF2 proteins during thawing suggests that they play a role in facilitating recovery through their control over the gene transcription of proteins involved in energy homeostasis and muscle repair. This finding warrants further investigation. An incongruency was observed between heart MEF2 transcript and protein levels and may be brought about by the energy stress that cells face. While MEF2 proteins are likely playing an important role, the synthesis of new proteins including MEF2 under freezing conditions is costly. Instead, the cell can make the existing pool of MEF2 proteins more active through the use of post-translational modifications, which can result in a more active transcription factor as evident by the increased expression of downstream genes under MEF2 control (Fig. 5). The lack of perfect correlation between MEF2 transcript and protein abundance levels could also potentially be a result of various posttranscriptional regulatory mechanisms such as microRNAs that have been shown to facilitate freeze tolerance in wood frogs (Bansal et al., 2016). However, Fig. 2 shows no changes in the active phosphorylated population of MEF2 proteins in heart during freezing, which is discordant with the downstream gene upregulation reported in Fig. 5. While phosphorylation is known as the main regulatory PTM affecting MEF2, other PTMs can also increase MEF2C binding to DNA and enhance transcriptional activity. These could include acetylation at K234, K239, K252, or K264 that were not examined in this study but might be involved in the MEF2-based upregulation of downstream genes (Ma et al., 2005). Furthermore, although the gene transcripts examined herein are documented MEF2 downstream gene targets, it is possible that their upregulation during freezing could also be affected by the action of other transcription factors. For example, glucose transporter 4 is also under transcriptional regulation by GLUT4 enhancer factor (GEF) and myogenic differentiation factor D (MyoD) (Knight et al., 2003; Zorzano et al., 2005), calreticulin is regulated by GATA binding protein 6 (GATA6) (Qiu et al., 2008), and creatine kinase is also regulated by MyoD (Bera and Ray, 2009). Our findings reveal that during freezing, MEF2A and MEF2C are post-translationally regulated by phosphorylation in skeletal muscle. To investigate whether the measured increase in nuclear localization and MEF2 phosphorylation corresponded with increased rates of transcription, we measured transcript levels of MEF2 downstream gene targets

two MEF2 transcription factors, MEF2A and MEF2C, over the freezethaw cycle in wood frog skeletal and cardiac muscle at transcriptional, translational, and post-translational levels. Furthermore, four downstream genes under MEF2 regulatory control – glucose transporter-4, calreticulin, and the brain and muscle isozymes of creatine kinase – were evaluated due to their potential involvement in modulating muscle metabolism during freeze/thaw. Our findings indicate that MEF2 proteins respond to freezing in a tissue-specific manner and suggest roles for them in coordinating cryoprotectant distribution, calcium homeostasis, balancing energy reserves, and potentially protecting skeletal muscles against atrophy. MEF2 transcription factors have recently been shown to play a significant role in muscle adaptation during hibernation in thirteenlined ground squirrels. They were activated in muscles of torpid squirrels and it was suggested that MEF2s coordinated the muscle remodelling required to prevent skeletal muscle disuse atrophy during hibernation and facilitate the cardiac hypertrophy that aids the heart in pumping cold viscous blood during torpor (Tessier and Storey, 2010, 2012). MEF2s are activated by phosphorylation in response to various extracellular signals that are transmitted from the cell membrane to the nucleus via signalling cascades that include the well-characterized mitogen-activated protein kinases (MAPKs) as well as phosphorylation by other protein kinases including Ca2+/calmodulin-dependent protein kinase (CaMK) (Zhao et al., 1999; Blaeser et al., 2000; Kato, 2000). The nature of such post-translational modifications makes them ideal candidates for facilitating and coordinating metabolic responses to stress since they are; [1] easily inducible, [2] able to elicit large changes in protein activity, [3] ATP-inexpensive, and [4] readily reversible (Storey, 2015). Indeed, previous studies have identified phosphorylation as a key regulator of metabolic activity and gene expression during periods of hypometabolism in various evolutionary distant species, further emphasizing the high degree of conservation of this regulatory mechanism in facilitating stress responses (Dieni and