The FASEB Journal article fj.14-267336. Published online March 20, 2015.

The FASEB Journal • Research Communication

Masticatory muscles of mouse do not undergo atrophy in space Anastassios Philippou,*,† Fabio C. Minozzo,‡ Janelle M. Spinazzola,†,§ Lucas R. Smith,†,§ Hanqin Lei,†,{ Dilson E. Rassier,‡ and Elisabeth R. Barton†,§,{,1 *Department of Physiology, Medical School, National and Kapodistrian University of Athens, GoudiAthens, Greece; †Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA; ‡Department of Kinesiology, McGill University, Montreal, Quebec, Canada; §Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA; and {Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida, USA Muscle loading is important for maintaining muscle mass; when load is removed, atrophy is inevitable. However, in clinical situations such as critical care myopathy, masticatory muscles do not lose mass. Thus, their properties may be harnessed to preserve mass. We compared masticatory and appendicular muscles responses to microgravity, using mice aboard the space shuttle Space Transportation System-135. Age- and sex-matched controls remained on the ground. After 13 days of space flight, 1 masseter (MA) and tibialis anterior (TA) were frozen rapidly for biochemical and functional measurements, and the contralateral MA was processed for morphologic measurements. Flight TA muscles exhibited 20 6 3% decreased muscle mass, 2-fold decreased phosphorylated (P)-Akt, and 4- to 12-fold increased atrogene expression. In contrast, MAs had no significant change in mass but a 3-fold increase in P-focal adhesion kinase, 1.5-fold increase in P-Akt, and 50–90% lower atrogene expression compared with limb muscles, which were unaltered in microgravity. Myofibril force measurements revealed that microgravity caused a 3-fold decrease in specific force and maximal shortening velocity in TA muscles. It is surprising that myofibril-specific force from both control and flight MAs were similar to flight TA muscles, yet power was compromised by 40% following flight. Continued loading in microgravity prevents atrophy, but masticatory muscles have a different set point that mimics disuse atrophy in the appendicular muscle.— Philippou, A., Minozzo, F. C., Spinazzola, J. M., Smith, L. R., Lei, H., Rassier, D. E., Barton, E. R. Masticatory muscles of mouse do not undergo atrophy in space. FASEB J. 29, 000–000 (2015). www.fasebj.org

ABSTRACT

Key Words: atrogenes • microgravity • muscle adaptations muscle atrophy



Abbreviations: AFC, atomic force cantilever; CBTM-3, Commercial Biomedical Testing Module-3; Egr1, early growth response-1;FAK,focaladhesionkinase;HSL,half-sarcomerelength; Igf1, insulin-like growth factor I; MA, masseter; Mstn, myostatin; MuRF-1, muscle ring finger-1; NASA, National Aeronautics and Space Administration; P, phosphorylated; SL, sarcomere length; STS, Space Transportation System; TA, tibialis anterior; TBS, Trisbuffered saline; TTBS, TBS plus 0.1% Tween 20

0892-6638/15/0029-0001 © FASEB

SKELETAL MUSCLE HAS THE remarkable ability to adapt to changes in workload. Numerous muscle properties can be modulated, including muscle mass, contractile properties, and metabolism. Changes in patterns of gene expression and shifts in the balance between protein synthesis and degradation are required for adaptational responses. How well the existing properties meet the demands on the tissue is coordinated by mechanical, chemical, and metabolic information to instigate the process of muscle adaptation. Identification of major pathways that directly regulate gene expression and protein synthesis/degradation demonstrate that multiple inputs can converge on final common pathways for muscle adaptation. Understanding the contribution of the wide variety of inputs on muscle adaptation has been challenging. Skeletal muscle mass generally is regulated by a dynamic balance between protein synthesis and degradation and a vital equilibrium between the signals driving these processes (1, 2). In skeletal muscle, sensors of mechanical loading are situated in the sarcolemma tethering the intracellular cytoskeleton to the extracellular matrix. Specifically, two major protein complexes—the focal adhesion complex and the dystrophin glycoprotein complex—are important for sensing mechanical stress at the membrane and are thought to coordinate the balance between muscle growth and atrophy (3–5). Both complexes transmit mechanical information to the cell nucleus via their association with specific nonreceptor protein tyrosine kinases such as focal adhesion kinase (FAK) (6). Phosphorylation of FAK affects its association with other signaling proteins, leading to the activation of the Ras-Raf-MEK-ERK pathway, as well as the phosphatidylinositol 3-kinase-Akt pathway, through which FAK mediates its signaling to promote muscle cell survival and muscle mass maintenance (7). In response to reduction of external mechanical loading, including disuse and microgravity, the dynamic balance is shifted in favor of protein degradation over synthesis (2, 8–10). Systematic muscle protein degradation occurs by the activation of muscle-specific ubiquitin ligases, 1 Correspondence: 1864 Stadium Rd., 124 Florida Gym, Gainesville, FL 32611. E-mail: erbarton@ufl.edu doi: 10.1096/fj.14-267336

