MicroRNAs facilitate skeletal muscle maintenance and metabolic suppression in hibernating brown bears

Hibernating brown bears, Ursus arctos, undergo extended periods of inactivity and yet these large hibernators are resilient to muscle disuse atrophy. Physiological characteristics associated with atrophy resistance in bear muscle have been examined (e.g., muscle mechanics, neural activity) but roles for molecular signaling/regulatory mechanisms in the resistance to muscle wasting in bears still require investigation. Using quantitative reverse transcription PCR (RT‐qPCR), the present study characterized the responses of 36 microRNAs linked with development, metabolism, and regeneration of skeletal muscle, in the vastus lateralis of brown bears comparing winter hibernating and summer active animals. Relative levels of mRNA of selected genes (mef2a, pax7, id2, prkaa1, and mstn) implicated upstream and downstream of the microRNAs were examined. Results indicated that hibernation elicited a myogenic microRNA, or “myomiR”, response via MEF2A‐mediated signaling. Upregulation of MEF2A‐controlled miR‐1 and miR‐206 and respective downregulation of pax7 and id2 mRNA are suggestive of responses that promote skeletal muscle maintenance. Increased levels of metabolic microRNAs, such as miR‐27, miR‐29, and miR‐33, may facilitate metabolic suppression during hibernation via mechanisms that decrease glucose uptake and fatty acid oxidation. This study identified myomiR‐mediated mechanisms for the promotion of muscle regeneration, suppression of ubiquitin ligases, and resistance to muscle atrophy during hibernation mediated by observed increases in miR‐206, miR‐221, miR‐31, miR‐23a, and miR‐29b. This was further supported by the downregulation of myomiRs associated with a muscle injury and inflammation (miR‐199a and miR‐223) during hibernation. The present study provides evidence of myomiR‐mediated signaling pathways that are activated during hibernation to maintain skeletal muscle functionality in brown bears.


