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TGF-β and myostatin are the two most important regulators of muscle growth. Both growth factors have been shown to signal through a Smad3-dependent pathway. However to date, the role of Smad3 in muscle growth and differentiation is not investigated. Here, we demonstrate that Smad3-null mice have decreased muscle mass and pronounced skeletal muscle atrophy. Consistent with this, we also find increased protein ubiquitination and elevated levels of the ubiquitin E3 ligase MuRF1 in muscle tissue isolated from Smad3-null mice. Loss of Smad3 also led to defective satellite cell (SC) functionality. Smad3-null SCs showed reduced propensity for self-renewal, which may lead to a progressive loss of SC number. Indeed, decreased SC number was observed in skeletal muscle from Smad3-null mice showing signs of severe muscle wasting. Further in vitro analysis of primary myoblast cultures identified that Smad3-null myoblasts exhibit impaired proliferation, differentiation and fusion, resulting in the formation of atrophied myotubes. A search for the molecular mechanism revealed that loss of Smad3 results in increased myostatin expression in Smad3-null muscle and myoblasts. Given that myostatin is a negative regulator, we hypothesize that increased myostatin levels are responsible for the atrophic phenotype in Smad3-null mice. Consistent with this theory, inactivation of myostatin in Smad3-null mice rescues the muscle atrophy phenotype.
The ability of skeletal muscle to regenerate is due to availability of adult muscle stem cells, termed satellite cells (SCs) 1. SCs are quiescent cells that are located between the basal lamina and sarcolemma. Upon injury to skeletal muscle, SCs are activated to enter the cell cycle and as a result rapidly proliferate to give rise to myoblasts, which then differentiate and fuse to give rise to multinucleated nascent muscle fibers 2. At the same time, some of the myoblasts re-enter the quiescent phase to self-renew the SC population. Among several growth factors, myostatin (GDF8) has been shown to be a profound regulator of the skeletal muscle during prenatal and postnatal growth. Disruption of the myostatin gene or inhibition of myostatin expression in mice leads to increased skeletal muscle growth, which is due to both muscle hyperplasia (increase in muscle fiber number) and hypertrophy (increase in muscle fiber size) 3, 4. In contrast, overexpression of myostatin in mice results in reduced skeletal muscle mass, and decreased muscle fiber size and myonuclear number 5. Moreover, systemic overexpression of myostatin in adult mice leads to the development of cachectic-like muscle wasting 6. Myostatin functions by inhibiting myoblast proliferation and differentiation 7, as well as negatively regulating SC activation and SC self-renewal 8, 9. In addition to myostatin, TGF-β has also been shown to inhibit myogenesis 10, 11. Mechanistically, the Smad2/3 pathway plays a significant role in myostatin and TGF-β signaling and inhibition of myogenesis 10, 11. Specifically, Smad3 is required for the inhibition of myoblast differentiation by myostatin 12 and for the activation of fibroblast proliferation by myostatin 13. The Smad3-dependent pathway begins when activated myostatin receptors (activin type IIB receptor/ALK5) form heterotetrameric complexes, which then recruit, phosphorylate and activate the receptor-regulated SMADS (R-Smads). The newly activated Smad3 complexes with Smad4 and then translocates to the nucleus to activate/repress the expression of genes regulating muscle growth and differentiation. Although Smad3 is a key signaling molecule/transcription factor, its role in SC biology is not studied in greater detail. Here, we make use of Smad3-null mice and show that Smad3 is essential for normal skeletal muscle growth, as the absence of Smad3 promotes dramatic skeletal muscle atrophy accompanied with defective SC functionality, impaired in vitro myogenesis and enhanced skeletal muscle protein degradation. Interestingly, loss of Smad3 results in the up-regulation of myostatin expression. Furthermore, the elevated level of myostatin plays a key role in the development of the skeletal muscle atrophy observed in Smad3-null mice.
Smad3-null mice had an overall reduction in body size; however, there was no significant decrease in the muscle weights when calculated as a percentage of body weight (Supplementary information, Figure S1A-S1C and Table S1). To evaluate muscle fiber histology, M. tibialis anterior (TA) muscles were sectioned and stained with hematoxylin and eosin (H & E; Supplementary information, Figure S1D). As shown in Supplementary information, Figure S1E, no significant difference in muscle fiber number was noted between wild-type (WT) and Smad3-null mice. However, Smad3-null mice had a greatly reduced muscle fiber cross-sectional area (CSA) when compared to WT mice (Figure 1A). Specifically, in WT mice, ~63% of muscle fibers had an area of ≥ 2 000 μm2; however, in the Smad3-null mice, only ~8% were found to be of similar diameter.
