Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85α, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-γ coactivator-1α and the transcription factor PPAR-α were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.
- hindlimb suspension
spaceflight has many consequences on the musculoskeletal system. Specifically, the unloading due to microgravity results in decreases in muscle mass and force production capability as well as shifts in muscle fiber type toward a faster contracting and less endurable phenotype (10). Qualitatively similar decreases in muscle mass and shifts in fiber type occur in both rats (35) and mice (14), making them an excellent model with which to elucidate the molecular signaling mechanisms involved in the skeletal muscle alterations in response to spaceflight. Thus exposure to microgravity results in decrements in muscle strength and endurance in both humans and rodents that in humans could adversely affect the ability to perform critical functions while in space, particularly on long missions.
Ground-based models also have been used to simulate aspects of the spaceflight environment in rodents. Hindlimb suspension has been used extensively in rats and mice to approximate the musculoskeletal unloading as well as the cephalic fluid shift that occurs during spaceflight (27). Rats subjected to hindlimb suspension approximate the reduction in cross-sectional area and slow-to-fast fiber type transitions in slow-twitch muscles that are observed in the rats flown in space (reviewed in Ref. 26). However, other studies suggest that spaceflight induces gene expression changes in rat skeletal muscle that are not elicited in suspension models (28). This is in some ways unsurprising given that there are significant differences between spaceflight and hindlimb suspension. Specifically, one major difference is that organisms flown in space typically experience a few hours of reloading before death, and this reloading may also influence early events such as signal transduction and gene transcription.
Recently, much progress has been made in elucidating the intracellular signaling pathways governing muscle mass and fiber type. Flux through the phosphatidylinositol 3 (PI3)-kinase/Akt/mammalian target of rapamycin (mTOR) pathway has been demonstrated to increase protein synthesis (reviewed in Ref. 11) and to decrease protein degradation through inactivation of members of the FoxO family of transcription factors (39). This pathway is activated by insulin-like growth factor-I (IGF-I) and can also be influenced by mechanical loading (15), but definitive studies have yet to be done on the effects of spaceflight on activity and/or expression of genes involved in PI3-kinase/Akt/mTOR signaling.
Skeletal muscle mass is also negatively regulated by the myostatin pathway. Inactivation of myostatin through naturally occurring or targeted mutations results in dramatic muscle hypertrophy in mice, cattle, and humans (16). Myostatin acts in a paracrine/autocrine loop by binding to members of the activin RII receptor family, most notably ActRIIb (19), and decreases myoblast proliferation (42), differentiation (18, 33), and protein synthesis (40) while increasing expression of protein degradation genes in vitro (24). Increased myostatin expression has been reported in a number of conditions of unloading atrophy, including spaceflight and hindlimb suspension in rats (7, 17), but to date little is known about the effects of unloading on other myostatin-associated genes.
In addition, an exciting recent finding has been the identification of microRNAs and their role in regulating gene expression in a number of species and a variety of tissues. MicroRNAs are small (∼21 nt), evolutionarily conserved, noncoding RNAs with the ability to regulate gene expression on a wide scale through the binding of the microRNA to sequence-specific sites within target mRNAs (6). Several microRNAs have been identified that regulate skeletal muscle differentiation and gene expression (6) and that demonstrate muscle fiber type-specific expression (23). Currently, however, little is known regarding the expression of microRNAs during conditions of unloading atrophy such as spaceflight.
The purpose of the present study was threefold. First, to better understand the gene expression changes underlying the functional decrements in response to spaceflight, we examined the effects of spaceflight on mRNA expression in gastrocnemius muscle by utilizing both a global microarray approach and a candidate gene approach using quantitative real-time polymerase chain reaction (QRT-PCR). Second, we compared the effects of spaceflight on global mRNA expression to those of hindlimb suspension for a comparable length of time. Third, because all spaceflight studies contain the possible confound of several hours of reloading upon reentry and landing, we evaluated the effects of the 3.5 h of reloading (experienced by the spaceflight mice before death) on mRNA expression of suspended mouse muscle. We hypothesized that both spaceflight and hindlimb suspension would be associated with shifts in mRNA expression that would favor myostatin signaling and decrease PI3-kinase/Akt/mTOR signaling and thus would favor protein degradation over protein synthesis and decreased myogenic cell proliferation and differentiation over cell growth, and that reloading would reverse these effects.
