Molecular Mechanism Underlying Muscle Mass Retention in Hibernating Bats: Role of Periodic Arousal

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1 ORIGINAL ARTICLE 313 Journal of Molecular Mechanism Underlying Muscle Mass Retention in Hibernating Bats: Role of Periodic Arousal KISOO LEE, 1 HYEKYOUNG SO, 1 TAESIK GWAG, 1 HYUNWOO JU, 1 JU-WOON LEE, 2 MASAMICHI YAMASHITA, 3 AND INHO CHOI 1,4 * 1 Division of Biological Science and Technology, College of Science and Technology, Yonsei University, Wonju, Gangwon-Do, Republic of Korea 2 Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, Jeonbuk, Republic of Korea 3 Department of Space Biology and Microgravity Sciences, Institute of Space and Astronautical Science (ISAS), Japan Aerospace Exploration Agency (JAXA), Sagamihara, Kanagawa, Japan 4 Institute of Biomaterials, Yonsei University, Wonju, Gangwon-Do, Republic of Korea Cellular Physiology Hibernators like bats show only marginal muscle atrophy during prolonged hibernation. The current study was designed to test the hypothesis that hibernators use periodic arousal to increase protein anabolism that compensates for the continuous muscle proteolysis during disuse. To test this hypothesis, we investigated the effects of 3-month hibernation (HB) and 7-day post-arousal torpor (TP) followed by re-arousal (RA) on signaling activities in the pectoral muscles of summer-active (SA) and dormant Murina leucogaster bats. The bats did not lose muscle mass relative to body mass during the HB or TP-to-RA period. For the first 30-min following arousal, the peak amplitude and frequency of electromyographic spikes increased 3.1- and 1.4-fold, respectively, indicating massive myofiber recruitment and elevated motor signaling during shivering. Immunoblot analyses of whole-tissue lysates revealed several principal outcomes: (1) for the 3-month HB, the phosphorylation levels of Akt1 ( p-akt1) and p-mtor decreased significantly compared to SA bats, but p-foxo1 levels remained unaltered; (2) for the TP-to-RA period, p-akt1 and p-foxo1 varied little, while p-mtor showed biphasic oscillation; (3) proteolytic signals (i.e., atrogin-1, MuRF1, Skp2 and calpain-1) remained constant during the HB and TP-to-RA period. These results suggest that the resistive properties of torpid bat muscle against atrophy might be attained primarily by relatively constant proteolysis in combination with oscillatory anabolic activity (e.g., p-mtor) corresponding to the frequency of arousals occurring throughout hibernation. J. Cell. Physiol. 222: , ß 2009 Wiley-Liss, Inc. Mammalian skeletal muscles exhibit significant loss of contractile proteins and myofibrillar integrity during prolonged unloading or disuse (e.g., denervation, hindlimb suspension, and spaceflight) (Fitts et al., 2001; Choi et al., 2005). Previous reports have shown that expression of many myofibrillar proteins, including alpha-actin, myosin light chain, and troponin T, are downregulated in unloaded antigravity muscles like the soleus muscle, resulting in a considerable reduction in force generation and work capacity (Isfort et al., 2000; Seo et al., 2006). In contrast to this general trend, most hibernators show only a marginal loss of muscle mass and contractility over the 4 7 months of winter dormancy (Yacoe, 1983; Lohuis et al., 2007; Hershey et al., 2008). A previous study from our group demonstrated that a bat species, Murina leucogaster, displays almost no change in total protein content, muscle mass, tetanic tension, and sarcomeric and metabolic protein contents during the 3 months of hibernation (Lee et al., 2008). As an underlying mechanism for such muscle retention, we proposed that periodic arousals play a functional role in boosting protein synthesis, and the ensuing hypothermia diminishes the degradation of the proteins even in the disuse state (also see Harlow et al., 2004). Upregulation (or balanced regulation) of several molecular chaperones (e.g., heat shock protein 70) examined during winter dormancy might further imply the significance of periodic arousals for muscle retention (Lee et al., 2008). Despite the results of these and other studies (van Breukelen and Martin, 2002; Carey et al., 2003), the molecular mechanisms that regulate the balance between muscle protein synthesis and degradation have not been clarified in this natural muscle system of hibernators. According to recent reports, muscle cells respond to loading stress primarily via a signaling pathway comprised of insulin/ insulin-like growth factor-1 (IGF-1) and phosphosinositide-3 kinase (PI3K, class I) (Stitt et al., 2004). Once activated, this pathway induces phosphorylation of Akt1/protein kinase B (PKB), which in turn phosphorylates and activates its downstream mammalian target rapamycin (mtor), leading to Contract grant sponsor: National Space Laboratory Program (Korea Science and Engineering Foundation); Contract grant number: *Correspondence to: Inho Choi, Division of Biological Science and Technology, College of Science and Technology, Yonsei University, 234 Maeji-ri, Heungup-myon, Wonju, Gangwon-do , Republic of Korea. Received 6 July 2009; Accepted 2 September 2009 Published online in Wiley InterScience ( 21 October DOI: /jcp ß W I L E Y - L I S S, I N C.