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most prominently Atrogin-1 (MaFbx) and muscle ringer finger-1 (MuRF-1) (11, 12). The expression of progrowth genes is down-regulated simultaneously (13–15). In the microgravity environment of space flight, absence of weight bearing has detrimental effects on skeletal muscle, including reprogramming of the expression pattern of various genes related to muscle growth/atrophy, transformation of muscle fiber types, and mass reduction (16–18). Most of these previous studies on mice subjected to microgravity have focused on limb muscles, where much has been revealed regarding adaptational responses of appendicular muscle to lack of external load. A differential response may occur in masticatory muscles, which has not been addressed. We have reported previously clear differences in terms of loading signals between the masseters (MAs) and limb muscles (19). Further, in clinical situations where there is severe muscle wasting, as seen in patients with acute quadriplegic myopathy in the intensive care unit, the masticatory muscles are spared. This suggests these muscles are equipped with a different load sensing program than limb muscles (20, 21). Animal models for acute quadriplegic myopathy recapitulate the protection against atrophy in MA muscles in stark contrast to the muscle atrophy in the rest of the body (22, 23). These studies raise the possibility that masticatory muscles have a unique loading set point and that they do not respond to unloading in the same manner as appendicular muscles. In the current study, we compared the signaling, expression, and functional responses of appendicular versus masticatory muscles to the microgravity environment of space flight. We obtained tibialis anterior (TA) and MA muscles from mice subjected to microgravity and age- and sex-matched ground controls on the last space shuttle mission, Space Transportation System (STS)-135, of the National Aeronautics and Space Administration (NASA). To evaluate the loading response thoroughly, we also compared the responses of the masticatory muscles in mice subjected to a liquid diet, which eliminates the loading from normal chewing but still affords muscle movement and activity. We hypothesized that the loading of MA muscles comes in part from normal chewing activity, and therefore the mouse MAs may be spared from atrophy in the weightlessness environment, yet they would still succumb to atrophy on a liquid diet.

MATERIALS AND METHODS Animals All experiments were approved by the University of Pennsylvania Institutional Animal Care and Use Committee, whereas handling of animals in all phases and conditions of the study (baseline and ground controls and flight animals) was in accordance with the principles described in the Guide for the Care and the Use of Laboratory Animals (Office of Science and Health Reports of the National Institutes of Health, Bethesda, MD, USA). This study was part of the Commercial Biomedical Testing Module-3 (CBTM-3) payload of the STS-135 shuttle mission by NASA, and its approval was requested and obtained by NASA. The CBTM-3 payload was developed by the NASA Ames Research Center (Moffett Field, CA, USA), and the STS-135/CBTM-3 mission characteristics are described on the NASA site (http://www.nasa.gov/ mission_pages/station/research/experiments/1016.html).

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The animals used for this study included wild-type C57Bl/6 female mice, 9 weeks of age at launch and ;11 weeks old at the time of dissection. Three groups of mice (N = 15 each) were planned for this study. Specifically, the flight group was housed aboard the Shuttle Atlantis (NASA mission STS-135, July 2011) for ;13 days (12 days, 18 hours, 28 minutes), the ground controls also were housed on the ground for 13 days in normal laboratory cages in CBTM-3 payload, and baseline controls were housed in normal laboratory cages, but they were ;13 days younger at the time of dissection. The animals of all groups were acclimated to NASA solid food bars and had full access to food and water. All mice were conditioned in the same manner (i.e., housed under the same environmental conditions including temperature, light/dark cycle, humidity, oxygen levels, and carbon dioxide levels, with the exception of gravity). The animal maintenance and muscle dissections of the on-ground experiments were carried out at the Space Life Sciences Laboratory and its support facilities in the Kennedy Space Center (Merritt Island, FL, USA). The authors of the present study were allowed access to the mice at the time of muscle tissue collection, and they were not involved in designing the study or performing the animal maintenance.

Liquid diet regimen Adult C57Bl/6 female mice (age 10–12 weeks) were subjected to a liquid diet (AIN-93G) or hard chow for 13 days (N = 6 per condition). Mice were housed in animal facilities at the University of Pennsylvania for this portion of the study. At the end of the treatment, mice were killed, and superficial MAs were dissected for morphologic and biochemical analysis. In addition, the mandible was removed intact to measure the length of the incisors and confirm the reduction of external load on the masticatory muscles.