MATERIALS AND METHODS
All animals were housed individually in animal enclosure modules [AEM; developed by National Aeronautics and Space Administration (NASA) Ames Research Center (Moffett Field, CA)] with access to food and water ad libitum. The muscles used in the spaceflight study came from the same mice as those previously described (12, 14, 30): female C57BL/6J mice were divided into AEM ground controls and spaceflight (SF) groups. Animals were maintained on a 12:12-h light-dark cycle with all animal care, treatment, and housing density within National Institutes of Health guidelines. All protocols were approved by the appropriate institutions (University of Colorado at Boulder, Amgen, Inc., NASA Ames Research Center, and NASA Kennedy Space Center). SF animals were flown on the middeck of the space shuttle Endeavour (STS-108/UF-1) for 11 days and 19 h beginning December 5, 2001. AEM animals were housed at Cape Canaveral Air Force Station's Hangar L orbital environmental simulator for 11 days and 19 h with conditions mimicking the shuttle's middeck temperature, humidity, and CO2 levels. As mentioned previously, the module containing the SF mice experienced an increase in temperature during flight that may have contributed to differences in eating and drinking behavior, as previously mentioned (12, 14, 30). AEM and SF animals were killed at 77 days of age, with SF animals being killed 3.5–4 h after landing. The triceps surae (gastrocnemius, plantaris, and soleus) of SF and AEM animals were removed and attached to corkboard with optimal cutting temperature medium (Sakura Finetek, Torrance, CA) and then frozen in liquid nitrogen-cooled isopentane for other purposes (12). The medial section of the gastrocnemius was isolated for RNA analysis. Gastrocnemius muscle (n = 4/group) was subsequently used for DNA microarray analysis.
Hindlimb suspension and reloading.
For the ground-based unloading/reloading studies, mice were divided into three groups (n = 5/group): control, suspension, and suspension plus reloading. The control group was normally housed three mice per cage. The suspension and reloading groups were suspended for 12 days to match the STS-108 flight. Tail suspension was performed using previously described methods (37). Briefly, the mice were suspended at an approximately 30° angle by taping the tail to a plastic dowel attached to a swivel apparatus. This apparatus was attached to a guide wire running the length of the cage. The mice were able to access all areas of the cage because of the swivel between the dowel and the wire. The suspension animals were killed while suspended at the end of the 12 days. Suspended mice were unsuspended for 3.5 h at the end of the 12 days of suspension and allowed to engage in normal loading for this period to match the time between landing and death for the STS-108 mice. All animals were 77 days of age at the time of death. This protocol was approved by the Clemson University Animal Care and Use Committee. The triceps surae of all animals were removed by dissection, weighed, and snap frozen in liquid nitrogen.
Preparation of total RNA.
Total RNA was prepared from tissue samples by Trizol extraction followed by purification using RNeasy columns (Qiagen, Valencia, CA). Quality and purity of the RNA preparations were assessed by spectrophotometric determination of the ratio of absorbance at 260/280 nm and by quantification of the ratios of 28S:18S ribosomal RNA using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Synthesis of biotin-labeled cRNA targets and hybridization to Affymetrix GeneChips.
Synthesis of double-stranded cDNA, in vitro transcription of biotin-labeled cRNA targets, and fragmentation of target cRNA were performed as outlined by Affymetrix protocols (Affymetrix, Santa Clara, CA). Fragmented cRNA samples were hybridized overnight at 45°C to Affymetrix Mouse Expression 430A GeneChips. Posthybridization washing and phycoerythrin-streptavidin staining and fluorescence scanning were performed using Affymetrix instrumentation in accordance with manufacturer protocols. The resulting scans (CEL files) were deposited in the MUSC DNA microarray database (2); these can be accessed from the database using the project identifiers _1112123225.418684 (gastrocnemius) and _1120601249.362999 (suspension/reload). Microarray data were also deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus repository under the accession number GSE10533.
Analysis of DNA microarray data.