2 314 LEE ET AL. protein synthesis. The phosphorylated Akt1 is also known to stimulate phosphorylation of forkhead box O (FoxO) proteins, which leads to their nuclear exclusion and degradation (Hoffman and Nader, 2004; Sandri et al., 2004). The overall consequence is an increase in muscle mass (hypertrophy). In contrast, when muscle cells are mechanically unloaded, the signaling responses are reversed. The activity of the Akt1-mTOR pathway is decreased and the muscle becomes atrophied (Hoffman and Nader, 2004). Muscle atrophy is regulated not only by the suppression of anabolic pathways, but also by activation of proteolytic pathways. Previous studies have reported that the ubiquitin-proteasome pathway, considered a powerful contributor to muscle proteolysis, is regulated by FoxO proteins (Sandri et al., 2004; Nader, 2005; Leger et al., 2006). These transcription factors bind to target genes and stimulate the expression of ubiquitin E3 ligases, and unnecessary or damaged proteins are degraded by the proteasomal system. Atrogin-1/muscle atrophy F-box (MAFbx) and muscle RING Finger 1 (MuRF1) are muscle-specific E3 ligases that are responsible for the ubiquitination required for proteasomal proteolysis (Sandri et al., 2004). In parallel to the FoxO-ubiquitin E3 ligase pathway, another transcription factor, NF-kB, which mediates the effects of the cytokine TNF-a has been found to be a potent molecule associated with protein degradation. This transcription factor is known to induce expression of MuRF1 and concomitant muscle atrophy (Cai et al., 2004). Moreover, recent findings suggest that the cellular level of phosphorylated FoxO can be repressed by ubiquitin ligase activities in unloaded cells. The FoxO1 protein was found to be a substrate of the F-box protein S-phase kinase-associated protein 2 (Skp2), a component of the Skp/Cullin/F-box (SCF) ubiquitin ligase complex, and phosphorylated FoxO1 is degraded due to ubiquitination by Skp2 (Huang et al., 2005; Huang and Tindall, 2007; Ward et al., 2008). Taken together, these studies indicate that FoxO proteins play a central role in ubiquitin E3 ligase-proteasomal proteolysis and that FoxO1-mediated proteolysis may be accelerated in part by the expression of Skp2 in unloaded muscles. Although FoxO proteasomal activities may be the major route of muscle proteolysis, they may not be sufficient to explain the whole atrophic process without including other relevant signaling pathways. For instance, oxidant-induced cell damage during unloading often causes a rapid rise in the cytoplasmic level of Ca 2þ ions released from the sarcoplasmic reticulum (McClung et al., 2007; Servais et al., 2007). This suggests that calpain, a Ca 2þ -dependent protease, could play a role in non-transcriptional proteolytic degradation in muscle loss (Servais et al., 2007). The aim of the current study was to investigate the signaling mechanisms that enable hibernators to retain myofibrillar proteins during prolonged dormancy. Our questions were as follows: (1) what role does interbout arousal play in the resistance of hibernator muscles to disuse atrophy and (2) what mediator(s) in signaling pathways contribute to the resistance? Having anticipated that mammalian skeletal muscles upregulate proteolytic pathways but downregulate anabolic pathways during unloading, we hypothesized that hibernators use periodic arousals to activate anabolic pathways to compensate for continued muscle proteolysis during the disuse. We tested the hypothesis by elucidating (1) the long-term gross effects of hibernation on muscle properties and (2) the short-term temporal effects of unloading on muscle properties. For the first approach, we compared the mass and signaling activities of pectoral muscles between summer-active and hibernating bats. For the second approach, we examined the mass and signaling activities of muscles during 7-day torpor between interbout arousals. Materials and Methods Subjects This study was approved by the Yonsei University Animal Care and Use Committee. A local environmental protection agency issued a permit to collect the bat species M. leucogaster ognevi from a natural cave located in Gangwon Province ( N, E), Republic of Korea. We used only male bats for the study. The summer-active bats (SA) were captured in August September using bird mist nets. Hibernating bats (HB) were collected with gloved hands in February (about 3 months into their 5-month hibernation season). All animals were collected between 21:00 and 24:00. The rectal temperature (T r ) of the individuals was recorded immediately after capture using a 30 American wire gauge (AWG) copper constantan thermocouple connected to an Omega thermometer (Cole-Parmer Instrument, Vermon Hills, IL). The SA and HB animals were then decapitated and immediately frozen in liquid nitrogen. Handling time from capture to freezing was minimized to 30 sec