One of the reasons for postnatal muscular atrophy is the impaired myogenic nature of SCs. In order to analyze myogenic potential, proliferation rate of myoblasts derived from Smad3-null SCs and WT SCs were compared in vitro. Data showed that Smad3-null primary myoblasts had a significantly reduced proliferation rate when compared to WT primary myoblasts (Figure 1B). In addition, we generated a Smad3-knockdown C2C12 cell line (shSmad3; Supplementary information, Figure S2), and analysis of proliferation demonstrated that short hairpin RNA (shRNA)-mediated knockdown of Smad3 also results in a reduction in the proliferation rate (Figure 1C). Consistent with the reduced proliferation, fluorescence-activated cell sorting (FACS) revealed that shRNA-mediated knockdown of Smad3 results in a reduction in the percentage of cells in S phase of the cell cycle, concomitant with increased percentages of cells in both G1 and G2/M phases of the cell cycle (Table 1). Cyclin-dependent kinase 2 (Cdk2), cyclin-E and retinoblastoma (Rb) protein have all been shown to be critical in regulating the G1/S transition in myoblasts 19. Therefore, we next analyzed the levels of these proteins via western blot analysis. As shown in Figure 1D, down-regulation or inactivation of Smad3 leads to reduced expression of both Cdk2 and cyclin-E (Figure 1D). Rb is the major substrate of G1 Cdks; moreover, Cdk-mediated phosphorylation of Rb relieves its repression on E2F/DP1, resulting in continued cell cycle progression 7. Subsequent western blot analysis revealed a reduction in the levels of hyperphosphorylated Rb (pRbPP) in both Smad3-null primary myoblasts and shSmad3 C2C12 cells, concomitant with an increase in hypophosphorylated Rb (pRb; Figure 1D).
In addition to the proliferation rate, we also measured the differentiation ability of Smad3-null myoblasts. The results demonstrate that Smad3-null myoblasts differentiated to form smaller myotubes with reduced branching, as compared to WT myoblasts (Supplementary information, Figure S3). Consistent with the histology, gene expression results showed that the critical myogenic genes, Myf5, p21, MyoD, myogenin and MyHC, are down-regulated in Smad3-null myotubes, especially in early (0 h) and late phases of differentiation (72 h and 96 h) when compared to WT cultures (Figure 1E and Supplementary information, Figure S4).
This defective differentiation can also result from inefficient fusion of myoblasts and myotubes. Therefore, we determined fusion index of Smad3-null myotubes. Our analysis showed that the Smad3-null myotubes had decreased fusion index and reduced average myonuclei number (Figure 1F and and1G).1G). This suggests that Smad3-null myoblasts and myotubes have fusion defects during differentiation. We also analyzed the expression levels of some of the genes that have been shown to be involved in fusion during myogenesis. Caveolin-3 is a scaffolding protein primarily associated with caveolae membrane domains in skeletal muscle tissue 20. β1D integrin is a muscle-specific membrane receptor, which appears to play a critical role in myoblast fusion 21, 22. SHP-2 (SH2 domain-containing protein tyrosine phosphatase) positively regulates myogenesis through activation of RhoA 23, and the resulting phosphorylation of focal adhesion kinase (FAK) is essential for myoblast fusion 24. Results from real-time PCR analysis showed that these fusion-related genes were down-regulated in Smad3-null myotubes at late time points (48 h and 72 h) (Figure 1H-1J), further supporting the fusion defects observed in the Smad3-null myoblasts.
As described earlier, Smad3-null mice show signs of muscular atrophy, which we hypothesized, that may be attributed to impaired SC function, namely SC self-renewal. We used two independent experimental systems, namely, tissue-dissociated SC and single muscle fiber culture to quantify SC self-renewal. The self-renewal ability of Smad3-null myoblasts was quantified through in vitro immunostaining analysis using antibodies directed against MyoD and Pax7 (Figure 2A). Cells that are positive for MyoD only (MyoD+/Pax7−) were considered fusion competent and cells positive for Pax7 only (MyoD−/Pax7+) were quantified as a measure of the fusion incompetent, self-renewing lineage (quiescent SCs). The results indicate that there was a significant decrease in the percentage of MyoD−/Pax7+ myoblasts (from 19 % ± 0.95 % to 9 % ± 0.63 %), with a concomitant increase in the percentage of the MyoD+/Pax7− myoblast population (from 27 ± 0.84 % to 40 ± 2.87 %) in Smad3-null myoblasts when compared to WT (Figure 2B) in the tissue-dissociated SC experimental system. Similarly in single muscle fiber cultures, we observed a significant reduction in the self-renewed population of SCs (Figure 2C and and2D).2D). We next quantified SC numbers through immunostaining with Pax7 antibodies (Supplementary information, Figure S5). Our results show that Smad3-null mice had significantly reduced SC number when compared to WT controls (Figure 2E and and2F).2F). Therefore, Smad3 appears to play a role in the regulation of SC function. Specifically, loss of Smad3 results in reduced SC self-renewal capacity, which may lead to a progressive decrease in the number of SCs.
In order to analyze the molecular basis for the observed atrophy, we performed real-time PCR analysis to measure the expression of candidate genes that regulate muscular atrophy, such as myostatin and IGF-1. The expression of myostatin was up-regulated in Smad3-null M. quadriceps (Quad) muscle (Figure 3A). In contrast, the expression of IGF-1 was significantly down-regulated in Quad muscles from Smad3-null mice when compared to WT controls (Figure 3B). In addition, we analyzed the expression of both myostatin and IGF-1 in primary myoblast cultures isolated from both Smad3-null and WT mice. Consistent with the results outlined above, we find increased expression of myostatin concomitant with reduced expression of IGF-1 in Smad3-null primary myotube cultures when compared to WT mice-derived cultures (Figure 3C and and3D3D).