Muscle analysis From the mice for the STS-135/BSP protocol, our laboratory had access to TA from 1 hindlimb of 7 animals from each experimental group and also both MA muscles from all (15) animals of each group. The mice were killed by carbon dioxide inhalation, and the dissections of the aforementioned muscles were performed at the Space Life Sciences Laboratory of the Kennedy Space Center, whereas the muscles from the flight mice were excised 3–5.5 hours after the Shuttle landing. Muscles were blotted, weighed, and rapidly frozen in liquid nitrogen for immunoblotting and expression analysis or myofibril isolation for function, or they were placed in 4% paraformaldehyde for up to 4 hours at 4°C, incubated with 20% sucrose overnight on a shaker at 4°C, and then mounted in a slightly stretched position, surrounded by optimal cutting temperature compound (Sakura, Torrance, CA, USA), and rapidly frozen in melting isopentane for morphologic analysis.

Morphologic analysis Frozen cross-sections (10 mm thick) were cut from the mid-belly of each left MA muscle and subjected to immunohistochemistry for laminin (rabbit Ab-1, RB-082-A; Neomarkers, Fremont, CA, USA), with an anti-rabbit secondary antibody (Alexa Fluor 555, no. A-21428; Life Technologies, Carlsbad, CA, USA), to measure the cross-sectional area of individual fibers. Stained sections were visualized on a Leica DMR microscope (Leica Microsystems, Bannockburn, IL, USA), and digital images were analyzed using semi-automatic muscle analysis (24).

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PHILIPPOU ET AL.

stained with Coomassie Brilliant Blue R-250 after immunoblotting to confirm equal protein loading.

Protein extraction and immunoblotting analysis TA and right MA muscles were removed from liquid nitrogen storage and ground with a mortar and pestle cooled with dry ice. Approximately half of the sample was homogenized in 10 volumes/muscle wet weight of modified RIPA lysis buffer containing protease and phosphatase inhibitors (50 mM Tris·HCl, pH 7.4, 1% w/v Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM NaVO4, 1 mM NaF, and 1 mM EGTA) for total protein extraction. The other half of the sample was placed in TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) for RNA isolation. For protein isolation, muscle tissue homogenates were centrifuged to pellet debris, and the total protein was measured in the supernatant using the Bradford procedure (Bio-Rad Protein Assay, Hercules, CA, USA) using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). To identify potential signaling differences between baseline control and flight mice in appendicular and masticatory muscles, equal amounts of protein (80 mg) from each muscle lysate were heated at 95°C for 5 min, separated by 12% Tris/HCI SDS-PAGE under denaturing conditions, and transferred to polyvinylidene fluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). Because the number of samples exceeded the lanes on the gels, several of the samples were loaded onto multiple gels to provide common references for comparisons across all conditions. Membranes were incubated in a blocking buffer [5% nonfat dry milk in Tris-buffered saline (TBS) plus 0.1% Tween 20 (5% milk-TTBS)] and then incubated in primary antibody diluted in 5% milk-TTBS overnight at 4°C. The following primary antibodies were used for the immunodetection of the various proteins of interest: rabbit phospho-Akt antibody (1:300 dilution) (no. 3787S; Cell Signaling, Beverly, MA, USA), mouse total Akt (1: 2,000 dilution) (no. 2920S; Cell Signaling), mouse phospho-FAK (1:500 dilution) (no. 05-1140; Millipore) and rabbit total FAK (1:500 dilution) (no. 06-543; Millipore), rabbit Egr1 (1:400 dilution) (no. 18-003-43935; GenWay Biotech, GenWay Biotech, San Diego, CA, USA), and mouse tubulin (1:2000 dilution, T5168; Sigma-Aldrich, St. Louis, MO, USA). Membranes then were washed in TTBS and incubated with horseradish peroxidase-conjugated secondary antibodies for 1.5 hours at room temperature: anti-rabbit IgG (goat antirabbit, 1:2000 dilution; no. 7074; Cell Signaling), or anti-mouse IgG (goat anti-mouse, 1:2000 dilution, no. 7076; Cell Signaling). After a series of washes in TTBS and TBS, specific bands were visualized by exposure the membrane to X-ray film after incubation with an enhanced chemiluminecent substrate according to the manufacturer’s protocol (Western lightningECL; PerkinElmer, Waltham, MA, USA). Analysis of band intensity was performed by the Kodak mm4000 detection system (Eastman Kodak, Rochester, NY, USA). Tubulin, total FAK, and Akt were used as internal controls to correct for potential variation in the protein loading. In addition, membranes were