or less. In February, additional bats were carried to our laboratory to examine electromyographic activities during the early phase of arousal (see below) and signaling activities at various time points from arousal through 7-day torpor (TP) to re-arousal (RA). The RA group consisted of subjects at a 30-min time point after the initiation of arousal. Some of the aroused animals (0 day) were decapitated and frozen in liquid nitrogen. The rest of the bats were divided into three TP groups (1 day, 3 days, 7 days) and one RA group. They were caged individually and induced into dormancy in a dark humid room at 5 18C (an average hibernacular temperature). To check for complete inactivity of the bats during the given torpor periods, we monitored the skin temperature and position of each bat in the cage every morning and night with an infrared thermometer (KM 848, Hertfordshire, UK) and a digital camera (DCR-TRV19, Tokyo, Japan). At the end of the assigned torpid period, individuals from 1-, 3-, and 7-day TP groups were quickly subject to T r, decapitated, and frozen in liquid nitrogen. Individuals from the RA group were induced to arouse for 30 min by gently shaking the cage and were subject to the same procedure as the TP groups. Electromyography The pectoral muscle was chosen for this study since it is the main tissue responsible for flying and climbing, which both require continuous antigravity activities. To assess the role of interbout arousal for tissue resistance to disuse atrophy, we collected two bats in February and recorded electromyograms (EMG) during the course of arousal. The bats were weighed and anesthetized with pentobarbital sodium (i.p., 0.01 mg g 1 ). Small cuts were made on the dorsal skin between the scapulars and on the ventral skin of the left central chest for implantation of thermocouples and electrodes. A Teflon-insulated duplex copper constantan thermocouple (0.025 mm diameter, California Fine Wire, Grover City, CA) was used to monitor pectoral muscle temperature. The sensing tip was inserted about 5 mm into the central spot of the muscle via a 26-gauge hypodermic needle. To obtain EMG signals, a pair of stainless steel bipolar electrodes and one ground electrode were used, each with 0.5 mm uninsulated tips. The bipolar electrodes were inserted directly below the thermocouple in the pectoral muscle via the same hypodermic needle. The ground electrode was placed in the intraperitoneal cavity. Once implantation was complete, the thermocouple and electrodes were passed underneath the skin to the dorsal cut. On the dorsal surface, the wires were gathered in a 5 mm long piece of Tygon tubing (O.D mm) that was sutured tightly to the skin of the subject. The bat was then kept warm (258C) for 8 h in a wiremesh cage (l w h ¼ 250 mm 250 mm 300 mm) for recovery and given free access to water. The bat was then moved to a dark and humid refrigerator (5 18C) for 1 day to induce deep dormancy.

3 PERIODIC AROUSAL AND MUSCLE RETENTION IN A HIBERNATOR 315 Every experiment began at 20:00 while the animals were perched in their normal (vertical) posture in the cages within the refrigerator. The thermocouple was connected to the Omega thermometer to check muscle temperature. The electrodes were connected to a Grass P511 preamplifier where EMG signals were amplified 10,000. A half-amplitude low filter and a half-amplitude high filter of the preamplifier were set to 100 Hz and 3 khz, respectively, and a 60-Hz notch filter was turned on. Approximately 10 min after preparation, the subjects were forcibly aroused by gentle shaking of the cage. Event times and muscle temperature were recorded simultaneously, while the EMG signals from the preamplifier were continually monitored on an oscilloscope and fed to a Biopac MP100 A/D converter. Signals, which were 1 sec in duration, were collected every 58C starting from 108C upwards until EMG activities were unable to be resolved. The digitized signals were transmitted to a Pentium compatible PC for further analyses. Tissue samplings The body mass of each frozen bat was measured to within 0.01 g. The pectoral muscle from the left thorax was collected and prepared for the immunoblot analysis as previously described (Choi et al., 2005). The body was then slowly thawed on ice. The pectoral muscle from the right thorax was weighed to the nearest 0.1 mg to determine the ratio of muscle mass to body mass (M m /M b ) for each subject. Immunoblot analysis The frozen tissues were homogenized in ten volumes of tissue lysis buffer [20 mm HEPES (ph 7.4), 75 mm NaCl, 2.5 mm MgCl 2, 0.1 mm EDTA, 0.05% (v/v) Triton X-100, 20 mm b- glycerophosphate, 1 mm Na 3 VO 4, 10 mm NaF, 0.5 mm DTT, and the protease inhibitor Complete Mini (Roche, Penzberg, Germany)]. The homogenized samples were incubated for 15 min on ice and centrifuged for 30 min at 12,000g at 48C. The supernatants were collected as the