Muscle atrophy contributes to morbidity and mortality in aging and chronic disease, emphasizing the need to gain understanding of the mechanisms involved in muscle atrophy and (re)growth. We hypothesized that the magnitude of muscle regrowth during recovery from atrophy determines whether myonuclear accretion and myogenic differentiation are required and that insulin-like growth factor (IGF)-I/Akt/glycogen synthase kinase (GSK)-3β signaling differs between regrowth responses. To address this hypothesis we subjected mice to hindlimb suspension (HS) to induce atrophy of soleus (−40%) and plantaris (−27%) muscle. Reloading-induced muscle regrowth was complete after 14 days and involved an increase in IGF-IEa mRNA expression that coincided with Akt phosphorylation in both muscles. In contrast, phosphorylation and inactivation of GSK-3β were observed during soleus regrowth only. Furthermore, soleus but not plantaris regrowth involved muscle regeneration based on a transient increase in expression of histone 3.2 and myosin heavy chain-perinatal, which are markers of myoblast proliferation and differentiation, and a strong induction of muscle regulatory factor (MRF) expression. Experiments in cultured muscle cells showed that IGF-I-induced MRF expression is facilitated by inactivation of GSK-3β and selectively occurs in the myoblast population. This study suggests that induction of IGF-I expression and Akt phosphorylation during recovery from muscle atrophy is independent of the magnitude of muscle regrowth. Moreover, our data demonstrate for the first time that the regenerative response characterized by myoblast proliferation, differentiation, and increased MRF expression in recovering muscle is associated with the magnitude of regrowth and may be regulated by inactivation of GSK-3β.
- glycogen synthase kinase-3β
- muscle growth
- muscle atrophy
muscle atrophy commonly occurs in the elderly and in chronic disease, partly because of an adaptive sedentary lifestyle. Muscle atrophy not only compromises physical functioning through adverse effects on muscle strength but has also been associated with increased mortality independent of body mass index. Therefore, understanding the mechanisms involved in muscle atrophy and muscle (re)growth is imperative for the development of approaches to maintain or improve muscle mass in an increasing susceptible population. Skeletal muscle is a highly plastic tissue, which is reflected in its ability to adapt to changes in functional demand. For example, muscle inactivity caused by a leg cast or prolonged bed rest in a disease condition results in muscle atrophy.
Muscle atrophy can be studied by unloading the weight-bearing skeletal muscles of mice with hindlimb suspension (HS). HS induces a rapid decline in weight and cross-sectional area (CSA) of skeletal muscle (atrophy), which results in muscle weakness. HS affects various muscles of the hindlimb musculature, including soleus and plantaris muscle, but the extent of atrophy varies between these muscles (9). Under physiological conditions, reloading of inactivity-atrophied skeletal muscle will restore muscle mass by induction of muscle regrowth, through hypertrophy and regeneration (36). Muscle growth can occur without myonuclear accretion (i.e., the addition of new myonuclei), as is the case during hypertrophy signaling (6). Muscle hypertrophy relies on increased mRNA translation and subsequent protein synthesis, resulting from signaling through the Akt/mammalian target of rapamycin (mTOR) pathway (44). However, after certain atrophic conditions, myonuclear accretion is a prerequisite for complete regrowth of skeletal muscle (11).
The number of myonuclei contained in a muscle fiber appears to be flexible, allowing muscle growth or atrophy beyond the margins defined by the myonuclear domain (38), which is the volume of cytoplasm within the myofiber regulated by the gene products of a single myonucleus (28, 43). Satellite cells have been shown to serve as a source of new myonuclei during muscle regeneration (48, 50) and muscle growth (17). Satellite cells proliferate upon activation, and the subsequent daughter cells, myoblasts (MB), differentiate into new fibers or fuse with existing fibers (5). MB differentiation and fusion are dependent on two members of the family of muscle regulatory factors (MRFs), MyoD and myogenin (41), and the expression of these MRFs is induced in some models of muscle growth (3) and regrowth (38). Insulin-like growth factor (IGF)-I plays a major role in skeletal muscle (re)growth in an autocrine/paracrine fashion (1). Downstream of IGF-I, the Akt pathway has been shown to be important in muscle regrowth following atrophy (44).