Gene hybridization intensities were normalized using Robust Multichip Average (5) implemented with the ArrayQuest web-based analysis system (3). Normalized hybridization values were imported into the analysis tool dChip 2005 (21) for subsequent analysis. mRNAs differentially expressed in response to spaceflight were identified using both statistical (P < 0.05 for unpaired Student's t-test) and fold change (fold change >1.5) thresholds. The fold change metric was chosen to ensure that the detected expression differences were sufficiently large to be validated by PCR-based approaches. False discovery rate, estimated by iterative comparison of randomized sample groupings, approximated 1.5% for these differential expression criteria. Differential expression in response to suspension, reloading, or the combination of suspension plus reloading was evaluated by pairwise comparison (Student's t-test, unpaired). Statistical power analysis using Power Atlas (29) determined that α (P) <0.05 provided the most robust outcomes in terms of proportion of expected differentially expressed genes discovered (>25%) versus proportion of true positives in the genes discovered (>75%). Gene functional categories were assigned following review of gene ontology annotations as well as functional information available through NCBI Entrez Gene. Hierarchical clustering of differentially expressed mRNAs was conducted using the centroid linkage model with distance metric 1-correlation.
Quantitative RT-PCR (QRT-PCR) was used to confirm expression changes observed in the microarray analysis. First-strand cDNA was prepared from total RNA (1 μg) using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's specifications. cDNA preparations were diluted to 200 μl, and 2 μl were used in 25-μl reactions with the iQ SYBR green Supermix reagent (Bio-Rad). Reactions were amplified in an iCycler real-time PCR detection system (Bio-Rad). The resulting data were analyzed with the Gene Expression Analysis macro (Bio-Rad), which derives an unscaled expression value for the gene of interest by first calculating a relative expression value and then normalizing it by division with the geometric mean of relative expression values found for control mRNAs (Hprt1 and β-actin).
QRT-PCR was also used to quantify mRNA levels of myostatin (MSTN), its receptor, ActRIIb, and its primary binding/inhibiting protein, FSTL3, in all of the SF and AEM samples, as previously described (1). The RT reaction was carried out using 0.5 μg of RNA with the cDNA Archive kit (Advanced Biosystems) according to the manufacturer's protocol. Primer and probe sets for MSTN, ActRIIb, FSTL3, and β-actin TaqMan analysis were obtained from Applied Biosystems (Foster City, CA). These real-time PCR procedures were run in triplicate to correct for variances in loading. In addition, a standard curve ranging from 10- to 0.001-μg dilutions of mouse TA cDNA was run in duplicate for every assay to produce a standard curve for quantification. All values are expressed as the mean of the triplicate measure for the experimental (MSTN, ActRIIb, or FSTL3) divided by the statistical mean of the triplicate measure of β-actin, which was run in duplex, as a normalization control. β-Actin mRNA levels were unaffected by spaceflight (data not shown).
Finally, for analysis of miR-1, miR-133a, and miR-206, RT was performed using a microRNA-specific primer according to the manufacturer's instructions (Applied Biosystems). After RT, samples were analyzed using a microRNA-specific real-time PCR kit according to the manufacturer's instructions (Applied Biosystems). All microRNA PCR results were determined using the relative (ΔΔCt) quantification method with the small nuclear U6 RNA (snRNA U6) run in singleplex as the normalization control, since this is a more appropriate control for normalization of microRNA levels than an mRNA. Levels of the snRNA U6 were not significantly altered by spaceflight (data not shown). For both the QRT-PCR for the myostatin-associated mRNAs and for the miRs, an independent t-test was used to evaluate statistical significance, with α < 0.05 taken as significant.
Muscle atrophy induced by unloading.
As previously reported (14), body weight was significantly decreased in the SF mice compared with both preflight (18.3 g preflight vs. 17.4 g postflight) and AEM mice (19.4 g for AEM vs. 17.4 g for SF). Similarly, body mass in suspended mice was also lower postsuspension compared with presuspension (18.2 g presuspension vs. 17.8 g postsuspension) and unsuspended mice (19.2 g for unsuspended vs. 17.8 g for suspended). Moreover, gastrocnemius samples from mice flown on STS-108 showed significant reductions in fiber cross-sectional area and a decrease in citrate synthase activity, as reported previously (14). Analysis of triceps surae collected from the hindlimb suspended mice and suspended plus reloaded mice in the present study confirmed that the ground simulation unloading models also experienced muscle atrophy: mean weights of the triceps surae taken from the ground-based models were 96.6 ± 1.1, 85.4 ± 2.9, and 88.4 ± 1.3 mg for control, suspended, and reloaded tissues, respectively, with both suspended and reloaded weights significantly smaller than control weights (P < 0.05).