whole-tissue soluble lysates, and their protein concentrations were determined using a Bradford assay kit (Bio-Rad, Hercules, CA). Whole-tissue lysates (50 mg) were subject to SDS PAGE to detect Akt1, phospho-akt1, mtor, phospho-mtor, FoxO1, phospho-foxo1, FoxO3, phospho-foxo3, mtor, Skp2, atrogin-1 (MAFbx), MuRF1, IkB, phospho-ikb, and calpain-1. The proteins were electrophoretically transferred from a gel to a nitrocellulose membrane. The membrane was incubated in blocking buffer (1 TBS, 0.05% Tween-20 with 5% (w/v) nonfat dry milk) for 1 h at room temperature and was washed three times for 5 min each with 15 ml of TBS/T. The membrane was subsequently incubated overnight at 48C with primary antibodies of appropriate dilution (1:500 1:10,000) against each indicated protein in 10 ml of primary antibody dilution buffer (1 TBS, 0.1% Tween-20 with 5% (w/v) nonfat dry milk). The membrane was then incubated with HRP-conjugated secondary antibody (1:1,000 1:10,000) for detection in 10 ml of blocking buffer with gentle agitation for 1 h at room temperature and washed three times for 5 min each with 15 ml of TBS/T between incubations. Immune complexes were detected with the ECL system (GE Healthcare, Fairfield, CT), and the bands were quantified using a densitometer (Bio-Rad). Densities of the proteins were normalized to GAPDH. For detection of an additional protein, the membranes were stripped using Restore TM Western Blot Stripping Buffer (Thermo Scientific, Rockford, IL; 15 ml/membrane). The membranes were incubated for 15 min at room temperature and were washed three times for 5 min each with 15 ml of TBS/T. The primary antibodies used for the assays were: mouse anti-akt1, rabbit anti-phospho-akt1 (Ser473), rabbit anti-mtor, rabbit anti-phospho-mtor (Ser2448), rabbit anti-foxo1, rabbit anti-phospho-foxo1 (Ser256), rabbit anti-calpain1, rabbit anti-ikb, and mouse anti-phospho-ikb (Ser32/36) monoclonal antibodies (Cell Signaling Technology, Beverly, CA); rabbit anti-atrogin-1, MuRF1, and Skp2p45 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); and a mouse monoclonal antibody to GAPDH (Abcam, Cambridge, UK). The secondary antibodies were HRP-conjugated anti-mouse IgG, anti-rabbit IgG (Cell Signaling), and anti-goat IgG (Santa Cruz). Data presentation All data are presented as means 1 SEM, unless otherwise noted. The significance of seasonal differences in bats (SA vs. HB) was examined by independent sample t-tests. The significance of time-course variations was examined using one-way analysis of variance (ANOVA) and Scheffe s post hoc multiple comparison test. Statistical tests were performed with SPSS/PCþ at a significance level P ¼ Results Subjects Mass and temperature data recorded from the bats are summarized in Table 1. Comparison of the muscle mass relative to body mass is illustrated in Figure 1. The relative pectoral mass did not statistically differ either between SA and HB (t-test, P > 0.05) or over the course of 1-week TP-to-RA period (one-way ANOVA, P > 0.05) (Fig. 1A,B). Muscle temperature and EMG activity To determine the extent of the mechanical and thermogenic activities of pectoral muscles during the torpor-to-arousal transition, the muscle temperatures (T m ) and EMG signals were simultaneously recorded from two arousing bats. Figure 2A illustrates a representative T m recording for a bat during the 40-min arousal period. The T m was C at torpor, but rapidly increased to C at 30 min following arousal. The T m tended to peak at times between 30 and 40 min post-arousal and was about 48C higher than T r in the aroused state (Table 1). Figure 2B displays typical 1-sec EMGs at the tissue temperatures 15, 18, 25, 30, and 358C. In Figure 2C, average peak amplitudes of the EMGs are illustrated at the corresponding temperatures. The EMG signals were first detected at C with a peak amplitude of mv. The peak amplitude then increased 3.1-fold as the muscle temperature reached 308C. At temperatures above 358C, the EMGs became weaker and appeared to be mixed with those of non-shivering activities, such as wing flapping or crawling. The median spike frequency, which was analyzed in bins of 100 ms, was Hz at 188C. This frequency gradually increased 1.4-fold at 308C (data not shown). Expression and activation of major signaling molecules Akt1 - The level of Akt1 was similar between the SA and HB bats, but the level of phosphorylated Akt1 was considerably lower in HB than in SA bats, resulting in a significant decrease ( 61%) in the p-akt1/akt1 ratio in HB bats (t-test, P < 0.05; TABLE 1. Body mass, muscle mass, and rectal temperature of the Murina leucogaster bats examined during the 7-day torpor to re-arousal period Variables Time (days) 0 day 1 day 3 days 7 days RA M b (g) M m (mg) T r (8C) Data: mean 1 SEM, n ¼ 4; M b,bodymass;m m, muscle mass; T r, rectal temperature; RA, rearousal state at 30 min after 7-day torpor.