Nearly all studies investigating mechanisms of muscle (re)growth to date have only addressed IGF-I/Akt signaling in the context of hypertrophy, e.g., the regulation of mRNA translation. Indeed, substantial evidence suggests that increased protein synthesis during muscle hypertrophy relies on stimulation of mRNA translation via the Akt/mTOR pathway. (40). A downstream target of IGF-I/Akt signaling is glycogen synthase kinase (GSK)-3β, which is phosphorylated and inhibited by activated Akt (16). GSK-3β suppresses protein synthesis by inhibiting eukaryotic transcription factor (eIF)2B-dependent translation (31). Conversely, IGF-I stimulates eIF2B-dependent translation through inactivation of GSK-3β (44). In addition to increasing mRNA translation during muscle growth, IGF-I has been shown to stimulate myogenic differentiation (21, 55) and muscle-specific gene transcription in an Akt-dependent fashion (55). Interestingly, inhibition of GSK-3β was sufficient to reproduce the stimulatory effect of IGF-I on myogenic differentiation (55).
As it remains unclear whether the requirement for myonuclear accretion during muscle regrowth is stimulus- or muscle specific, we hypothesized that the magnitude of the regrowth response determines whether skeletal muscle requires myonuclear accretion and myogenic differentiation. Moreover, considering that Akt/GSK-3β signaling during muscle regrowth has only been studied in relation to hypertrophy, we investigated whether Akt/GSK-3β signaling differs if muscle regrowth also requires myonuclear accretion for complete recovery. To address these hypotheses we compared soleus and plantaris muscle regrowth during recovery from HS-induced muscle atrophy.
MATERIALS AND METHODS
Animals and hindlimb suspension.
All protocols and procedures involving mice were approved by the University of Vermont Institutional Animal Care and Use Committee. Animals were housed in a temperature-controlled room on a 12:12-h light-dark cycle with food pellets and water provided ad libitum.
Forty-eight C57/BL6 mice (4–5 mo) were randomly assigned to one of six groups: 1) baseline, 2) HS for 14 days, and 3–6) HS followed by reloading (RL) for 3, 5, 7, or 14 days. The animals were subjected to HS as described previously (34). This model causes atrophy of the postural muscles, and subsequent RL of the hindlimbs evokes muscle regeneration, resulting in the restoration of muscle mass (38). After the experimental procedures, mice were euthanized by halothane overdose and body weight and length were determined. Soleus and plantaris muscles were collected with standardized dissection methods, weighed, frozen in liquid nitrogen, and stored at −80°C.
The murine skeletal muscle cell line C2C12 was obtained from the American Type Culture Collection (ATCC no. CRL1772, Manassas, VA). These cells are able to undergo differentiation into spontaneously contracting myotubes (MT) after growth factor withdrawal (60). MB were cultured in growth medium (GM), composed of low-glucose Dulbecco's modified Eagle's medium (DMEM) containing antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) and 9% (vol/vol) fetal bovine serum (FBS) (all from GIBCO, Rockville, MD). Cells were plated at 104/cm2 on Matrigel (BD Biosciences, Bedford, MA)-coated (1:50 in DMEM) dishes as described previously (33) and cultured in GM for 24 h. To induce differentiation, GM was replaced with differentiation medium, which contained DMEM with 0.5% heat-inactivated FBS and antibiotics. Murine IGF-I (Calbiochem, La Jolla, CA) was added 24 h after induction of differentiation or 5 days after induction of differentiation for 15 or 30 min or 24 h.
Phosphorylated (Ser473) Akt (p-Akt), Akt, phosphorylated (Ser9) GSK-3β (p-GSK-3β), and GSK-3β protein abundance was evaluated by Western blotting. Protein lysates were prepared by mincing muscle tissue in cold lysis buffer [mM: 50 Tris, 150 NaCl, 1 EDTA, 1 DTT, 1 Na3VO4, 1 PMSF, 1 Na-pyro-PO4, and 1 β-glycerophosphate, with 10% (vol/vol) glycerol, 0.5% (vol/vol) Nonidet P-40, 10 μg/ml leupeptin, and 1% (vol/vol) aprotinin] immediately followed by homogenization (Polytron, Kinematica). Alternatively, adherent cultured muscle cells were washed in PBS, and protein lysates were prepared by the addition of lysis buffer [mM: 20 Tris, 150 NaCl, 1 DTT, 1 Na3VO4, and 1 PMSF, with 1% (vol/vol) Nonidet P-40, 10 μg/ml leupeptin, and 1% (vol/vol) aprotinin].