| INTRODUCTION
Hibernators are able to endure extreme environmental conditions that cannot be tolerated by most mammals. Typically, for small mammal hibernators, seasonal torpor is characterized by a prolonged state of inactivity, a strong decrease in core body temperature to near-ambient values, and the suppression of basal metabolic rate to levels often less than 10% of euthermic values (Heldmaier, Ortmann, & Elvert, 2004;Jastroch et al., 2016;Ruf & Geiser, 2015;. However, the iconic hibernators, bears (Ursidae), stand out from these others in several characteristics. Their large body mass makes it difficult to substantially decrease their body temperature such that core body temperature remains at 30-35°C during hibernation and they have the impressive ability to endure up to 6 months of inactivity without arousing and with no food or water intake, no urination or defecation, no muscle wasting, and no accumulation of nitrogen-containing waste products due to an active recycling program (Berg von Linde, Arevström, & Fröbert, 2015). Previous studies of bears also showed no statistically significant changes in skeletal muscle fiber size between hibernating and nonhibernating animals (Hershey, Robbins, Nelson, & Lin, 2008;Tinker, Harlow, & Beck, 1998). In mammals, weight-bearing activities are crucial for the maintenance of skeletal muscle mass, morphology, and protein composition (Baldwin & Haddad, 2001). Yet despite drastically reduced weight-bearing activity during hibernation, the skeletal muscle of bears retains most of its strength, with only a 23% decrease in maximal force (elicited by electrical stimulation; Harlow, Lohuis, Beck, & Iaizzo, 2001), whereas reduction in force and size as high as 60% is common in other nonhibernating mammals (Baldwin & Haddad, 2001). In small hibernating mammals, up to 20-40% of muscle protein content is lost at low body temperature (Steffen, Koebel, Musacchia, & Milsom, 1991;Wickler, Hoyt, & van Breukelen, 1991;Yacoe, 1983). However, hibernating bears lose only 4-11% of muscle protein content, and the size and number of their muscle fibers remain unchanged (Tinker et al., 1998).
In an attempt to better understand these observations, another study investigated the importance of the signaling pathways that underlie the resilience of skeletal muscle in hibernating brown bears (Ursus arctos). The authors challenged the concept that mechanical loading and neural activity of the muscles are the primary factors in maintaining skeletal muscle size (Lin, Hershey, Mattoon, & Robbins, 2012). Indeed, studies on other nonhibernating mammals have demonstrated that regular neural activation or electrical stimulation of muscles is not enough to curtail muscle atrophy induced by mechanical unloading (Haddad, 2006;B. T. Zhang et al., 2010). Given that there is decreased muscle loading during hibernation, these authors studied the differences between the skeletal muscles (long distal extensor and cranial tibial) of hibernating and nonhibernating U. arctos that had undergone transections of the common peroneal nerve (Lin et al., 2012). Their results showed that active brown bears with denervation exhibited significantly higher muscle mass loss as compared to hibernating bears with denervation (Lin et al., 2012). The authors, therefore, suggested that a third factor is involved, proposing that differential regulation of signaling pathways, such as those that mediate the ubiquitin-mediated catabolism of contractile proteins, must be involved in atrophy resistance (Lin et al., 2012).
The observed resistance to muscle atrophy in hibernating bears could be elicited by signaling pathways that mediate changes in regulatory microRNAs. MicroRNAs are a class of highly conserved small noncoding RNAs that are capable of mediating the fate of mRNA transcripts in response to diverse signaling pathways in cells (Bartel, 2004;. As a component of RNA-induced silencing complexes, microRNAs contribute to posttranscriptional regulation by binding to complementary mRNAs to promote their degradation or inhibit their translation into functional proteins. MicroRNAs are differentially regulated in a number of small mammal hibernator models in a tissue specific manner, contributing to metabolic control, antioxidant defenses, and muscle maintenance networks during hibernation (Hadj-Moussa et al., 2016;Kornfeld, Biggar, & Storey, 2012;Luu, Biggar, Wu, & Storey, 2016; but, as yet, have not been evaluated in the context of bear hibernation. One direct regulator of skeletal muscle microRNA (myomiR) expression is the myocyte enhancer factor-2A (MEF2A) transcription factor, and its targets include miR-1, miR-133, and miR-206 (Liu et al., 2007;Rao, Kumar, Farkhondeh, Baskerville, & Lodish, 2006). The MEF2A transcription factor is known to be crucial for skeletal muscle development, maintenance, and regeneration (Taylor & Hughes, 2017). Previous studies have demonstrated that MEF2A signaling is activated in the skeletal muscle of 13-lined ground squirrels (Ictidomys tridecemlineatus) during hibernation and may contribute to muscle maintenance (Tessier & Storey, 2010). Likewise, a study that profiled 10 microRNAs known to affect muscle-specific factors revealed that expression of these small noncoding transcripts is also differentially regulated in favor of skeletal muscle maintenance of hibernating bats (Myotis lucifugus; Kornfeld et al., 2012).
The present study explores whether signaling pathways that impede muscle wasting are activated during hibernation in U. arctos.
To address this, U. arctos mRNA and microRNA gene transcript sequences were obtained from available genomic data. RNA was isolated from skeletal muscle biopsies of six brown bears that were sampled during summer active and winter hibernating conditions. Specifically, mRNA transcript levels of MEF2A were measured by RT-qPCR along with a group of 36 myomiRs that are known to control skeletal muscle regeneration, metabolism and/or be under the control of MEF2A (Diniz & Wang, 2016;Horak, Novak, & Bienertova-Vasku, 2016;Lima et al., 2017). Furthermore, selected downstream mRNA targets including myostatin (mstn), AMP-activated protein kinase (AMPK) catalytic subunit alpha 1 (prkaa1), paired box protein 7 (pax7), and inhibitor of DNA 2 (id2) that are known targets of the subset of microRNAs assessed herein were also quantified to better understand the potential roles of these microRNAs during bear hibernation. | 3985 counties, Sweden. Their immobilization was performed as previously reported (Evans et al., 2012), to allow sampling during hibernation in late February 2015 or 2016 and again during the active summer period in early June of the same years. After darting in their den (winter) or from a helicopter (summer), immobilized individuals were moved onto an insulated blanket before tissue sampling. Biopsies of the vastus lateralis muscle were collected and immediately frozen on dry ice before transfer to storage at −80°C until analysis.