4 316 LEE ET AL. Fig. 1. The muscle-mass-to-body-mass ratio of the bat Murina leucogaster. The right-side pectoral muscle was weighed to the nearest 0.1 mg. Data are presented as means W 1SEM.A:Therelative pectoral muscle mass did not differ between summer-active (SA) and hibernating (HB) bats (n U 6; independent samples t-test, P > 0.05). B: Time-course examinations of the relative muscle mass at 0 day (arousal), 1-, 3-, and 7-day TP, and RA (n U 4; one-way ANOVA, P >0.05). Fig. 3A,B). The p-akt1/akt1 ratio did not vary significantly during the 7-day TP-to-RA period (P > 0.05; Fig. 3D,E). mtor: The levels of mtor and p-mtor displayed a similar pattern to that observed for Akt1, leading to a significant decrease ( 48%) in the ratio of p-mtor/mtor in HB compared to SA bats (Fig. 3A,C). The time-course study revealed that the p-mtor/mtor ratio increased slightly at 1-day TP (P > 0.05), decreased 44% at 7-day TP ( P < 0.05), and then returned closely to the level of previous arousal (0 day) at RA ( P > 0.05) (Fig. 3D,F). FoxO1: Unlike the patterns of Akt1 and mtor, neither the levels of FoxO1 and p-foxo1 nor the p-foxo1/foxo1 ratio differed between SA and HB bats ( P > 0.05; Fig. 4A,B). During the time-course study, levels of FoxO1, p-foxo1, and the p-foxo1/foxo1 ratio changed little over the 7-day TP-to-RA period (Fig. 4C,D). Ubiquitin E3 ligases: Expression of atrogin-1, MuRF1, and Skp2 did not differ statistically between SA and HB bats ( P > 0.05; Figs. 5A D). Levels of the three molecules remained relatively stable during the 7-day TP-to-RA period ( P > 0.05; Fig. 5E H). Calpain-1: The protease was expressed at similar levels in the SA and HB groups ( P > 0.05; Fig. 6A,B). In the time-course study, the level of calpain-1 did not vary significantly over the 7-day TP-to-RA period (P > 0.05; Fig. 6C,D). IkB: Levels of IkB, p-ikb, and p-ikb/ikb did not differ between SA and HB bats ( P > 0.05). The protein levels and phosphorylation states also did not change significantly during the 7-day TP-to-RA period ( P > 0.05) (data not shown). Discussion Periodic arousal has been observed in all mammalian hibernators studied so far (Park et al., 2000; Epperson and Martin, 2002; van Breukelen and Martin, 2002). A striking similarity was observed even in wintering Notothenia coriiceps, an Antarctic fish, which displayed short bouts (1 3 h) of arousal every 4 12 days during winter dormancy (Campbell et al., 2008). Such observations of arousal from a wide diversity of Fig. 2. Muscle temperature and electromyographic activities during arousal. A: Muscle temperature increased rapidly during the course of arousal of a bat. B: Typical EMG signals of the pectoral muscle were shown at five tissue temperatures. C: Average peak amplitudes of the pectoral EMGswereillustratedatthe corresponding temperatures; data arepresented asmeans W 1SEM(nU 2). Bipolar electrodeswereinserted intothe pectoral muscle via a hypodermic needle, with a ground electrode in the intraperitoneal cavity. For simultaneous measurement of the muscle temperature, a copper constantan thermocouple (0.025 mm D) was inserted into the muscle near the EMG electrodes. T m : muscle temperature; T a : ambient temperature.

5 PERIODIC AROUSAL AND MUSCLE RETENTION IN A HIBERNATOR 317 Fig. 3. Effect of dormancy on Akt1 and mtor phosphorylation in the bat pectoral muscle. Means W 1 SEM. Whole-tissue soluble lysates were used for immunoblot analysis with the respective antibodies. A C: p-akt1/akt1 and p-mtor/mtor decreased 61% and 48%, respectively, in HB compared to SA bats (n U 6, P < 0.05). D F: p-akt1/ Akt1 varied only slightly between 0 d and RA (n U 4, P > 0.05), but the p-mtor/mtor changed significantly (P < 0.05), depicting biphasic oscillation during this period. Fig. 5. Regulation of ubiquitin E3 ligases during dormancy. A D: Expression of atrogin-1, MuRF1, and Skp2 did not differ between SA and HB (n U 6, P > 0.05). E H: The expression of the E3 ligases also did not vary significantly during the 7-day TP-to-RA period (n U 4). Error bars represent 1 SEM. Fig. 4. FoxO1 regulation during dormancy. A,B: The p-foxo1/ FoxO1 ratio did not differ between SA and HB bats (n U 6, P > 0.05). C,D: The time-course study also showed little variation in the p- FoxO1/FoxO1 ratio during the 7-day TP-to-RA period (n U 4, P >0.05). Error bars represent 1 SEM. species suggest it is a generally adaptive feature, possibly due to the advantage of restoring internal homeostasis during the prolonged inactivity (Carey et al., 2003). With respect to muscle properties, the periodic arousal would provide a critical mechanism to elevate mechanical loading and thereby recover sarcomeric proteins, similar to strenuous exercise (Leger et al., 2006; Lee et al., 2008). In agreement with this view, the oscillatory regulation of p-mtor across the TP-RA period (Fig. 3F) implies that the anabolic mechanism potentially operates in the hibernator s muscle. This observation apparently supports our original hypothesis. The steady-state regulation of proteolytic markers (e.g., FoxO1 and atrogens; Figs. 4 6), irrespective of the effects of the short-term torpor-arousal cycle or the long-term hibernation, was also a novel observation implying an important mechanism for muscle homeostasis. This latter outcome in fact deviated from our hypothesis, which shall be discussed below. Specifically, our results demonstrate that bat pectoral muscles exhibit almost no atrophy over 3 months of hibernation or during 1 week of torpor following arousal (Fig. 1). Minimal muscle atrophy has also been found in other mammalian hibernators (Yacoe, 1983; Lohuis et al., 2007;