Global microarray analysis.
Analysis of DNA microarray data for STS-108 gastrocnemius revealed that 272 mRNAs were differentially expressed compared with AEM controls (fold change >1.5, P < 0.05), with 151 mRNAs upregulated and 121 mRNAs downregulated (Fig. 1A and Table 1). To evaluate how these 272 mRNAs responded to unloading and reloading stimuli, we performed DNA microarray analysis on calf muscle prepared from ground-based mouse models subjected to hindlimb suspension or hindlimb suspension plus brief reloading. Hierarchical clustering of the expression data for the 272 spaceflight-affected mRNAs revealed many similarities in the responses to spaceflight and hindlimb suspension (Fig. 1, A–C). Many of the mRNAs significantly affected by spaceflight showed similar trends in response to suspension, as indicated by the heat map (Fig. 1A), particularly in the area highlighted by the bracket at right. Examination of microarray data revealed that most mRNAs upregulated by spaceflight showed upregulation in response to hindlimb suspension (113 of the 151; Fig. 1B), whereas most mRNAs downregulated by spaceflight were also downregulated by suspension (92 of 121; Fig. 1C). Furthermore, it can be seen that reloading counteracted the effect of hindlimb suspension for many of these mRNAs such that mRNAs that were upregulated by suspension tended to be downregulated by reloading, and vice versa; only 22 upregulated mRNAs and 8 downregulated mRNAs did not follow this pattern (Fig. 1, B and C).
Further examination of the expression data revealed that 88 of the mRNAs significantly affected by spaceflight were also significantly affected by one or more of the ground-based stimuli (Table 1). Specifically, 46 of the spaceflight-affected mRNAs were significantly affected by suspension, 42 by reloading, and 37 by the combination of suspension plus reloading, with overlaps apparent among these groups (Fig. 1D and Table 1). The majority of these mRNAs (72 of 88) showed a response to suspension that matched the direction of change observed in the SF samples. Furthermore, most of these mRNAs (74 of 88) showed expression patterns that were consistent with sensitivity to both unloading and reloading stimuli, with reloading causing a change in expression that opposed the change induced by hindlimb suspension.
Validation of differential expression
Expression changes observed by microarray experimentation were confirmed for selected representative genes by QRT-PCR analysis. Generally, QRT-PCR results closely matched the microarray findings in terms of magnitude and direction of change (Table 2). Specifically, QRT-PCR supported the effect of spaceflight for all nine mRNAs. Furthermore, all mRNAs that were identified by microarray analysis as significantly affected by suspension or reloading also showed significant changes by QRT-PCR (Table 2).
Spaceflight and PI3-kinase/Akt/mTOR signaling genes.
Spaceflight was associated with significant shifts in expression of mRNAs involved with the PI3-kinase/Akt/mTOR pathway. Levels of mRNA of the PI3-kinase regulatory subunit polypeptide 1, pi3kr1/p85α (2.13-fold), the forkhead box O1 (FoxO1) transcription factor (2.31-fold), the muscle-specific ubiquitin ligase F-box only protein 32 (MAFbx/atrogin1; 1.61-fold), and the ubiquitin-conjugating enzyme E2 variant 2 (1.57-fold) genes were significantly increased in SF muscle compared with control. Conversely, insulin receptor substrate-1 (IRS-1) mRNA levels were significantly decreased 1.55-fold in SF gastrocnemius.
Spaceflight and expression of myostatin-associated genes.
Expression of mRNA for the myostatin-associated gene activin Ib receptor (Acvr1b; also known as ALK4), was significantly increased 1.59-fold with spaceflight (Table 1). The myostatin mRNA is not represented on the 430A GeneChip. However, the primary myostatin receptor ActRIIb and the myostatin binding/inhibitory protein FSTL3 were both present on the GeneChip, although neither were detected as differentially expressed. Nevertheless, given the established importance of these three genes in regulating muscle mass, we examined their mRNA expression using QRT-PCR as an alternate means of analysis. As shown by QRT-PCR, myostatin mRNA levels tended to increase in the SF samples, whereas mRNA levels of FSTL3 tended to decrease in the SF samples, although neither reached statistical significance (Fig. 2). Conversely, mRNA levels of the myostatin receptor ActRIIb were significantly decreased in SF gastrocnemius muscle compared with AEM control (Fig. 2).