Gene expression analysis Each muscle sample was ground as described above (protein extraction and immunoblotting analysis), and total RNA was isolated from the resultant powdered tissue using TRIzol (Invitrogen), according to the manufacturer’s recommendations. The extracted RNA was dissolved in RNAase-free water (Invitrogen), and the concentration and integrity were determined by 260:280 nm absorbance and gel electrophoresis, respectively. Reverse transcription was performed on 1 mg total RNA from each muscle sample (Applied Biosystems, Foster City, CA, USA), and the resultant cDNA was used in real-time PCR. Real-time PCR analyses were performed using Applied Biosystems 96-well Thermal Cyclers (Applied Biosystems), 7300 Real-Time PCR System, Step One Plus PCR System, and reagents (Power SYBRgreen PCR Master mix). The primer set sequences used for the specific detection of insulin-like growth factor-1 (Igf1), myostatin (Mstn), MuRF-1, atrogin-1, and the transcription factor early growth response-1 (Egr1) are shown in Table 1. The ribosomal 18S RNA housekeeping gene was used as an internal standard. Transcript levels of the genes of interest were assessed by automatically calculating the threshold cycle (Ct). Each sample was analyzed in duplicate, and the resulting data were averaged. Primer specificity was confirmed by melting curve analysis and by electrophoresis of the real-time PCR products. Negative controls included cDNA-free (RNA not reverse transcribed), and template-free (water) reactions.

Myofibril mechanics Frozen muscles were transferred into a rigor/glycerol (50:50) solution and stored overnight at 10°C. The next day, these were placed in a fresh rigor/glycerol (50:50) solution containing protease inhibitors (Roche Diagnostics, Basel, Switzerland) and stored at 220°C for $7 days for the skinning process to be complete. Before each experiment, a smaller piece of muscle (;1 mm3) was cut and defrosted at 10°C in rigor solution for ;1 hour. The same piece was homogenized in rigor solution using the following sequence: twice for 5 seconds at 7500 rpm and once for 3 seconds at 18,000 rpm. The homogenate containing single myofibrils was transferred into a temperature-controlled bath (10°C) and placed on the stage of an inverted microscope (Eclipse TE 2000U; Nikon, Tokyo, Japan). The bath was filled with rigor solution, which was replaced with a relaxing solution after 5 minutes. Myofibrils were selected for mechanical measurements based on intact striation pattern.

TABLE 1. Sequence of the specific sets of primers used in quantitative RT-PCR analyses of the various genes of interest Oligonucleotides (59-39) Gene

Atrogin1 Egr1 Igf1 Myostatin Murf1 18s

Sense

Antisense

AACAAGGAGGTATACAGTAAGG GAGCGAACAACCCTATGAGC CTTCACACCTCTTCTACCTGGCGCTCTGC CTGTAACCTTCCCAGGACCA AGGGCTCCCCACCACCTGTGT CTCTGTTCCGCCTAGTCCTG

AAATGTTCATGAAGTTCTTTTG GGTTCAGGCCACAAAGTGTT TCAGAGGAGTCTAGTGTCGAGGC GCAGTCAAGCCCAAAGTCTC TGCCCTCTCTAGGCCACCG AATGAGCCATTCGCAGTTTC

MUSCLE-SPECIFIC ADAPTATIONS CAUSED BY SPACE FLIGHT

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The rigor solution (pH 7.0) was composed of (in mM) 50 Tris, 100 NaCl, 2 KCl, 2 MgCl2, and 10 EGTA. The activating (pCa2+ of 4.5) and relaxing (pCa2+ of 9.0), pH 7.0, solutions contained (in mM) 20 imidazole, 14.5 creatine phosphate, 7 EGTA, 4 MgATP, 1 free Mg2+, and free Ca2+ in 2 concentrations adjusted to obtain pCa2+ of 4.5 (32 mM) and 9.0 (1 nM). KCl was used to adjust the ionic strength to 180 mM for all solutions. The functional measurements used in this study were described previously in detail (25). In brief, myofibrils were attached between an atomic force cantilever (AFC) and a glass microneedle using micromanipulators. A multichannel fluidic system connected to a double-barreled pipette was used for activation (pCa2+ 4.5) and relaxation (pCa2+ 9.0) of the myofibrils. Changes in length were performed by the glass microneedle, which was attached to a piezoelectric motor. Under high magnification (360 objective), the contrast between the dark bands (A-bands) and the light bands (I-bands) provided a detectable variation in light intensity profile that allowed measurement of the myofibril sarcomere lengths (SLs) during the experiments. Because the AFC stiffness (K) is known, F is calculated based on the AFC displacement (Dd) during contraction (F = K 3 Dd). To detect Dd, a laser beam was shined onto the AFC and then reflected in a photo-quadrant detector. After the myofibrils were attached between the AFC and microneedle, the SL was adjusted to ;2.8 mm and subsequently flushed with activating solution. Once force was fully developed, the myofibrils were either kept isometric or shortened by a nominal amplitude of 20% SL (19.3 6 1.2%) at speeds ranging between 0.008 and 8.0 SL×s21, during which force declined and redeveloped to reach a new steady state. Considering a half-SL (HSL) of ;1.4 mm, a velocity of 8.0 SL×s21 (;11.2 mm×s21×HSL21) is fast enough to overcome the unloaded cross-bridge cycling velocity (25, 26), which causes the force to transiently drop to zero before taking up the shortening. The rate of force redevelopment (ktr) was calculated after the myofibrils were shortened by 8.0 SL×s21. The ktr was calculated using a double-exponential equation: F = {a 3 [1 2 exp(2ktr 3 t) 2 exp(2l 3 t)] + b}, where F is force, t is time, ktr is the rate constant for force redevelopment, l is the second rate constant, a is the amplitude of the exponential, and b is the initial force value. When myofibrils were shortened at slower speeds, forces decreased by different amounts; the ratio between the lowest force achieved in the end of shortening and the highest steady-state force from each contraction at different velocities was used to construct a force-velocity relationship. The force-velocity data were fitted with Hill’s equation: (F + a) 3 (V + b) = (Fo + a), where a and b are constants, F is force at a given velocity, V is the velocity, and Fo is the maximal isometric force when V = 0. Vmax occurs when F = 0, where Vmax = (b 3 Fo)/a. Statistical analysis All data are presented as means 6 SEM . Unpaired Student t tests were used for comparisons of MA muscle fiber size