6 318 LEE ET AL. Fig. 6. Regulation of calpain-1 during dormancy. A,B: The protease levels did not differ between SA and HB bats (n U 6, P >0.05).C,D:The expression of calpain-1 did not change significantly between 0 d and RA (n U 4, P >0.05) Error bars represent 1 SEM. Hershey et al., 2008). As a working hypothesis, we proposed in our prior (Lee et al., 2008) and current studies that the remarkable motor activity in every arousal is the key stressor for muscle retention. Very impressively, shivering was so intense during the early phase of arousal that the whole body of the bat was shaken at tissue temperatures of C. The functional significance of arousal in microchiropteran species has not been fully appreciated to date, leaving significant questions regarding its causes and effects on muscle properties. Our EMG analysis observed a 3-fold increase in spike amplitude and 1.4-fold increase in spike frequency during the torpor-to-arousal transition (Fig. 2). This indicates massive recruitment of muscle fibers, possibly with increased synchronization of motor activities, and concurrent augmentation of efferent neural transmission (Rome et al., 1992). Our next concern was to elucidate the impact of this motor upheaval in the middle of dormancy on protein metabolism. The Akt1-mTOR pathway is known to be the major signaling activity associated with protein synthesis, with activation of Akt1 inducing phosphorylation and activation of mtor and its downstream signals (e.g., 70 kda ribosomal S6 protein kinase, p70/s6k1) (Zanchi and Lancha, 2008). Conversely, inhibition of this pathway readily reduces muscle protein expression and myofiber size (Bodine et al., 2001). In the current study, decreased activity of this pathway was evident in the pectoral muscle during the 3 months of hibernation (Fig. 3A C). Interestingly, regulation of the anabolic signaling was different for Akt1 and mtor during the 7-day TP-to-RA period (Fig. 3D F). Differential regulation of Akt and mtor has been reported previously, with modulation by various factors, including exercise intensity and endurance (Leger et al., 2006; Deldicque et al., 2008), contractile modes (concentric vs. eccentric) (Eliasson et al., 2006), and nutritional status (Deldicque et al., 2008). In the bat muscle, the phosphorylation state of Akt1 ( p-akt1) remained constant for the TP-to-RA period. In contrast, the level of p-mtor was biphasic, with an apparent oscillatory pattern over the TP-to-RA period. This observation suggests that p-mtor oscillation might correspond to the frequency of arousals throughout hibernation. We expect the level of p-mtor at RA to be more elevated if re-arousal durations exceed 30 min. In the bat species studied so far, arousal has been observed to last 2-to-9 h (Park et al., 2000), which is much longer than the duration (30 min) examined in our study. In terms of temporal effects, our data show a couple of discrepancies with regards to anabolic signaling. First, the phosphorylation state of Akt1 and mtor returned closely to the previous levels at RA, while the level of these molecules actually decreased during the 3-month hibernation (Fig. 3). In this case, we cannot exclude the possibility that the short-term, small change results in significant cumulative consequences during the long time span (3 months) of hibernation. Second, the muscle-mass-to-body-mass ratio remained unaltered throughout the 3-month hibernation, whereas the Akt1-mTOR pathway was downregulated during this period (Figs. 1A and 3B,C). This discrepancy suggests that the Akt1-mTOR pathway is not sufficient to provide anabolic products for muscle mass retention. This further implies that, unless proteolysis is suppressed as much as the anabolic process to balance the muscle mass, alternative signals such as the mitogen-activated protein kinase (MAPK) and/or protein kinase C (PKC) cascades might also stimulate protein synthesis in the muscular system (Korzick et al., 2004; Choi et al., 2005; Deldicque et al., 2008). For instance, resistance exercise and overloading conditions both result in stimulation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) with concurrent muscle protein synthesis (Deldicque et al., 2008). Our previous study also reported that the activation of