Spaceflight and other growth-associated genes.
Spaceflight also affected mRNA levels for other growth regulatory genes. For example, mRNA levels of suppressor of cytokine signaling 2 (Socs2), p21, and Btg2 were significantly increased in gastrocnemius in response to spaceflight (1.51-, 2.68- and 2.95-fold, respectively). Other pathways that have been linked to muscle growth and adaptation include the TNF-α/NF-κB signaling pathway and the calcineurin/nuclear factor of activated T cells (NFAT) pathway. The TNF-α downstream target TNF-α-induced protein 2 mRNA was significantly increased in SF gastrocnemius (1.68-fold), but the NF-κB inhibitor nuclear factor κB light chain gene enhancer in B cells inhibitor α (Nfkbia/IκBα) mRNA was also significantly increased (2.28-fold). Moreover, mRNA levels of the NFAT cytoplasmic, calcineurin-dependent 3 (Nfatc3) transcription factor were significantly decreased (−1.57-fold) in SF gastrocnemius. Finally, mRNAs for three members of the CAAT/enhancer binding protein (C/EBP) family of transcription factors, C/EBP-α (1.72-fold), C/EBP-β (1.58-fold), and C/EBP-δ (3.03-fold) were significantly increased in SF gastrocnemius.
Spaceflight and fiber type genes.
The gastrocnemius muscle of the STS-108 mice examined in this study exhibited a decrease in citrate synthase activity consistent with a shift toward a less oxidative phenotype (14). Among the 272 mRNAs differentially expressed in response to spaceflight, two encode proteins associated with regulating oxidative phenotype through activation of oxidative gene expression. Peroxisome proliferator activated receptor (PPAR)-α and the PPAR-γ coactivator 1α (PGC1-α) were both significantly decreased (1.83-fold and −2.38-fold, respectively).
mRNAs affected by spaceflight, suspension, and reloading.
As mentioned above, the majority of mRNAs affected by spaceflight showed a qualitatively similar response to suspension, and reloading tended to diminish or even reverse the effects of suspension. This was true for pi3kr1/p85α, p21, Socs2, C/EBP-α and -γ, and PPAR-α, which were affected by spaceflight and suspension in a matching fashion and for which reloading counteracted the suspension effect (Table 1). Moreover, 13 other mRNAs affected by spaceflight were significantly by suspension and by reloading (Fig. 1D). Notably, all 13 of these mRNAs showed counteracting responses to suspension versus reloading. Furthermore, all except one showed a significant response to suspension that matched the direction of the significant response to spaceflight, the exception being Btg2, which significantly increased with both spaceflight and reloading but significantly decreased with suspension. Among the 13 mRNAs was the key growth and metabolism regulator IRS-1, which significantly decreased in response to spaceflight and suspension and significantly increased in response to reloading. Conversely, Acvr1b/ALK4 mRNA levels were significantly increased by spaceflight and suspension but significantly decreased in response to reloading (Table 1). Finally, levels of PGC-1α mRNA were significantly decreased by spaceflight and, although not significantly affected by suspension, were significantly increased by reloading relative to suspension (Table 1).
MicroRNA affected by spaceflight.
As shown in Fig. 3, levels of miR-206 relative to the endogenous control snRNA U6 were significantly decreased by nearly 50% in gastrocnemius muscle from SF mice compared with AEM controls (Fig. 3C). In contrast, neither miR-1 nor miR-133a were significantly affected by spaceflight, although both showed a trend toward a decrease, with the decrease in miR-133a nearly reaching significance (Fig. 3, A and B). However, the ratio of miR-1 to miR-133a was significantly increased in SF gastrocnemius (Fig. 3D).
The data previously published for mice from STS-108 showed that murine skeletal muscle exhibited several adaptations in response to spaceflight (14). All fiber types analyzed showed a reduction in cross-sectional area. Moreover, the gastrocnemius showed a decrease in citrate synthase activity consistent with a shift toward a less oxidative phenotype. In this study, analysis of mRNA expression by both gene array and QRT-PCR has shown that spaceflight affects mRNA levels of genes involved in numerous cellular processes, including metabolism, cell cycle, apoptosis, and cytoskeletal and mitochondrial function. Moreover, spaceflight impacts expression of several signal transduction pathways affecting both muscle growth, including those related to growth signaling via PI3-kinase/Akt/mTOR, calcineurin/NFAT, and myostatin and those related to muscle fiber type determination, such as PPAR-α and PGC1-α.