| Sampling of bear muscle
The mean body mass of hibernating bears (46.8 ± 8.7 kg; 25-74 kg) was not significantly different (p = .062; paired t test) from that of the same bears when resampled as summer-active animals (54.4 ± 7.3 kg; 37.8-83.6 kg).
All procedures complied with Swedish laws and regulations.

| Total RNA isolation
Total RNA was isolated from frozen tissue using TRIzol reagent (Invitrogen, Carlsbad, CA), as previously described (Luu, Green, Childers, Holahan, & Storey, 2017). Briefly, samples of frozen bear muscle (~50 mg) were homogenized 1:20 w/v in TRIzol reagent and then combined with 300 µl of chloroform. Samples were vigorously shaken by hand and incubated at room temperature for 5 min before centrifugation at 10,000g for 15 min at 4°C. The aqueous phase of each sample was then combined with 500 µl isopropanol, incubated at room temperature for 15 min, and then centrifuged at 12,000g for 15 min at 4°C to facilitate RNA precipitation. RNA pellets were washed with 70% ethanol and air dried before resuspension in autoclaved RNase-free water. To standardize concentrations across samples, RNA concentration and purity (260/280 nm ratio) was assessed using a BioTek Take3 plate in a spectrophotometer (BioTek, Winooski, VT). The integrity of RNA was assessed by the appearance of 28S and 18S ribosomal RNA bands on a 1% agarose gel stained with SYBR Green dye.

| RNA polyadenylation and cDNA synthesis
For analysis of microRNAs, samples of bear muscle RNA were polyadenylated and then cDNA synthesis was conducted as previously described ) using a Poly(A) polymerase tailing kit (Epicenter, Madison, WI) with a modified adapter primer (Table S1) for use with short RNA molecules.
Synthesis of cDNA was performed separately for mRNA quantification as previously described (Holahan, Smith, Luu, & Storey, 2018). Standard oligo-dT (5′-TTTTTTTTTTTTTTTTTTTTTTV-3′; where V = A, G, or C) was combined with RNA (1 μg) in a total volume of 10 μl of RNase-free water. Samples were incubated at 65°C for 5 min in an Eppendorf thermocycler, and then cooled on ice.
Next, 4 μl of 5× first strand buffer, 2 μl of 0.1 M DTT, 1 μl of 25 mM dNTPs, and 1 μl of MMLV reverse transcriptase enzyme (Invitrogen) were combined with the RNA/oligo-dT samples. Reactions were incubated at 42°C for 60 min in a thermocycler to facilitate cDNA synthesis.

| Primer design
MicroRNAs investigated in this study were selected from the literature based on their expression in skeletal muscle and their roles in regulating muscle cell processes (Diniz & Wang, 2016;Horak et al., 2016;Lima et al., 2017). To determine the equivalent microRNA sequences in U. arctos, Mus musculus premicroRNA sequences were obtained from miRBase (miRBase v.22; http:// www.mirbase.org). Each M. musculus pre-microRNA sequence was aligned against the U. arctos WGS genome (SRA Accession No. SRX156136) using Sequence Read Archive Nucleotide BLAST (NCBI SRA BLAST; https://www.ncbi.nlm.nih.gov/sra). The conserved U.
From these alignments, the sequences of mature U. arctos microRNA were determined. Primers for relative microRNA quantification were designed as previously described  and are listed in Table S1.

| RT-qPCR
Relative quantification of gene transcripts used a BioRad CFX96 Connect System (BioRad, Hercules, CA). Reagents for RT-qPCR were prepared as previously described (Pellissier, Glogowski, Heinemann, Ballivet, & Ossipow, 2006). Briefly, each amplification reaction a total volume of 20 μl. Each RT-qPCR reaction was analyzed with a melt curve analysis to ensure that the reaction was not amplifying more than one product and primers that amplified multiple products were not used for quantification. For relative quantification of microRNAs, reactions were brought to 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, as previously described . Relative quantification of mRNAs used a modified protocol, where thermocycler conditions were 50 cycles of 95°C for 10 s, 57°C for 20 s, and 72°C for 20 s. Standard curves were performed with pooled complementary DNA samples and primer sets to assess the reaction specificity and efficiency for each gene transcript, and the efficiency of the primer pair was always used for quantification calculations.