PKCa by phorbol 12-myristate 13-acetate (the PKCa activator) accompanies a simultaneous increase in sarcomeric alpha-actin expression (Choi et al., 2005). These alternative anabolic mechanisms will be investigated further in order to better understand the protein metabolism of the hibernators muscle. Turning to the proteolytic signaling pathways, it was surprising to find that the activities of FoxO1, three ubiquitin E3 ligases (atrogin-1, MuRF1, Skp2), calpain, and NF-kB (estimated by p-ikb/ikb) varied little over the 3-month HB and the 7-day TP-to-RA period (Figs. 4 6). By keeping muscle proteolysis no greater than that of SA bats (via the balanced regulation of FoxO1 and proteases), dormant bats may at least have a molecular mechanism for retaining muscle mass for the extended period of disuse. A novel finding of the present study is the difference in the phosphorylation levels of FoxO1 and Akt1 over the 3-month hibernation, that is, the balanced regulation of p-foxo1/foxo1 versus downregulation of p-akt1/akt1 in the HB bats (Figs. 3 and 4). How could FoxO1 phosphorylation be maintained at similar levels between SA and HB bats, despite a decrease in the phosphorylation of its upstream kinase, Akt1? To answer this question, we looked at the signaling networks related to FoxO1, in which Skp2 is a potential candidate for the regulation of FoxO1 (Huang and Tindall, 2007). As a component of SCF ubiquitin ligase complexes, this protein is known to promote ubiquitination and proteasomal degradation of p-foxo1 (Dehan and Pagano, 2005). According to our prior work on the unloaded rat soleus muscle, p-foxo1 is downregulated and Skp2 and other E3 ligases (atrogin-1, MuRF1) are upregulated in unloaded muscle (I. Choi, unpublished data). In contrast, the current study showed the level of p-foxo1 to be nearly unaffected during the 3-month hibernation and 7-day torpor, which seems to be consistent with the balanced regulation of Skp2 (Figs. 4 and 5). The unexpected discrepancy between p-akt1 and p-foxo1 levels in the HB bats may be explained further by the relationship of atrogin-1 to FoxO proteins previously reported in cardiomyocytes (Li et al., 2007). According to that study, Akt-dependent phosphorylation of FoxO is blocked by atrogin-1, while insulin-mediated Akt activation per se is not affected by the ligase. This finding suggests a feed-forward mechanism in which atrogin-1 is activated by and in turn co-activates the

7 PERIODIC AROUSAL AND MUSCLE RETENTION IN A HIBERNATOR 319 Fig. 7. Summary diagrams illustrating signaling mechanisms for protein breakdown (A) and protein synthesis (B) in the bat pectoral muscle. The atrophy resistance of the muscle might be attained by relatively constant catabolic activities (i.e., FoxO1, ubiquitin E3 ligases, and calpain-1), along with oscillation of anabolic activities (e.g., p-mtor) caused by arousals that periodically interrupt the dormancy. SA: summer-active bats; HB/TP: hibernating/torpid bats; AR: arousal. FoxO proteins, eventually suppressing cardiac hypertrophy (Li et al., 2007). This cardiac outcome may be applicable to skeletal muscle since the two types of muscle share the same myofibrillar structure and contractile mechanism. Because the expression of atrogin-1 did not differ between SA and HB bats, the balanced regulation of p-foxo1, which could be partly uncoupled from the influence of Akt1, may be viable in the HB bat muscle. In conclusion, the resistance of bat muscles to atrophy may be attained by relatively constant proteolysis in combination with oscillatory regulation of the anabolic marker (i.e., p-mtor) corresponding to the frequency of arousals during hibernation (Fig. 7). The FoxO1 protein is likely to act as the principal mediator keeping proteolysis stable throughout the year, possibly in cooperation with Skp2 and atrogin-1. The heterothermic regime and its associated signaling mechanisms may aid in muscle retention and survival of these hibernators over the prolonged dormancy. Understanding the mechanisms of hibernation may be useful for long-term space missions, during which muscle atrophy and payload problems need to be overcome. Acknowledgments We would like to thank two anonymous reviewers for their critical comments to improve the article. Mr. Haksup Shin and Min Jae Park kindly assisted in laboratory procedures, including animal management. This research was supported by the NSL (National Space Lab) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (grant No ) awarded to I. Choi. Literature Cited Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, Glass DJ Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: Cai D, Frantz JD, Tawa NE, Jr., Melendez PA, Oh BC, Lidov HG, Hasselgren PO, Frontera WR, Lee J, Glass DJ, Shoelson SE IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell 119: Campbell HA, Fraser KP, Bishop CM, Peck LS, Egginton S Hibernation in an Antarctic fish: On ice for winter. PLoS ONE 3:e1743. Carey