Spaceflight and genes associated with PI3-kinase/Akt/mTOR signaling.
Numerous studies have now demonstrated that a primary pathway regulating muscle protein metabolism, and therefore muscle growth, is the PI3-kinase/Akt/mTOR pathway. This pathway represents a primary node for regulating both protein synthesis and protein degradation in response to various stimuli (11, 20, 32). In the present study, we observed that spaceflight altered mRNA expression of several genes involved in this pathway: the PI3-kinase regulatory subunit p85α, which negatively regulates PI3-kinase signaling by sequestration of the insulin receptor substrate adaptor-1 (13), was increased, IRS-1 was decreased, and mRNA levels of the FoxO1 transcription factor were increased, as were those of one of its primary targets, MAFbx/atrogin1. Together these data support the hypothesis that spaceflight results in a shift in gene expression that favors reduced flux through the PI3-kinase/Akt/mTOR pathway.
Spaceflight and myostatin signaling.
Both the gene array and QRT-PCR data suggested that spaceflight is associated with a shift in gene expression favoring increased myostatin signaling. Levels of myostatin mRNA tended to increase in spaceflight gastrocnemius (Fig. 2), consistent with previous findings from both spaceflight and hindlimb suspension muscle (7, 17). Similarly, mRNA levels of the myostatin binding/inhibiting protein gene FSTL3 also tended to decrease in spaceflight gastrocnemius. Expression of the myostatin-ActRIIb partnering receptor Acvr1b /ALK4 was significantly increased by both spaceflight and hindlimb suspension (and decreased by reloading), although ActRIIb mRNA levels significantly decreased in spaceflight. With the exception of the decrease in ActRIIb levels, these shifts, if translated into changes in the associated proteins, would be consistent with a shift toward increased myostatin signaling in spaceflight muscle. Consistent with this interpretation, levels of p21, a cell cycle inhibitor that is a target of myostatin signaling (42), were significantly upregulated in response to spaceflight, consistent with a previous gene array study on spaceflight rat muscle (41).
In addition, there is considerable feedback between the PI3-kinase/Akt/mTOR/FoxO pathway and the myostatin pathway. Myostatin activates protein degradation by inhibiting Akt phosphorylation of the FoxO transcription factors, resulting in increased expression of ubiquitin ligases (24). Moreover, myostatin expression is itself positively regulated by the FoxO transcription factors (1). Thus the changes in mRNA expression associated with reduced Akt activity described above, as well as the increase in FoxO1 mRNA expression, may also have contributed to the increase in myostatin transcription observed in this study.
Spaceflight and other growth pathways.
Spaceflight significantly affected mRNA levels of several other genes associated with growth regulation. mRNA encoding Socs2, a suppressor of cytokine and growth hormone signaling, was increased, as were mRNAs encoding the cell cycle inhibitors p21 and Btg2. These are consistent with a shift in gene expression favoring reduced cell proliferation, consistent with results from previous gene array studies on spaceflight and hindlimb suspension (41, 44). In addition, expression of mRNAs encoding TNF-α-induced protein 2 and Nfatc3 were altered in ways consistent with shifts in activity of the NF-κB and calcineurin/NFAT pathways, respectively, that would favor muscle atrophy. Finally, there were increases in mRNAs encoding three members of the C/EBP family of transcription factors: C/EBP-α, C/EBP-β, and C/EBP-δ. Recent evidence has suggested that members of the C/EBP family are regulated by glucocorticoids, particularly C/EBP-δ (25), and their binding activity is increased during periods of muscle catabolism (31). Given the present results, the potential role of these transcription factors in regulating muscle atrophy in response to spaceflight and/or other modes of unloading needs to be further addressed.
Spaceflight and metabolism.