After homogenization of muscle tissue or lysis of cells, lysates were incubated on ice for 30 min, followed by 30-min centrifugation at 16,000 g. A portion of the supernatant was saved for protein determination, before the addition of 4× Laemmli sample buffer [0.25 M Tris·HCl pH 6.8, 8% (wt/vol) SDS, 40% (vol/vol) glycerol, 0.4 M DTT, and 0.04% (wt/vol) bromophenol blue]. Next, samples were boiled for 5 min and stored at −20°C. Total protein was assessed with the Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. For SDS-PAGE, 10–40 μg of protein was loaded per lane and separated on a 10% polyacrylamide gel (Mini Protean 3 System, Bio-Rad), followed by transfer to a 0.45-μm nitrocellulose membrane (Bio-Rad) by electroblotting. The membrane was blocked for 1 h at room temperature in 5% (wt/vol) nonfat dried milk diluted in Tris-buffered saline (TBS)-Tween 20 (0.05%). Nitrocellulose blots were washed in TBS-Tween 20 (0.05%), followed by overnight incubation (4°C) with a polyclonal antibody specific for p-Akt (no. 9271), Akt (no. 9272), p-GSK-3β (no. 9336), or GSK-3β (no. 9332), all at 1:1,000 dilutions (Cell Signaling, Beverly, MA), or a monoclonal antibody (1:2,500 dilution) specific for GSK-3β (no. 610201, BD Biosciences, Lexington, KY) diluted in TBS-Tween 20 (0.05%). After three wash steps of 20 min each, the blots were probed with a peroxidase-conjugated secondary antibody (nos. PI1000 and PI200, Vector Laboratories, Burlingame, CA) and visualized by chemiluminescence with Supersignal WestPico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL) according to manufacturers' instructions and exposed to film (Biomax light film, Kodak).
GSK-3β immunoprecipitation and kinase activity assay.
Muscle homogenates were prepared as described under Western blotting. An input of 100 μg of total protein was precleared with protein G-Sepharose (Invitrogen, Carlsbad, CA) and subsequently incubated with 1 μg of monoclonal anti-GSK-3β under gentle agitation for 1 h at 4°C. The immune complexes were isolated by the addition of 25 μl of a suspension of protein G-Sepharose in lysis buffer [50% (vol/vol)] and incubation under gentle agitation overnight at 4°C. Immunoprecipitates were washed twice in lysis buffer and divided in two. To assess equal GSK-3β content, one half of the immunoprecipitate was used for Western blotting as described above and the other half was used for GSK-3β activity measurement. For this purpose, the immunoprecipitate was washed twice in kinase reaction buffer [mM: 8 MOPS, pH 7.4, 0.2 EDTA, 10 magnesium acetate, 1 Na3VO4, 1 DTT, and 2.5 β-glycerophosphate, with 10 μg/ml leupeptin and 1% (vol/vol) aprotinin]. The kinase activity assays were performed in 40 μl of total reaction buffer containing 62.5 μM GSK-3β peptide substrate that corresponds to the ε-subunit of eIF2B (BioMol, Plymouth, PA), 20 mM MgCl2, 125 μM ATP, and 10 μCi of [γ-32P]ATP. The reaction mixture was incubated for 30 min at 30°C, and 25 μl of the supernatant was spotted onto Whatman P81 phosphocellulose paper. The filter squares were washed five times for 5 min each in 0.75% phosphoric acid. Next, the filters were briefly rinsed in acetone, dried at room temperature, and subjected to liquid scintillation counting.
RNA isolation and assessment of mRNA abundance for ribonuclease protection assay.
Muscle tissue was homogenized (Polytron, Kinematica) in 0.75 ml of solution D. RNA was extracted by adding 0.1 vol of 2 M sodium acetate pH 5.0, 1 vol of water-saturated phenol, and 0.2 vol of chloroform-isoamyl alcohol (50:1). After 15-min incubation and centrifugation, RNA was precipitated with an equal volume of isopropanol. The pellet was resuspended in 0.5 ml of solution D and precipitated again with isopropanol. After a wash step in 70% ethanol, the RNA was dissolved in nuclease-free water and stored at −80°C.