between controls and flight or liquid diet samples for the same muscle group. Gene expression, protein analysis, and force generation were compared across all muscle groups using 2-way ANOVA followed by Bonferroni’s post hoc test. Hill’s equation was used to extrapolate V max from each experiment separately and to fit the force-velocity data in all experiments. The level of statistical significance was set at P , 0.05.

RESULTS Muscles exhibit differential susceptibility to microgravity Body and muscle masses were measured in microgravity, ground control, and baseline mouse groups. After 13 days of space flight, the mean body mass of mice in the flight group did not show significant differences compared with that of the baseline or ground control animals, exhibiting a 3.6% reduction from the age-matched ground control group (Table 2). The wet weight of the TA muscles from flight mice was significantly reduced by ;15% compared with baseline control muscles and ;19% compared with the muscles of the age-matched ground control group. This led to a significant 20% reduction in the TA muscle/body mass ratio in the flight mice. In contrast, MA muscle mass was not affected by microgravity, nor was there a change in MA muscle/body mass ratio (Table 2). There was no significant change in MA muscle fiber size distribution of flight compared with ground control animals (Fig. 1A, B), consistent with the mass results. To determine whether loss of external load for masticatory muscles resulted in fiber atrophy, mice were subjected to a liquid diet (27). There was no effect of the diet on body weight. To confirm that the mice were not using their muscles during the feeding process, the frontal incisor length was measured before and after liquid diet. Incisors increased by ;0.5 mm after 2 weeks on a liquid diet, which is consistent with the normal rate of incisor growth and indicates that the masticatory muscles were not subjected to the load of chewing hard food for that period (28). After 2 weeks on a liquid diet, the superficial MA fiber size decreased significantly (Fig. 1C). Median fiber size was reduced by .40% (Fig. 1D). These results support that external load is a significant factor in determining muscle fiber size independent of the developmental origin.

TABLE 2. Body and muscle mass of control and flight mice after space flight for 13 days Parameter

Age (wk) Body mass (g) MA mass (mg) MA/body (mg/g) TA (mg) TA/body (mg/g)

Baseline control

19.7 74.6 3.91 38.1 1.92

9 6 6 6 6 6

0.4 2.2 0.08 1.1 0.10

Ground control

19.7 86.0 4.70 41.0 2.11

11 6 6 6 6 6

0.6 2.0 0.10 2.0 0.07

Flight

19.0 84.2 4.55 32.5 1.70

11 6 6 6 6 6

0.6 3.6 0.19 2.2* 0.10*

Mass ratio (flight/ground)

0.96 0.98 0.97 0.81 0.80

6 6 6 6 6

0.03 0.04 0.02 0.05* 0.03*

Values represent the means 6 SEM for N = 7 mice, N = 4 TA muscles, and N = 7 MAs for each group. Significantly different from baseline control for unpaired Student’s t tests: *P , 0.05.

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Figure 1. MA fiber size does not change after space flight but decreases with liquid diet. A) Cumulative fiber size distribution calculated from cross sections subjected to immunohistochemistry with anti-laminin for N = 7 MAs per condition. There was no statistical difference in the distribution between MAs from mice in flight vs. those on the ground. B) Median fiber areas for the same groups of MA muscles show there is no change in this parameter after space flight. C) Cumulative fiber size distribution from cross sections subjected to immunohistochemistry with anti-laminin for N = 4 MAs per condition, shows a leftward shift in fiber size. D) Median fiber areas for the same groups of MA muscles exhibited a significant reduction after liquid diet compared with control animals. Means 6 SEM. *Significantly different between control and liquid diet muscles by unpaired Student’s t test.