Muscle adaptation to disuse is often characterized by fiber type alterations in both contractile and metabolic proteins and protein isoforms, and previous gene array studies on rat soleus have demonstrated metabolic gene expression shifts with spaceflight and/or hindlimb suspension (8, 38, 43). In the present study, expression of both PPAR-α and PGC1-α mRNA was decreased in spaceflight gastrocnemius and was significantly increased in suspended plus reloaded muscle. Dapp et al. (8) also showed a significant decrease in PPAR-α expression with 7 days of hindlimb suspension in the soleus, and expression of both these transcription factors is increased by endurance exercise (36). In addition, PPAR-α agonists induce a fiber-selective transcriptional response in rats (9), whereas transgenic expression of PGC1-α in type II fibers has been shown to stimulate changes in muscle morphology, gene expression, and transition toward a type I fiber phenotype in mice (22). Thus the decrease in expression of these two genes may contribute to the shift toward a less oxidative phenotype observed in response to spaceflight (14).
Spaceflight and microRNA expression.
One of the most exciting breakthroughs in modern biology has been the identification of microRNAs and their role in regulating cell function. MicroRNAs regulate RNA stability and/or translation in a wide variety of species and cells, including muscle (reviewed in Ref. 6). More recent data have demonstrated that several microRNAs are expressed in a muscle type-specific and/or differentiation-specific manner (6) and that these microRNAs appear to play critical roles in muscle gene expression, proliferation, and differentiation (6). In the present study, we found that expression of miR-206 was significantly decreased in spaceflight gastrocnemius compared with ground-based control, whereas levels of miR-1 and miR-133a were not significantly altered (Fig. 3, A–C). Interestingly, however, the ratio of miR-1 to miR-133a was significantly increased with spaceflight (Fig. 3D). The significance of this is that miR-1 and miR-133a act in opposition to one another with respect to their effect on skeletal muscle, with miR-1 inhibiting proliferation and miR-133a promoting proliferation via their effects on HDAC4 and serum response factor, respectively (43). This increase in the ratio of miR-1 to miR-133a suggests a microRNA expression profile favoring inhibition of cell proliferation, a finding that is consistent with the shift in expression of key regulatory genes such as p21 and Socs2 that appears to favor decreased proliferation, as well. To our knowledge these are the first data to examine the levels of microRNAs in muscle in response to spaceflight. To date, only one study has investigated the in vivo effects of altering muscle activity/load on skeletal muscle microRNA expression (23). Seven days of functional overload (FO) of the plantaris resulted in a ∼50% decrease in miR-1 and miR-133a combined with an ∼18-fold increase in the miR-206 precursor but no change in mature miR-206 expression (23).
At present, the functional consequences of decreased miR-206 expression with microgravity exposure are unknown. miR-206 expression is known to be higher in slow, oxidative muscle than in fast, glycolytic muscle (23), and thus the decrease in spaceflight muscle might reflect the slow-to-fast shift that characterizes this state. Furthermore, miR-206 expression is low in proliferating myoblasts and increased during myoblast differentiation (6), and overexpression of miR-206 promotes cell cycle withdrawal and differentiation (6), and thus the decrease in miR-206 observed in the present study may also represent a shift in differentiation capabilities of the satellite cells and/or muscle fibers. FSTL1 is a target gene negatively regulated by miR-206 (34); however, in the present study FSTL1 mRNA levels were decreased in response to spaceflight, which is at odds with the results observed for miR-206 expression. Thus at present it is unclear what role, if any, miR-206 plays in regulating muscle gene expression during spaceflight.
Comparison of spaceflight and ground-based unloading models.
A majority of the 272 mRNAs affected by spaceflight (171 mRNAs) in the present study showed a similar trend toward increased or decreased expression with hindlimb suspension (Fig. 1B). Furthermore, 88 of these mRNAs were significantly affected by one or more of the ground-based stimuli. Generally, there was an overriding reciprocal relationship between the effect of suspension versus the effect of reloading: only 14 of the 88 mRNAs affected by one or more of the ground-based stimuli did not follow this pattern. Although the sets of mRNAs affected significantly by the ground-based stimuli are only partially overlapping, our results suggest that significance for any of the stimuli appears to act as a strong indicator of sensitivity to unloading/reloading factors. A previous study of rat muscle (28) reported that spaceflight resulted in at least an 8-fold change to 257 total genes, whereas hindlimb suspension resulted in at least an 8-fold change to just 74 genes (see Table 3 of Ref. 28), percentages fairly similar to those observed in the present study, suggesting that spaceflight appears to affect more genes than ground-based unloading in both mice and rats.