Ribonuclease protection assay (RPA) was performed to determine MyoD, myogenin, Myf-5, MRF4, and proliferation marker histone 3.2 (H3.2) mRNA levels as previously described (34) with templates developed in our laboratory. Briefly, a radiolabeled multiprobe panel was prepared by a transcription reaction with the MaxiScript Kit (Ambion, Houston, TX) with [α-32P]UTP (800 Ci/mmol) followed by hybridization with 10 or 2 μg of RNA (8 × 104 cpm per probe) per sample at 42°C for 18 h. After incubation, unhybridized RNA was digested (1:100 diluted RNase A/RNase T1 cocktail) with the RPA III kit (Ambion), and protected fragments were resolved on a 5% denaturing polyacrylamide gel. Gels were dried and exposed to film (Biomax MR-1, Kodak) or an imaging screen for quantification in a Personal Molecular Imager FX (Bio-Rad), and band intensity was analyzed with Quantity One software (Bio-Rad).
RNA isolation and assessment of mRNA abundance for quantitative PCR.
Total RNA from muscle tissue or C2C12 cells was isolated with the Totally RNA kit (Ambion) according to the manufacturer's instructions. After isolation RNA was dissolved in Tris-EDTA buffer and stored at −80°C. One microgram of RNA was reverse transcribed to cDNA with the Reverse iT First Strand Synthesis kit (ABgene, Epsom, UK) with oligo(dT) primers.
Myosin heavy chain-perinatal (MyHC-p) and β-actin mRNA were determined by quantitative RT-PCR (Q-PCR). Q-PCR primers were designed with Primer Express 2.0 software (Applied Biosystems, Foster City, CA) and obtained from Sigma Genosys (Haverhill, UK). Skeletal muscle IGF-IEa and MyHC-p were amplified with the following primers: IGF-IEa FP 5′-CAAGACTCAGAAGGAAGTACATTTGAA-3′, RP 5′-GCGGTGATGTGGCATTTTC; MyHC-p FP 5′-gaggagcgggctgacatc-3′, RP 5′-acccagagaggcaagtgacc-3′. PCR reactions (25-μl total volume) contained 1× qPCR MasterMix Plus for SYBR Green I (Eurogentec, Seraing, Belgium) and primers (300 nM). Standard curves were made in duplicate by performing serial dilutions of pooled cDNA aliquots. Real-time PCR reactions were performed in an ABI PRISM 7700 Sequence Detector (Applied Biosystems). Cycle threshold values were obtained for each sample, and the relative DNA concentrations were derived from the standard curve. The expression of the genes of interest was normalized to β-actin (primers obtained from Ambion).
Raw data were entered into SPSS (version 11.0) for statistical analysis. Values as expressed in Figs. 1–5 are means ± SE. To test for statistically significant differences (P < 0.05), data were subjected to Mann-Whitney test, one-way ANOVA analysis, or a paired Student's t-test as applicable.
Changes in muscle mass after HS and RL.
Soleus and plantaris muscle weights after HS and during the course of RL-induced regrowth are shown in Fig. 1. Muscle weight of soleus was more reduced than that of plantaris after HS (60 ± 3.7% vs. 73 ± 3.9% of baseline; P = 0.01) (Fig. 1). After 14 days of RL soleus muscle recovered to 92 ± 2.9% and plantaris muscle to 91 ± 3.1% of baseline muscle mass. Body weight showed a 15% decrease compared with control animals after 14 days of HS and was recovered completely after 7 days of RL (data not shown).
IGF-IEa mRNA expression after HS and RL.
IGF-IEa mRNA expression was assessed after HS and RL in soleus and plantaris muscle. Under basal conditions there was no difference in IGF-IEa expression between the two muscles (data not shown). IGF-IEa mRNA expression was not affected in soleus and plantaris muscle after HS (Fig. 2A). However, after 3 days of RL, IGF-IEa mRNA expression was significantly increased in both soleus (620 ± 40% of baseline) and plantaris (202 ± 13% of baseline) muscle, with the strongest increase observed in the soleus muscle. After 7 days of RL IGF-IEa expression in soleus returned to baseline, whereas at that point in time IGF-IEa expression peaked (247 ± 10% of baseline) in plantaris muscle, to return to baseline after 14 days of RL.