Storey, 2009; Humphrey et al., 2015). MEF2 proteins can be phosphorylated by kinases from a number of signalling pathways including p38, ERK5, and Ca2+ signalling (McKinsey et al., 2002; Potthoff and Olson, 2007). Studies ranging through yeast, plants, and mammals demonstrate that p38 MAPK responds to osmotic stress and hypothermia (Roberts et al., 2002; Clanachan et al., 2003; Sheikh-Hamad and Gustin, 2004). The ERK5 pathway has been shown to respond to oxidative stress and hyperosmotic stress (Nishimoto and Nishida, 2006). In fact, the p38 MAPK pathway is known to be activated in response to anoxia in the anoxiatolerant red-eared slider turtle and freezing in the wood frog (Greenway and Storey, 2000a, 2000b). The post-translational modification sites investigated in this study (MEF2AThr312 and MEF2CThr300) are activating phosphorylation sites for p38, as well as ERK5 MAPK (Zhao et al., 1999; Kato, 2000). Interestingly, p38 MAPK activation has also been observed in response to hyperosmotic and hypothermic conditions in the heart of the marsh frog, Rana ridibunda (Aggeli et al., 2001, 2002). Thus, it is highly possible that the phosphorylations of MEF2A and MEF2C described below are a result of p38 MAPK signalling activation in response to freezing. Herein, we demonstrate a novel freeze-response by MEF2A/C proteins through phosphorylation, nuclear localization, and the subsequent transcriptional upregulation of downstream genes. An initial examination mef2a and mef2c transcript levels in skeletal muscle showed that these remained constant in frozen frogs (Fig. 4). Likewise MEF2C protein levels were unchanged, whereas MEF2A protein levels increased significantly, relative to the control, during skeletal muscle thawing (Fig. 1). However, total MEF2 transcript and protein levels alone are not indicative of their functionality since MEF2 proteins must first be phosphorylated to be activated. As such, we investigated changes in the relative phosphorylation of p-MEF2AThr312 and pMEF2CThr300, both of these sites being activating phosphorylations. Relative levels of p-MEF2AThr312 increased during both freezing and 6

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months, despite protein levels remaining constant over the freeze-thaw cycle in skeletal muscle, and that this increased activity was regulated by protein phosphorylation (Dieni and Storey, 2009). Thus, it can be suggested that the observed upregulation of ckb and ckm may be an anticipatory response in muscles to support creatine kinase protein synthesis during freezing and thawing. It also shows that creatine kinase is regulated at various levels and that MEF2s are helping to preserve muscle functionality and metabolism during cold hypometabolic periods. Indeed, the energy stress introduced during the interruption of heartbeat and breathing must be stabilized and one such mechanism is the appropriate regulation of creatine kinase to sustain the adenylate pool in these high ATP-turnover tissues (Dieni and Storey, 2009). This observed transcriptional regulation of creatine kinase implicates MEF2 signalling as an important player in the maintenance of high phosphagen levels in wood frog muscle as an adjunct to ATP production via glycolysis in the frozen state. In conclusion, the present study describes a novel freeze-responsive activation of the MEF2 transcription factor family in the skeletal muscles of the freeze tolerant wood frog. We demonstrate that this activation is primarily brought about by increased levels of phosphorylated MEF2 proteins in skeletal muscle and their translocation to the nucleus. This was evident in the freeze-induced significant upregulation of the MEF2 downstream gene targets, glut4, calr, ckb, and ckm in both muscle types. However, the mechanisms by which the downstream genes were upregulated in heart warrant further investigation. The regulation of MEF2 activity in the freeze tolerant frog provides insights into the molecular mechanisms of cryoprotectant glucose transportation, calcium homeostasis, and phosphagen energy reserves essential for successful freezing survival. The tissue-specific activation pattern observed herein suggests that MEF2 proteins may be involved in protecting against muscle atrophy and mitigating permanent tissue damage in skeletal muscles, which are more susceptible to atrophy than cardiac muscles. Understanding the regulation of the essential gene networks necessary to survive freezing, such as MEF2, in a vertebrate model of natural freeze tolerance can provide us with potential candidate targets for the development of better organ cryopreservation techniques and provides us with novel insights regarding MEF2implicated muscle disuse atrophy conditions.