Thus the present data suggest that spaceflight elicits qualitatively similar but quantitatively different effects on muscle mRNA levels compared with hindlimb suspension. It is also possible that some or all of the mRNAs that were significantly affected by spaceflight but not by hindlimb suspension were affected by a component of spaceflight other than the unloading due to microgravity, such as radiation or hypergravity upon reentry, among others, which may have potentiated the effects of microgravity-induced unloading on spaceflight muscle. In addition, as mentioned in materials and methods, slightly different tissue preparations were used; for the spaceflight studies, the medial portion of the gastrocnemius was used, whereas in the suspension studies, the entire calf was used. It is therefore possible that at least some of the differences in mRNA expression between spaceflight and suspension are a consequence of differences in how one portion of the gastrocnemius responds compared with the combined muscles of the calf. However, although the three calf muscles differ appreciably in their fiber type composition in normal animals, we previously demonstrated (14) that all three calf muscles undergo qualitatively similar shifts in fiber size in response to spaceflight. Thus it is unlikely that changes in mRNA expression were masked by differential shifts in signaling pathways regulating fiber size between the different calf muscles. Nevertheless, differences in responses between spaceflight and suspension may reflect a dilution of the mRNAs affected in the medial gastrocnemius used in the spaceflight studies by the additional calf musculature used in the suspension studies.
Spaceflight and reloading.
An important consideration for analysis of any spaceflight data is the reloading period that occurs after landing and before any potential examination. In the present study, we found that 42 of the 272 mRNAs affected in spaceflight samples were significantly affected by the reloading stimulus, and most of these showed an antagonistic response to reloading relative to suspension. This is consistent with a previous study on acute (12 h) suspension and 4-h reloading on rat soleus muscle (4), which also showed that reloading tended to counteract the effects of suspension. In the present study, notable genes whose mRNA was significantly affected by reloading relative to spaceflight and/or suspension were IRS-1, Acvr1b/ALK4, and PGC1-α.
As described above, the general effect of reloading was to counteract the effect of suspension. Among 13 genes independently affected by suspension and by reloading, only 1 exhibited a significant difference for combined suspension plus reloading relative to the control state, and only 3 showed a net change of >1.06-fold compared with control. Therefore, reloading tended to diminish and thus mask the effect of suspension. Despite this, mRNA expression changes in spaceflight samples were qualitatively most similar to the effects of suspension, suggesting that the predominance of gene expression changes occurring in spaceflight samples are likely due to the 12 days of unloading and not the 3.5 h of reloading. Nevertheless, our finding that 3.5 h of reloading were sufficient to cause some transcriptional changes, combined with the finding that reloading may mask some portion of the unloading response, indicates that care must be taken in the interpretation of mRNA expression data taken from reloaded spaceflight samples.
In summary, spaceflight induces changes in expression of mRNAs involved in a number of cellular processes and in particular appears to alter the mRNA expression of many genes involved with regulating muscle growth and fiber type. Surprisingly, few of these were significantly altered in both spaceflight and hindlimb unloading, which suggests key differences in how muscles respond on the transcriptional level to these stimuli. Moreover, reloading is associated with changes in several mRNAs, some of which respond differentially compared with spaceflight and/or hindlimb suspension. At present it is not clear to what extent these mRNA changes are translated into differences in protein levels for these genes. Future studies are needed to follow up on exploring the effects of spaceflight and suspension/reloading on protein levels of a selected group of candidate genes from the present study.
This work was funded by NASA Grant NCC8-242 (to L. S. Stodieck; BioServe Space Technologies), Amgen, Inc., South Carolina Space Grant Consortium NASA Grant (SCSGCNG) NCC5-575 (to T. A. Bateman), National Space Biomedical Research Institute NASA Grant NCC 9-58 (to T. A. Bateman), a NASA Graduate Student Research Program fellowship from Kennedy Space Center (to E. R. Bandstra), National Center for Research Resources Grants P20 RR016434 (to W. S. Argraves), SCSGCNG NCC5-575 (to W. S. Argraves), and South Carolina INBRE Bioinformatics Core P20 RR16461 (to W. S. Argraves).
We acknowledge Mark Rupert (BioServe/U of Colorado) for excellent payload management, support from Dr. Beverly Girten [National Aeronautics and Space Administration (NASA) Ames Research Center], and Ramona Bober (Kennedy Space Center) and resource support from the Medical University of South Carolina Proteogenomics Facility, including exceptional technical assistance from Victor Fresco.
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- Copyright © 2009 the American Physiological Society