Akt phosphorylation after HS and RL.
p-Akt relative to total Akt was assessed after HS and RL in soleus and plantaris muscle. Under basal conditions there was no difference in p-Akt relative to total Akt (data not shown). Akt phosphorylation was not affected in soleus and plantaris muscle after HS (Fig. 2B). A marked and significant induction of Akt phosphorylation was observed in both muscles after RL. After 3 days of RL a maximal increase in Akt phosphorylation (540 ± 73% of baseline) was observed in soleus muscle, whereas the maximal phosphorylation of Akt in plantaris muscle (382 ± 28% of baseline) was apparent after 5 days of RL. Despite the differing kinetics of induction, p-Akt returned to baseline in both muscles after 7 days of RL.
GSK-3β phosphorylation and activity after HS and RL.
GSK-3β is a downstream target of Akt (16). Therefore we investigated p-GSK-3β relative to total GSK-3β protein abundance and GSK-3β kinase activity in soleus and plantaris after HS and RL. Under basal conditions p-GSK-3β relative to total GSK-3β was ∼50% more abundant in plantaris muscle compared with soleus muscle (data not shown). After HS, the ratio of p-GSK-3β to total GSK-3β was not affected in either soleus or plantaris (Fig. 2C). During RL GSK-3β phosphorylation strongly increased in soleus muscle (250 ± 38% of baseline) (Fig. 2C, top and bottom), whereas GSK-3β phosphorylation was not affected in plantaris muscle compared with control (Fig. 2C, top and middle). Subsequently, GSK-3β kinase activity was assessed in soleus and plantaris muscle. HS did not significantly affect GSK-3β activity in soleus or plantaris (Fig. 2D). Consistent with increased GSK-3β phosphorylation during RL in soleus muscle, GSK-3β activity was significantly decreased after 5 days of RL (67 ± 2% of baseline) and returned to baseline levels at the later time points of RL. In contrast, GSK-3β activity in plantaris muscle was not affected during RL compared with baseline, which was in line with the constant p-GSK-3β-to-total GSK-3β ratio during RL. Despite a lower GSK-3β abundance, baseline values of GSK-3β activity were approximately twofold higher in soleus muscle compared with plantaris muscle (data not shown). To ensure equal input, we subjected immunoprecipitation lysates used for GSK-3β activity assay to Western blot analysis (data not shown).
mRNA abundance of proliferation and differentiation markers after HS and RL.
Soleus and plantaris muscles recover differently from atrophy (38). To evaluate MB proliferation and differentiation, mRNA levels of H3.2 and MyHC-p were assessed during HS and RL (Fig. 3) with RPA and Q-PCR, respectively (MyHC-p was not detectable in plantaris by RPA). MyHC-p is a sensitive differentiation marker (49), whereas the expression of H3.2 mRNA is restricted to the S phase of the cell cycle (58) and absent once MB differentiate (59). H3.2 and MyHC-p were not statistically affected after HS in soleus, whereas after 3 days of RL a strong increase in H3.2 expression (636 ± 32% of baseline) was observed. This increase was sustained up to 7 days of RL. MyHC-p mRNA abundance in soleus muscle increased after RL, reaching maximal expression at day 7 (426 ± 49% of baseline). Although a slight but significant increase in H3.2 and MyHC-p expression was also observed in plantaris muscle at days 3 and 5 of RL, respectively, these changes were three- to fourfold smaller compared with the increases observed in the soleus muscle during RL. These data suggest active participation of regeneration in muscle regrowth in soleus, but not plantaris, muscle.
Myogenic mRNAs after HS and RL.