HV, Andrews MT, Martin SL Mammalian hibernation: Cellular and molecular responses to depressed metabolism and low temperature. Physiol Rev 83: Choi I, Lee K, Kim M, Lee M, Park K Differential activation of stress-responsive signalling proteins associated with altered loading in a rat skeletal muscle. J Cell Biochem 96: Dehan E, Pagano M Skp2, the FoxO1 hunter. Cancer Cell 7: Deldicque L, Atherton P, Patel R, Theisen D, Nielens H, Rennie MJ, Francaux M Decrease in Akt/PKB signalling in human skeletal muscle by resistance exercise. Eur J Appl Physiol 104: Eliasson J, Elfegoun T, Nilsson J, Kohnke R, Ekblom B, Blomstrand E Maximal lengthening contractions increase p70 S6 kinase phosphorylation in human skeletal muscle in the absence of nutritional supply. Am J Physiol Endocrinol Metab 291:E1197 E1205. Epperson LE, Martin SL Quantitative assessment of ground squirrel mrna levels in multiple stages of hibernation. Physiol Genomics 10: Fitts RH, Riley DR, Widrick JJ Functional and structural adaptations of skeletal muscle to microgravity. J Exp Biol 204: Harlow HJ, Lohuis T, Anderson-Sprecher RC, Beck TDI Body surface temperature of hibernating black bears may be related to periodic muscle activity. J Mammal 85: Hershey JD, Robbins CT, Nelson OL, Lin DC Minimal seasonal alterations in the skeletal muscle of captive brown bears. Physiol Biochem Zool 81: Hoffman EP, Nader GA Balancing muscle hypertrophy and atrophy. Nat Med 10: Huang H, Tindall DJ Dynamic FoxO transcription factors. J Cell Sci 120: Huang H, Regan KM, Wang F, Wang D, Smith DI, van Deursen JM, Tindall DJ Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation. Proc Natl Acad Sci USA 102: Isfort RJ, Hinkle RT, Jones MB, Wang F, Greis KD, Sun Y, Keough TW, Anderson NL, Sheldon RJ Proteomic analysis of the atrophying rat soleus muscle following denervation. Electrophoresis 21: Korzick DH, Laughlin MH, Bowles DK Alterations in PKC signaling underlie enhanced myogenic tone in exercise-trained porcine coronary resistance arteries. J Appl Physiol 96: Lee K, Park JY, Yoo W, Gwag T, Lee JW, Byun MW, Choi I Overcoming muscle atrophy in a hibernating mammal despite prolonged disuse in dormancy: Proteomic and molecular assessment. J Cell Biochem 104: Leger B, Cartoni R, Praz M, Lamon S, Deriaz O, Crettenand A, Gobelet C, Rohmer P, Konzelmann M, Luthi F, Russell AP Akt signalling through GSK-3beta, mtor and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy. J Physiol 576: Li HH, Willis MS, Lockyer P, Miller N, McDonough H, Glass DJ, Patterson C Atrogin-1 inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin-dependent coactivation of Forkhead proteins. J Clin Invest 117: Lohuis TD, Harlow HJ, Beck TD Hibernating black bears (Ursus americanus) experience skeletal muscle protein balance during winter anorexia. Comp Biochem Physiol BBiochemMolBiol147: McClung JM, Kavazis AN, Whidden MA, DeRuisseau KC, Falk DJ, Criswell DS, Powers SK Antioxidantadministration attenuates mechanical ventilation-inducedrat diaphragm muscle atrophy independent of protein kinase B (PKB Akt) signalling. J Physiol 585: Nader GA Molecular determinants of skeletal muscle mass: Getting the AKT together. Int J Biochem Cell Biol 37: Park KJ, Jones G, Ransome RD Torpor, arousal and activity of hibernating greater horseshoe bats (Rhinolophus ferrumequinuum). Funct Ecol 14: Rome LC, Choi IH, Lutz G, Sosnicki A The influence of temperature on muscle function in the fast swimming scup. I. Shortening velocity and muscle recruitment during swimming. JExpBiol163: Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH, Goldberg AL Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117: Seo Y, Lee K, Park K, Bae K, Choi I A proteomic assessment of muscle contractile alterations during unloading and reloading. J Biochem (Tokyo) 139: Servais S, Letexier D, Favier R, Duchamp C, Desplanches D Prevention of unloadinginduced atrophy by vitamin E supplementation: Links between oxidative stress and soleus muscle proteolysis? Free Radic Biol Med 42: Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M, Yancopoulos GD, Glass DJ The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophyinduced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 14: van Breukelen F, Martin SL Invited review: Molecular adaptations in mammalian hibernators: Unique adaptations or generalized responses? J Appl Physiol 92: Ward EC, Hoekstra AV, Blok LJ, Hanifi-Moghaddam P, Lurain JR, Singh DK, Buttin BM, Schink JC, Kim JJ The regulation and function of the forkhead transcription factor, Forkhead box O1, is dependent on the progesterone receptor in endometrial carcinoma. Endocrinology 149: Yacoe M Maintenance of the pectoralis muscle during hibernation in the big brown bat Eptesicus fuscus. J Comp Physiol 152: Zanchi NE, Lancha AH, Jr Mechanical stimuli of skeletal muscle: Implications on mtor/ p70s6k and protein synthesis. Eur J Appl Physiol 102:

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