Load-related signaling pathways are preserved in MAs after space flight The signaling responses of appendicular and masticatory muscles to microgravity were examined by assessing the phosphorylation of specific signaling proteins associated with load-sensing and muscle growth pathways. The TA muscles exhibited a .3-fold lower FAK phosphorylation compared with MAs in both ground and flight conditions (Fig. 2A, D). However, there was no effect of flight on phosphorylated (P)-FAK in either muscle. Consistent with P-FAK levels, TA muscles displayed significantly lower PAkt levels than MAs (Fig. 2B, D). Microgravity caused a further reduction of P-Akt in TA muscles but not in the MAs. Finally, the mechano-responsive transcription factor EGR1 showed no difference between TA and MA on control animals but was significantly elevated in MAs compared with TA in flight mice, as well as to MAs from ground control mice (Fig. 2C, D). In all proteins, the differences between muscles achieved significance by 2-way ANOVA (P , 0.01). Overall, MAs displayed elevated load signaling in both flight and control conditions in phosphorylation of FAK and Akt, whereas TA muscles exhibited reduced signaling of Egr1 in response to microgravity. Gene expression changes are not apparent in MAs after space flight To evaluate potential differences in gene expression in the appendicular and masticatory muscles caused by the microgravity environment, classic mechano-sensitive and muscle atrophy- or growth-related genes were examined by MUSCLE-SPECIFIC ADAPTATIONS CAUSED BY SPACE FLIGHT

quantitative RT-PCR. The atrogenes atrogin1 and Murf1 displayed significance for flight and interaction between muscle and flight. Post hoc analysis revealed significantly increased atrogin1 and MuRF1 in TA flight muscles compared with control TA muscles and with MAs from flight mice (Fig. 3A, B). The pro- and antigrowth factors, Igf1 and Mstn, were expressed at higher levels in MAs than in TA muscles in the flight mice, but there was no response to flight in either muscle group (Fig. 3C, D). Likewise, Egr1 expression differed only between muscle groups in the flight mice where levels were higher in the MAs (Fig. 3E). For all genes of interest, the muscle type was a significant effect by 2-way ANOVA (P , 0.01). Taken together, gene expression followed expected patterns in response to space flight in the TA muscles, but MAs exhibited no sensitivity to anti-gravity. Reducing load in MAs by liquid diet alters signaling but not gene expression Because the MAs were atrophic after a liquid diet, the muscles were examined for potential differences in both signaling responses and gene expression as those muscles subjected to microgravity. MAs exhibited 2.7-fold lower FAK phosphorylation following a liquid diet (Fig. 4A, B), as well as a 1.6-fold reduction in P-Akt levels (Fig. 4A, C). However, there was no significant effect on gene expression of the atrogenes (atrogin1 and Murf1), pro- and antigrowth factors (Igf1 and Mstn), or the mechano-responsive transcription factor, Egr1 (Fig. 4D). Therefore, although MAs appear to have intact load-sensitive signaling pathways, they do not follow the expected patterns of gene expression following load reduction. 5

Figure 2. Load-associated signaling pathways differ between limb and masticatory muscles. A) FAK phosphorylation is elevated in MA muscles compared with TA muscles, but it does not change in space flight. B) Akt phosphorylation is higher in control MAs compared with TA muscles. Space flight caused a reduction in P-Akt in TA muscles but not MA muscles. C) Egr1 protein levels do not differ between MA and TA muscles in control mice but are significantly higher in MAs of space flight mice. D) Representative Western blot for the phosphorylated and total FAK and Akt and in the level of the transcription factor Egr1. Tubulin served as a loading control. Normalization between the TA and MA muscle samples as achieved by running TA muscle lysates on MA lysate immunoblots (right MA panel, TA sample between control and flight) and vice versa to have common samples between blots. Data shown are means 6 SEM for N = 4 TA and N = 6 MA muscles per condition, where the protein intensities are normalized to the values of TA control muscles. *Significantly different between control and flight; †Significantly different between MA and TA under same condition. Comparisons by 2-way ANOVA followed by Bonferroni multiple comparison test.

Contractile properties of myofibrils depend on loading The study design prevented our assessment of wholemuscle function in the mice. Therefore, to gain insight on the effects on space flight on the contractile properties of the muscles, we directly measured myofibrillar force production in ground control and flight muscles. Typical maximal isometric forces were significantly higher in control TA myofibrils compared with any other group (Fig. 5A, B). Similar to other parameters, specific force in TAs dropped almost 3-fold with space flight, whereas there was no significant change in strength occurred in MA myofibrils. The maximal shortening velocity (Vmax) in TA myofibrils was twice as fast as that in control MA myofibrils (Fig. 5C), and with flight, TA Vmax slowed to MA speeds. In contrast, the Vmax of MA myofibrils was not affected by flight. The rate constant for force redevelopment (ktr) displayed similar trends as strength and kinetic measurements but were not statistically significant (Fig. 5D). To compare the force generation properties of each group of myofibrils, force-velocity and power curves were generated from all accumulated measurements (Fig. 5E, F). TA controls were not only faster than the flight group, but produced higher forces at submaximal velocities, which became evident when the force-velocity curves were plotted against absolute forces, resulting in the construction of power curves (Fig. 5E, F). TA controls were able to 6