Recently it was shown that inhibition of GSK-3β in cultured muscle cells results in increased expression of various myogenic mRNAs (55). To evaluate whether changes in GSK-3β phosphorylation and activity are also inversely related to myogenic mRNA expression during regrowth of skeletal muscle, the abundance of the mRNA transcripts encoding the MRFs MyoD, Myf-5, myogenin, and MRF4 were determined after HS and RL in soleus and plantaris muscle (Fig. 4). Table 1 shows the baseline mRNA transcripts for the MRFs. After HS, MyoD, Myf-5, myogenin and MRF4 were not significantly affected in soleus or plantaris muscle. However, after 3 days of RL, MyoD (375 ± 61% of baseline; Fig. 4A), myogenin (1,250 ± 43% of baseline; Fig. 4C), and MRF4 (281 ± 40% of baseline; Fig. 4D) mRNA levels were strongly upregulated in soleus muscle. In contrast, at day 3 of RL in the plantaris muscle MyoD was not induced, and increases in myogenin (455 ± 229% of baseline) and MRF4 (159 ± 50% of baseline) were less pronounced compared with the soleus muscle. In fact, the induction of MyoD and myogenin expression after 3 days of RL was four- and threefold stronger in soleus compared with plantaris muscle. Myf-5 mRNA levels (Fig. 4B) were not significantly affected during HS or RL in either muscle.
GSK-3β phosphorylation and MRF expression in cultured myoblasts and myotubes.
IGF-I is an important mediator in skeletal muscle (re)growth and signaling by Akt, and its downstream mediators are essential for (re)growth of skeletal muscle (25, 26). To evaluate whether the differential regulation of GSK-3β phosphorylation and activity in reloaded soleus and plantaris muscle, despite similar activation of Akt, could be related to the differentiating MB, Akt and downstream signaling was induced in differentiating MB and fully differentiated MT by treatment with IGF-I. Both MB and MT displayed a similar phosphorylation of Akt after IGF-I (data not shown). In contrast, despite similar basal levels of GSK-3β phosphorylation, IGF-I treatment of differentiating MB resulted in a significantly higher GSK-3β phosphorylation status compared with IGF-I-treated MT (Fig. 5A).
Since we and others reported previously that the phosphorylation of GSK-3β in cultured muscle cells in response to IGF-I corresponds with GSK-3β inactivation, we next addressed whether the differential phosphorylation/inactivation of GSK-3β by IGF-I in MB and MT corresponded with a differential response of MRF expression to IGF-I. Treatment of differentiating MB with IGF-I resulted in a robust increase of MyoD and myogenin compared with control (Fig. 5B). In contrast, MyoD and myogenin expression in fully differentiated MT did not change in response to IGF-I. Myf-5 expression did not respond to IGF-I in MB or in MT. MRF4 was not detectable in C2C12 cells. These data suggest that the inactivation of GSK-3β after IGF-I/Akt signaling is associated with MB during regrowth of atrophied skeletal muscle, facilitating the induction of MyoD and myogenin expression and subsequent differentiation of these cells as part of skeletal muscle regeneration.
It is well established that the IGF-I signaling pathway is an important regulatory conduit of skeletal muscle growth (40). In this study molecular events during muscle regrowth, including the activity of the Akt-GSK-3β signaling module of the IGF-I pathway, were compared between two muscles recovering from different extents of atrophy. Despite almost similar patterns of IGF-IEa expression and Akt phosphorylation during regrowth of soleus and plantaris muscle after atrophy, this study revealed differences between these muscles in GSK-3β phosphorylation status and activity that were related to the extent of atrophy and the presence or absence of a regenerative response. Specifically, inactivation of GSK-3β was observed during regrowth of soleus muscle only, which involved muscle regeneration based on markers of MB proliferation and differentiation and a strong induction of MRF expression. Experiments in cultured muscle cells suggested that the induction of MRF expression is facilitated by inactivation of GSK-3β and selectively occurs in the MB population. Inactivity is an important determinant of muscle atrophy in the elderly and in chronic wasting diseases (24). HS is a well-established model for skeletal muscle atrophy (39). In accordance with other studies, a significant decrease in both soleus and plantaris muscle weight was observed after 14 days of HS (22, 37), which was most pronounced in the soleus muscle. Reloading of skeletal muscle after a period of disuse will initiate a muscle regrowth program that may involve elements of muscle hypertrophy and regeneration. In both soleus and plantaris muscle CSA is decreased after disuse-induced atrophy, but interestingly the myonuclear number is reduced only in soleus muscle and in plantaris muscle it is not affected (38). Consequently, full recovery of soleus muscle from HS-induced muscle atrophy requires myonuclear accretion by incorporation of satellite cell-derived MB into existing muscle fibers, in addition to elements of