| Statistical analyses
Since sampling of tissues was done in a paired manner where each bear was sampled (and tagged) in the winter and then sampled again the following summer, paired t-tests were used to determine statistically significant differences (p < .05) in microRNA and mRNA levels. Amplified transcripts were normalized against the expression of reference genes (U6 small nuclear RNA) for microRNA and gapdh for mRNA). The expression of these reference genes was determined to be stable between summer and winter hibernation conditions, as instructed (Schmittgen & Livak, 2008). The comparative ΔΔC t method was used for relative expression calculations (Schmittgen & Livak, 2008). Expression values for microRNA and mRNA were presented as mean ± standard error of mean with n = 6 bears for microRNA analysis and n = 5 bears for mRNA analysis). SigmaPlot v.12.5 was used for statistical analysis and bar graph generation (Systat Software Inc., San Jose, CA).
A subset of 11 myomiRs that are implicated in the atrophy and regeneration of skeletal muscles (Diniz & Wang, 2016) were also assessed. Specifically, miR-23a-5p, miR-23a/b-3p, miR-125b-3p, miR-199a-5p, miR-199a-3p, miR-221-3p, miR-222-5p, miR-222-3p, miR-223-5p, miR-223-3p, and miR-31-5p were analyzed. Three micro-RNAs, miR-23a-5p, miR-221-3p, and miR-31-5p were elevated during hibernation by 1.32 ± 0.12, 1.31 ± 0.11, and 1.26 ± 0.09 fold, respectively ( Figure 3a). Oppositely, miR-199a-5p and miR-223-5p decreased to 0.66 ± 0.04 and 0.70 ± 0.06, respectively, of the levels in summer active bears (Figure 3a). Separately, transcripts of the MicroRNA and mRNA transcripts were standardized against the expression of U6 and gapdh, respectively. Data are means ± standard error of mean, n = 6 for microRNA and n = 5 for mRNA individuals sampled in both winter hibernating and summer active states. Statistically significant differences are denoted with an asterisk (*) and were determined with a paired t-test (p < .05) F I G U R E 3 Expression of microRNAs linked with skeletal muscle regeneration and maintenance, and one downstream mRNA target, in skeletal muscle of the hibernating brown bear, U. arctos. (a) Relative abundance of microRNAs implicated in muscle maintenance during hibernation, and (b) transcript levels of the downstream target, mstn. MicroRNA and mRNA transcripts were standardized against the expression of U6 and gapdh, respectively. Data are means ± standard error of mean, n = 6 for microRNA and n = 5 individuals sampled in both winter hibernating and summer active states. Statistically significant differences are denoted with an asterisk (*) and were determined with a paired t-test (p < .05) result in more MEF2A-mediated gene transcription. Indeed, the MEF2A transcription factor is well known for its role in skeletal muscle development, maintenance, and regeneration (Taylor & Hughes, 2017), and its upregulation at the mRNA and protein levels have been shown to facilitate skeletal muscle maintenance during hibernation in ground squirrels (Tessier & Storey, 2010.
Therefore, enhanced mef2a transcript levels may be an important underlying mechanism of skeletal muscle resilience in hibernating U. arctos.
Downstream targets of the MEF2A transcription factor were also assessed to elucidate the activated networks in skeletal muscles of bears during hibernation. MEF2A is known to directly regulate skeletal muscle microRNAs, which includes miR-1, -133, and -206 (Liu et al., 2007;Rao et al., 2006) and thereby has important roles in the regulation of skeletal muscle metabolism (Horak et al., 2016). The present study found that miR-1b-5p, miR-133a-3p, and miR-206-3p all increased during hibernation (Figure 1b), which is consistent with the observed increase of mef2a transcripts (leading to elevation of MEF2A protein). Downstream consequences on myogenic signaling pathways by miR-1 and miR-206 include downregulation of the pax7 gene, that encodes a transcription factor that is responsible for upregulating id2 mRNA (J.-F. Chen et al., 2010;Dey et al., 2011). In turn, the ID2 protein is responsible for downregulating the myogenic transcription factor MYOD1 that, similarly to MEF2A, has a role in facilitating skeletal muscle growth and development through broad roles including the expression of miR-1 and miR-206 (Horak et al., 2016). Together, this suggests that activation of the MEF2Amediated signaling pathway in hibernating bear skeletal muscle occurs through the upregulation of miR-1 and miR-206 and consequent downregulation of pax7 and id2 (Figure 1c).