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generate almost 5 times as much power (;900 fW) as the flight group (;200 fW). In MA, power generation was substantially diminished in controls (130 fW/mm2), and flight caused a further ;40% reduction to 80 fW/mm2. Thus, power output appeared to be the only parameter negatively altered in MAs subjected to flight. DISCUSSION Gaining mechanistic insight into muscle atrophy associated with microgravity may contribute to finding ways to counteract disuse atrophy in many situations. In this study, we extended previous work by comparing the responses of masticatory with appendicular muscles because these muscles operate under different loading conditions even when subjected to the same microgravity environment. This experimental approach probed how load signaling and gene expression patterns that are associated with muscle cell survival and growth are modified when skeletal muscle acts in weightlessness with and without mechanical stress. We found that limb muscles underwent atrophy in response to microgravity, evident by the loss of mass, alterations in mechanical loading signaling pathways, and in the balance between pro- and antigrowth gene expression of TA muscles. The functional measurements of TA myofibrils revealed a dramatic reduction in strength following flight, including loss of force output, speed of contraction,

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Figure 3. Gene expression changes in response to space flight are evident in limb muscles but not masticatory muscles. The atrogenes Atrogin1 (A) and Murf1 (B) significantly increased in TA muscles in response to space flight by 12- and 4-fold, respectively, but no change was observed in the MA muscles. In both cases, expression of both atrogenes was significantly lower in MAs compared with TA muscles of mice subjected to space flight. In contrast, space flight did not alter expression of Igf1 (C) or Mstn (D) in either muscle group. MA muscles exhibited increased expression of both growth factors compared with TA muscles, which was significant in mice from the space flight group. Finally, expression of Egr1 (E) was modestly elevated in MAs but did not achieve significance. Egr1 did not change in response to space flight in either muscle group. Data shown are means 6 SEM for N = 4 muscles per condition, where the fold-change expression is normalized to the values of TA control muscles. *Significantly different between control and flight; †significantly different between MA and TA under same condition. Comparisons by 2-way ANOVA followed by Bonferroni multiple comparison test.

and, ultimately, power. Virtually all of these responses were absent from MA muscles. Further, MAs exhibited greater phosphorylation of FAK and Akt and an increased progrowth drive in control and flight conditions. Musclespecific adaptations caused by space flight have been reported between fast and slow muscles in rats (29–32), humans (33), and mice (18), where fast muscles are less responsive to unloading than slow muscles. However, myosin heavy chain analyses have shown that murine MA and TA (19) are all composed of fast-twitch fiber types. Therefore, fiber-type differences cannot explain the divergent responses to flight. Previous studies investigating the effects of exercise loading on human skeletal muscle during a prolonged (;180 days) exposure to space weightlessness have shown that mass loss in limb muscles is exponential with the duration of flight and that aerobic and low intensity resistance exercise countermeasures fail to adequately protect muscle mass, especially of the antigravity soleus muscle (33, 34). The lack of mass loss in MA muscles revealed in this study could be a protective effect, at least for the short period of an ;13 days of space flight, by chewing-induced loading of these muscles, the specific physiological characteristics that masticatory muscles possess, or a combination of both. Indeed, removal of load from MAs through a liquid diet results in atrophy, supporting that mechanisms that protect MUSCLE-SPECIFIC ADAPTATIONS CAUSED BY SPACE FLIGHT

against loss of mass, such as loading, are shared by masticatory and appendicular muscles. Although there is little difference in whole muscle function of MA muscles compared with limb muscles (35), this is not the case for myofibrils. It is surprising that, even though there was no significant response to microgravity of masticatory specific force, contractile properties of MA myofibrils in both conditions approximated those in TA myofibrils from flight, not control, suggesting that at the contractile protein level, there are also divergent features between these muscle groups. The loss of force and power in flight TA muscles is consistent with the preferential loss of actin and associated thin filament proteins following hindlimb suspension and space flight (36–39).This may hold true to a lesser degree for MAs as well, causing the reduction in power in our samples from flight. MA myosin/actin ratios appear normal up to 9 days in a rat model of critical illness myopathy (21), but these ratios have not been determined following space flight. It may be the case that control masticatory muscles have additional deficits in contractile proteins that cause diminished function on land, such as filament organization, which have yet to be identified. Consistent with previous studies of limb muscles (17), Akt phosphorylation dropped in TA muscles with space 7

flight. Because this pathway is a nodal point for the regulation of both protein synthesis and degradation, the reduction of activity correlates well with the increased atrogene expression and loss of mass. The fact that MAs did not exhibit the same drop is not surprising in the context of continued loading from mastication, but the significantly higher basal P-FAK and P-Akt in MAs suggest that normal mechanical stress is heightened in these muscles, or that there is a different setpoint for flux through this pathway. One contribution could be the heightened Igf1 expression in MA muscle in both conditions, whose activity also drives P-Akt. However, we did not explore the proportion of

Figure 4. Load-associated signaling pathways but not gene expression changes respond to liquid diet in MAs. A) Western blots for the phosphorylated and total FAK and Akt, and tubulin. Both P-FAK (B) and P-Akt (C) were significantly reduced in MAs following 2 wk of liquid diet. However, no significant changes in gene expression were observed (D). Data shown are means 6 SEM or N = 3 muscles per condition. *Significantly different between control and liquid by unpaired t test.