hypertrophy, including increased mRNA translation, that also occur in plantaris muscle regrowth. This notion is supported by studies using γ-irradiation of the hindlimb muscles to inactivate the satellite cells present in soleus and plantaris muscle, which demonstrated that in contrast to plantaris muscle γ-irradiated soleus muscle recovered only half of its lost mass (38). However, in a model of compensatory hypertrophy resulting in a 40% increase in plantaris muscle weight proliferative markers were upregulated (3), indicative of satellite cell activation and proliferation. This suggests that myonuclear accretion during muscle (re)growth can also occur in plantaris muscle and is not fiber type dependent but rather determined by the magnitude of the (re)growth response to meet increased functional demand imposed on a particular muscle. Previous studies have shown that muscle RL induces fiber injury and inflammation (32, 52). However, because these studies were performed in soleus muscle only, the contribution of potential differences in muscle injury during RL to the differential regrowth response observed in soleus and plantaris muscle cannot be excluded. After release from HS followed by RL both muscles recovered almost completely, in accordance with the literature (38). IGF-I is an important growth factor that can function in a autocrine/paracrine fashion to regulate muscle mass (30). In this study muscle IGF-IEa expression was not affected after HS in both soleus and plantaris. This has also been reported not to change in the gastrocnemius muscle in a similar model (27). Intracellularly, the protein kinase Akt is an essential mediator of IGF-I signaling toward growth and maintenance of muscle mass. Although Akt phosphorylation appeared to be attenuated after HS, this effect did not reach statistical significance in either muscle. This may be attributed to the low baseline levels of Akt phosphorylation in both muscles, because previous studies showed that skeletal muscle unloading or inactivity is accompanied by decreased Akt phosphorylation (29, 53). Subsequently, consequent to decreased Akt activity, reduced mRNA translation (2, 10) and increased protein degradation resulting from decreased suppression of atrogin expression (46) likely contributed to the loss of muscle mass. It has been shown that Akt plays a role in preservation of muscle mass not only in mice; a recent study in human subjects revealed a reduction in Akt activity in amyotrophic lateral sclerosis, a degenerating muscle disease (35).
Akt negatively regulates protein kinase GSK-3β (16). In accordance with a previous study, HS did not affect GSK-3β phosphorylation in soleus muscle (12) or in plantaris muscle. In line with unchanged GSK-3β phosphorylation, GSK-3β kinase activity was not affected by HS in soleus and plantaris muscle. Baseline levels of protein abundance of GSK-3β were ∼50% less in soleus muscle compared with plantaris muscle, although activity levels in soleus were significantly higher. Similar observations have been made for the protein phosphatase calcineurin, which demonstrates highest activity in soleus muscle despite far greater calcineurin expression levels in fast-twitch muscles compared with soleus (20, 51). Reloading of atrophied muscle resulted in a rapid and strong increase in IGF-IEa expression and Akt phosphorylation in both muscles. Although some differences in induction and kinetics between the soleus and plantaris muscles were observed, the IGF-IEa expression coincided with the Akt phosphorylation, which both returned to baseline values at later time points of RL in both muscles. To our knowledge, we report for the first time that IGF-IEa expression is enhanced during muscle regrowth after atrophy. Various studies have demonstrated the growth-promoting effect of IGF-I overexpression in muscle (4, 15, 40). In addition, muscle IGF-IEa expression is increased during compensatory overload after synergist ablation in soleus or plantaris muscle (3, 8), suggesting a local role of muscle-produced IGF-IEa in muscle growth. In line with this, Akt phosphorylation is increased in the overloaded muscle in these models, coinciding with muscle growth (10). In our study we measured Akt1 phosphorylation, because this isoform is required for growth (14), whereas Akt2 has mostly been associated with the regulation of glucose metabolism (13, 47), although some reports suggest a role for Akt2 in muscle differentiation (54, 56). Various studies have linked increased Akt phosphorylation to stimulated mRNA translation and protein synthesis in hypertrophic muscle via its downstream targets p70s6k and 4E-BP (10, 12). Phosphorylation and inactivation of GSK-3β by Akt also contributes to muscle hypertrophy (44, 57) by facilitating translation through eIF2B (31). Additionally, our group (55) recently showed that IGF-I/Akt signaling stimulates myogenic differentiation and MT formation, which was recapitulated by the inactivation of GSK-3β, suggesting a central role for GSK-3β in the regulation of muscle growth, as it may be involved in muscle hypertrophy and regeneration.