Analyses of other microRNAs in hibernating U. arctos included a subset of 15 microRNAs that target metabolic processes (Figure 2a; Lima et al., 2017). Of these, six (miR-27b-5p, miR-27a/b-3p, miR-29a-5p, miR-29a-3p, miR-29b/c, and miR-33-5p) were elevated during hibernation, compared with the summer active condition. The increases in miR-27, miR-29, and miR-33 found in hibernating bears suggest that these microRNAs may be potentially facilitating decreased glucose utilization. In a previous study, miR-27 action was shown to decrease glucose consumption when overexpressed in rat myoblast cell lines, and this was associated with decreased GLUT4 protein levels (Zhou et al., 2016). Indeed, when antisense sequences were introduced to suppress the endogenous miR-27 activity in these cell lines, glucose consumption and uptake were enhanced and GLUT4 protein levels increased (Zhou et al., 2016).
Thus, it can be postulated that elevated miR-27 in hibernating bears may be downregulating GLUT4 protein in skeletal muscle. In another study, a role for miR-29a/b/c in skeletal muscle glucose metabolism was indicated because these microRNAs were upregulated in a model of diabetic rats (He, Zhu, Gupta, Chang, & Fang, 2007). Further experiments with cell lines showed that overexpression of miR-29a/ b/c inhibited insulin-stimulated glucose uptake (He et al., 2007).
These previous studies support the notion that increased miR-27 and miR-29 in hibernating bear muscle facilitate decreased glucose metabolism in part by reducing glucose uptake, which may also contribute to lowering basal metabolic rate.
Levels of prkaa1 mRNA (that encodes the catalytic subunit of the nutrient sensor AMPK) were also measured in hibernating U. arctos skeletal muscle because expression of this gene known to be downregulated by miR-33 (Davalos et al., 2011;Rottiers et al., 2011). Skeletal muscle AMPK has several regulatory roles in glucose and lipid metabolism, two of these being to increase glucose uptake and promote fatty acid oxidation as a means of enhancing ATP production under energy-stressed conditions (Collier, Bruce, Smith, Lopaschuk, & Dyck, 2006;Jeon, 2016;O'Neill et al., 2014). As mentioned above, increases in miR-27, miR-29, and miR-33 in the bear muscle may suggest that both glucose uptake and fatty acid oxidation are suppressed in hibernating bears. Thus, levels of muscle prkaa1 mRNA were assessed to determine how this downstream gene of microRNA action responded in hibernation.
However, mRNA levels of prkaa1 did not change in muscle of hibernating bears, compared with summer active animals, suggesting that AMPK is not differentially regulated in summer vs winter, at least at the transcript level ( Figure 2b). The AMPK activity increased in some tissues of other hibernating species (e.g., in adipose of hibernating ground squirrels) during hibernation but was unchanged in other tissues including skeletal muscle (Horman, Hussain, Dilworth, Storey, & Rider, 2005), The differentially expressed microRNAs identified in this study also have implications for skeletal muscle regeneration and integrity.
In particular, miR-206, miR-221, and miR-31 that were elevated in hibernating bear muscle are known promoters of skeletal muscle regeneration (Figures 1b and 3a; Diniz & Wang, 2016;Greco et al., 2009;Togliatto et al., 2013). Two of the microRNAs that were reduced during hibernation, miR-199a and miR-223, are associated with inhibited myogenic differentiation, muscle disruption, injury, and inflammation ( Figure 3a; Alexander et al., 2013;Y. Chen, Melton, Gelfond, McManus, & Shireman, 2012). Similarly to miR-27b and miR-29b (discussed above), miR-23a increases in hibernating bear muscle, and these three myomiRs not only increase in other species during hibernation (e.g., the little brown bat, Myotis lucifugus), but are also associated with muscle atrophy resistance, increased MEF2A activity, and decreased transcript levels of mstn that encodes myostatin, an inhibitor of muscle growth (Figure 2a and 3a; Kornfeld et al., 2012;Miretti et al., 2013). In the present study, mstn mRNA levels were assessed in U. arctos skeletal muscle, but these did not change between summer active and hibernation conditions LUU ET AL.