that could be implicated in freezing, such as glut4 that has been described as being positively regulated by MEF2 factors (Thai et al., 1998). Glucose is the main cryoprotectant produced by wood frogs as a result of freeze-stimulated activation of glycogenolysis in liver which is activated within 2–5 min after ice formation is triggered on the skin surface (Storey and Storey, 1992). As a result, plasma glucose levels rise quickly from ~5 mM in control frogs to 150–300 mM and glucose is quickly taken up by all tissues. As such, the efficient cellular uptake of glucose is crucial for survival, further emphasizing the importance of glucose transporters during freezing (Storey, 1990). Glut4 was found to significantly increase in both skeletal muscle and heart after a 24 h freezing period (Figs. 4 and 5). Indeed, glucose transport has been documented to increase in the liver of autumn wood frogs preparing for the repeated freeze-thawing of winter, as compared to frogs collected in summer, that have adjusted their metabolic needs to the season (King et al., 1995). Moreover, microarray screens of frozen wood frog hearts revealed that glut4 was upregulated following 24 h of freezing (Storey, 2004). Here, we validate previous glucose transporter results and demonstrate a likely mechanism for the observed upregulation and transcriptional activity of MEF2 factors in skeletal muscle. This increase does not appear to be limited to just the glut4 isoform since a study reported increased abundance and activity of glut2 in the liver of Alaskan wood frogs as compared to the less freeze tolerant Ohioan wood frog and R. pipiens, emphasizing its important role in freeze survival (Rosendale et al., 2014). MEF2 factors have also been described as transcriptional regulators of the chaperone protein, calreticulin (Lynch et al., 2005). Calreticulin is a Ca2+-binding protein involved in many cellular functions such as Ca2+ transport, Ca2+ storage, and Ca2+-dependent transcription, ensuring proper protein folding, and in the ER unfolded protein response (Ellgaard and Helenius, 2003; Groenendyk et al., 2004). Calreticulin mRNA transcript levels displayed a pronounced freezeresponsive increase in both skeletal (2.35 ± 0.39 fold) and cardiac (4.85 ± 0.80 fold) muscle gene expression, an increase likely facilitated by the MEF2 transcriptional activation observed during freezing (Figs. 4 and 5). The upregulation of calreticulin suggests a potential increase in MEF2 activity during freezing as MEF2 and calreticulin regulate each other in an intricate feedback mechanism. MEF2C controls the expression of calreticulin which in turn regulates the release of Ca2+ from the ER, thereby altering the levels of Ca2+ in the cell and affecting the status of MEF2 activity (Barry and Townsend, 2010). Additionally, calreticulin has also been implicated in wound healing, suggesting that its increased expression levels could be contributing to the defence against mechanical ice damage (Gold et al., 2006). Furthermore, the exaggerated increase in calreticulin measured during freezing in cardiac tissue could be a result of the cardioacceleration that accompanies the initiation of freezing (Fig. 5) (Layne et al., 1989). The strong increase of heart rate during the early minutes and hours of freezing helps to ensure the distribution of the cryoprotectant, glucose, from the liver to all other organs (Layne et al., 1989). Calreticulin is responsible for maintaining the Ca2+ homeostasis required to sustain rapid muscle contractions and activity, therefore the observed large abundance of calreticulin mRNA transcripts could possibly be a residue of the short burst of high activity that the wood frog heart experiences during cardioacceleration in the early stages of freezing. Creatine kinase brain and muscle isozymes (ckb and ckm) are also under the transcriptional control of MEF2 transcription factors (Amacher et al., 1993; Shen et al., 2002). Creatine kinase buffers phosphagen energy reserves by catalyzing the transfer of ATP to creatine, generating phosphocreatine, which serves a cellular energy reserve. Creatine kinase is present in tissues with high ATP-turnover such as muscles that expend a lot of energy during contractions (Wallimann et al., 1992). Both creatine kinase brain and muscle isozyme mRNA transcript levels were found to be significantly upregulated in skeletal muscle and heart (Figs. 4 and 5). Previous work has shown that muscle creatine kinase activity increases during the winter

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