Consistent with the large increase observed in Akt phosphorylation, GSK-3β phosphorylation was increased after RL in soleus muscle. Surprisingly, despite similar increases in Akt phosphorylation, GSK-3β phosphorylation was not affected in plantaris muscle after RL. In soleus muscle the IGF-IEa induction was significantly stronger compared with plantaris muscle. Possibly, the IGF-IEa levels in the plantaris muscle were adequate to induce Akt activation but not sufficient to establish GSK-3β phosphorylation and inactivation, although this would be somewhat surprising considering that GSK-3β is a direct substrate of Akt. Another explanation for the dissociation of increased IGF-IEa and Akt phosphorylation levels with GSK-3β phosphorylation and activity in plantaris muscle could be that there appear to be two distinct pools of GSK-3β, which determine the availability of GSK-3β for phosphorylation by Akt (23). In contrast to free GSK-3β, GSK-3β residing in the Wnt signaling complex cannot be phosphorylated by Akt (19). Interestingly, in plantaris muscle the interaction of GSK-3β with this complex is associated with overload-induced hypertrophy (7).
In accordance with its phosphorylation status, GSK-3β activity was decreased in soleus and unaffected in plantaris during RL. This demonstrates that in addition to the hypertrophy response observed in both reloaded muscles, characterized and probably regulated by increased IGF-IEa expression and Akt activity, soleus regrowth is accompanied by additional signal transduction that involves GSK-3β. Since regrowth of inactivity-induced atrophied soleus, but not plantaris, muscle requires muscle regeneration based on the finding that myonuclear accretion is obligatory for full recovery of atrophied soleus muscle (38), the differential regulation of GSK-3β may be associated with proliferating and differentiating MB during soleus regrowth. Indeed, H3.2 and MyHC-p mRNA transcripts were dramatically increased in soleus compared with plantaris during muscle regrowth, reflecting MB proliferation and differentiation, respectively (34), and indicative of myonuclear accretion and muscle regeneration that have been described for the soleus muscle under these conditions (38).
The family of MRFs, particularly MyoD and myogenin, govern myogenic differentiation and are essential for muscle regeneration (45). In line with other studies (18, 38), a large increase in mRNA transcripts encoding MyoD, myogenin, and MRF-4 was observed during RL in soleus muscle. In addition, our study demonstrates that these increases were absent or much less pronounced in plantaris muscle that displayed no evidence of muscle regeneration. Increased MRF expression probably stems from differentiating MB, because γ-irradiation, which prevents MB proliferation and differentiation in regenerating soleus muscle, inhibits the induction of MyoD and myogenin expression (38). Experiments in cultured muscle cells further supported the notion that the induction of MRF expression occurred in differentiating MB and not in hypertrophying myofibers, because only cultured MB but not MT responded to IGF-I treatment with increased MyoD and myogenin expression, despite identical induction of Akt phosphorylation. In contrast to Akt, the extent of GSK-3β phosphorylation after IGF-I differed markedly, suggesting that GSK-3β is more receptive to inactivation by Akt in differentiating MB compared with fully differentiated MT, which recapitulates the differences between reloaded soleus and plantaris muscle. Moreover, the inverse relationship between GSK-3β activity and MRF expression observed in regenerating soleus muscle is in line with recent findings by our group (55) showing that GSK-3β is a negative regulator of myogenic differentiation, which may occur through suppression of MRF expression. Although the mechanisms by which GSK-3β contributes to the regulation of muscle growth are not entirely known, recent reports implicate nuclear factor of activated T cells and translation initiation factor eIF2B as relevant substrates of GSK-3β (42).
In conclusion, our study demonstrates that reloading of skeletal muscle after a period of disuse induces IGF-IEa mRNA expression and Akt signaling that is independent of the magnitude of muscle regrowth and likely initiates a muscle regrowth program involving elements of hypertrophy. Depending on the extent of atrophy, muscle regrowth also involves a regenerative response characterized by MB proliferation, differentiation, and increased MRF expression, which may be negatively regulated by GSK-3β. These findings indicate that the development of therapeutic strategies aimed at restoration and preservation of muscle mass in health and disease should be based on the signal transduction of both muscle hypertrophy and muscle regeneration.
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