| 3989
( Figure 3b). However, a lack of microRNA interaction with mstn mRNA cannot be ruled out because microRNAs function not only to facilitate mRNA degradation, but also manage mRNA storage, during which transcript levels can remain constant but their translation into protein is inhibited (Bartel, 2004). Indeed, mRNA storage appears to be a common feature of metabolic rate depression in multiple species and in response to a wide variety of environmental stress conditions, including hibernation Tessier, Audas, Wu, Lee, & Storey, 2014). MiR-23a also promotes resistance to muscle atrophy by disrupting protein degradation. The mechanism by which this occurs is the direct suppression of two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1, that are known to be induced during muscle atrophy and atrophy-associated protein degradation (Wada et al., 2011). Protein levels of ubiquitin ligases such as these are known to be modulated throughout the torpor- shown that miR-29 inhibits SREBP-1 expression in cancer cells (Ru et al., 2016). Although SREBP-1 has mostly been studied in the liver for its roles in metabolic regulation, studies have shown that it is significantly expressed in muscle cells (Ducluzeau et al., 2001;Guillet-Deniau et al., 2002), and furthermore, SREBP-1 nuclear protein content induces muscle atrophy in both in vitro and in vivo models (Lecomte et al., 2009). Many experiments have identified that SREBP regulates many myogenic and metabolic genes in skeletal muscle (Abiola et al., 2009;Lecomte et al., 2009;Rome et al., 2008).
Of interest among SREBP targets are myogenic transcription factors MYOD1, MYOG, and MEF2C, which have shown to decrease with SREBP overexpression (Dessalle et al., 2012). Furthermore, SREBP is believed to induce skeletal muscle atrophy by leveraging the ubiquitin ligase system, in part by upregulating MuRF1 mRNA (Lecomte et al., 2009). The present results suggest that a similar effect might occur in bears, where downregulation of SREBP by miR-33 and/or miR-29 facilitates a cytoprotective effect in skeletal muscle.

| CONCLUSION
The present study sought to assess the role of microRNAs as regulatory factors in limiting atrophy of U. arctos skeletal muscle during winter hibernation. Analysis of selected muscle-specific microRNAs and mRNA transcripts indicated broad modulations occurring at both transcriptional levels, which were suggestive of posttranscriptional mechanisms activating myogenic pathways, suppressing glucose and lipid metabolism, and decreasing atrophic signaling during hibernation (Figure 4). The results support a F I G U R E 4 Proposed microRNA-mediated mechanism facilitating metabolic suppression and skeletal muscle maintenance in U. arctos during hibernation. Perforated boxes denote areas of the model supported by this study. Red font and asterisks denotes targets and areas warranting further investigation hypothesis made previously that the modulation of cellular signaling pathways is crucial for the resilience of skeletal muscles against atrophy in hibernating brown bears (Lin et al., 2012). These current results warrant continuing investigation into other regulatory controls on signaling networks (such as SREBP and/or ubiquitin ligases) to further our understanding the complex molecular mechanisms involved in muscle maintenance in the hibernating brown bear.

ACKNOWLEDGMENTS
The authors thank J.M. Storey for editorial review of the manuscript.