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which either integrin or growth factor receptor pathways led to increase P-Akt. However, because IGF-I has been shown to prevent the expression of muscle atrophyinduced ubiquitin ligases (40), and atrogene expression is lower in MAs, it is likely that this pathway is elevated in the masticatory muscles. Unlike other genes of interest we assessed, neither Mstn nor Egr1 expression was altered in TA muscles after space flight. Because increased myostatin activity prevents muscle growth, in part through blockade of Akt phosphorylation (41–43), our results present a dissociation of myostatin from the atrophy observed. Specifically, the reduction of P-Akt in TAs from flight was not correlated with an increase in myostatin. Further, the MAs exhibited increases in both Mstn expression and P-Akt. Recent evidence from other studies suggests that not all models of atrophy share the same pathways. Myostatin inhibition in a variety of conditions is differentially effective for prevention of muscle mass loss (44–46) Thus, although myostatin blockade may be beneficial in unloading-induced atrophy, it does not appear to be a central player in our model. For Egr1, which is an early response gene sensitive to mechanical loading, the major difference we observed was an increase in both transcription and translation of this transcription factor in MAs from flight. However, although Egr1 is responsive to mechanical stretch (47) and is thought to play a role in myogenesis and muscle regeneration (48, 49), or to be a potential key regulator of muscle cachexia (50), it is not clear that it plays a role in the response to a lack of external load. Thus, we attribute the elevated Egr1 in MAs to heightened mastication/activity in contrast to that in the TA muscles of both conditions. Similar to limb muscles after space flight, a liquid diet resulted in a reduction of P-Akt in MAs but also lower P-FAK, supporting that the signaling pathways involved in load sensing are common to both muscle groups. However, these signaling changes did not lead to alterations in gene expression in MAs after a liquid diet, even though muscle fiber size was affected. Masticatory muscles are morphologically and physiologically distinct from axial muscles, and, unlike the latter, masticatory muscles might be slow to remodel (19, 35, 51, 52). They also have reduced ability to hypertrophy and an increased propensity to undergo apoptosis (19). Whether alternate pathways are responsible for the loss of fiber area in MAs or that we did not capture the time point at which atrogene expression is changing was not addressed. Regardless, it is becoming clearer that communication between sarcolemmal load sensors and the nucleus is not a shared property between masticatory and appendicular muscles. In conclusion, the present study sheds more light on a mechanistic understanding of muscle responses to microgravity, demonstrating differential signaling, gene expression, and functional responses in the limb muscles of the space flown mice compared with the chewing-loaded MAs of the same animals. These findings indicate that continued mechanical loading of skeletal muscle in a weightlessness environment can

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PHILIPPOU ET AL.

Figure 5. Myofibrillar force generation parameters are altered by space flight. A) Superimposition of isometric contractions produced by myofibrils isolated from TA (left) and MA (right) muscles in control and flight groups. Mean values (6SEM) of specific forces (B), Vmax (C), and KTR (D) calculated after experiments with myofibrils isolated from TA (open bars) and MA (hatched bars) muscles from control (white bars) and flight (gray bars) groups. *Significantly different from the same muscle under different conditions. †Significantly different between muscle groups within the same condition by 2-way ANOVA followed by Bonferroni multiple comparison test. Velocity and power plotted against absolute forces compiled from all measurements for TA (E) and MA (F). Solid lines are control, and dotted lines are flight.

prevent atrophy but may not be sufficient to protect against loss of power. The authors gratefully acknowledge NASA for allowing access to the animals of the CBTM-3 payload of the STS-135 shuttle mission and for providing technical support and equipment in the Space Life Sciences Laboratory in Kennedy Space Center. The study was supported, in part, by NASA Space Biology Grant NNX09AH44G (to E.R.B.), U.S. National Institutes of Health Grant R052646 (to E.R.B.), the Canadian Institutes for Health Research (to D.E.R.), and

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the Natural Science and Engineering Research Council of Canada (to D.E.R.).

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Masticatory muscles of mouse do not undergo atrophy in space.

Muscle loading is important for maintaining muscle mass; when load is removed, atrophy is inevitable. However, in clinical situations such as critical...
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