Since Smad3-null mice have increased levels of myostatin and decreased levels of IGF-1, we next investigated if there was an increase in ubiquitination of intracellular proteins in Smad3-null muscles. Indeed proteins of Quad muscle in the molecular weight range of 22-60 kDa appear to be highly ubiquitinated as compared to WT muscles (Figure 4A), which is consistent with the increased muscle atrophy observed. However, despite the increase in myostatin, there was no change in the expression and phosphorylation status of FoxO1 (Figure 4A). In addition, both mRNA expression and protein level of MuRF1 were up-regulated, while no change in the expression of Atrogin1 was observed, in the muscle tissue from Smad3-null mice when compared to controls (Figure 4A and and4B).4B). MuRF1 has been previously demonstrated to interact with and promote the degradation of MyHC protein in myotubes treated with dexamethasone (DEX) 25. Consistent with this, we find decreased expression of MyHC at the protein level in Smad3-null mice, with no appreciable change in mRNA expression of MyHC detected between WT and Smad3-null muscle tissue (Figure 4A and and4B).4B). Muscle mass is maintained through a balance of protein synthesis and protein degradation. Therefore, we next analyzed the expression of Akt, a key signaling intermediate involved in protein synthesis. Western blot analysis demonstrated that the expression and the level of active phosphorylated Akt remained unaltered between Smad3-null and WT muscle tissue (Figure 4A).
Smad3-null skeletal muscle demonstrated elevated protein ubiquitination and MuRF1 expression level, which suggested the involvement of the ubiquitin-proteasome system in Smad3-null muscle atrophy. A proteasome inhibitor, epoxomicin (epox), was used to determine whether inhibition of the ubiquitin-proteasome system will ameliorate the loss of MyHC protein in Smad3-null myotubes. Smad3-null primary myotubes showed significantly reduced MyHC expression as compared to WT primary myotubes. However, 10 h treatment with epox partially improved the MyHC expression in Smad3-null myotubes (Figure 4C and and4D).4D). This partial rescue of MyHC expression in Smad3-null myotubes with the treatment of epox suggested that the proteasome system might be involved in myotube atrophy in Smad3-null mice.
In order to genetically prove that increased myostatin is responsible for the observed muscular atrophy in Smad3-null mice, we crossed the Smad3-null mice into a myostatin-null genetic background to generate a double-knockout mouse model (DKO; Smad3−/−/myostatin−/−). Inactivation of mature myostatin expression and expression of an inactive Smad3 allele are shown in Supplementary information, Figure S7.
DKO mice have reduced body size and decreased musculature when compared to myostatin-null mice (Supplementary information, Figure S6A). Skeletal muscle weights were similar between DKO mice and WT mice; however, when muscle weights were normalized to total body weight, we found increased skeletal muscle mass when compared to that of WT mice (Supplementary information, Table S1 and Figure 5A, 5B). Histological analysis of muscle sections (Supplementary information, Figure S6B) indicated that muscle CSA of DKO was similar to that of WT (Figure 5C). However, quantification of muscle fiber number revealed an increase in fiber number in muscle sections from DKO mice when compared to WT controls, which suggests that the increased muscle mass observed in the DKO mice may be due to muscle fiber hyperplasia (Figure 5D), consistent with the phenotype observed in myostatin-null mice. Despite increased muscle fiber number, DKO mice had decreased average muscle fiber area when compared to WT controls, which is similar to that observed in the Smad3-null mice (Figure 5E). Therefore, inactivation of myostatin in Smad3-null mice rescues, at least in part, the atrophic phenotype observed in the Smad3-null mice, resulting in an increase in muscle mass back to levels comparable to WT mice. Furthermore, it appears that the improvement in muscle mass results from an increase in muscle fiber number (hyperplasia), rather than an increase in individual muscle fiber area (hypertrophy).
We next isolated primary myoblasts from DKO mice and analyzed their myogenic potential in vitro. DKO myoblasts had a significantly increased proliferation rate when compared to WT myoblasts (Figure 5F). Analysis of myogenic differentiation also revealed an enhanced fusion index and an increase in average myonuclei number in the primary myotubes from DKO mice when compared to Smad3-null controls (Figure 5G, 5H and Supplementary information, Figure S6C). These data therefore suggest that myoblasts isolated from DKO mice demonstrate improved myogenesis when compared to myoblasts isolated from Smad3-null mice, which is consistent with the enhanced proliferation and differentiation observed in myostatin-null mice. Therefore, loss of myostatin appears to partially rescue the defective myoblast proliferation and differentiation phenotype observed in Smad3-null primary myoblast cultures.
Loss of myostatin partially rescues muscle atrophy in Smad3-null mice. In order to further confirm the postnatal role of myostatin in Smad3-null muscle atrophy, we neutralized endogenous myostatin by treating WT and Smad3-null primary culture with the soluble extracellular domain of activin type IIB receptor (ActRIIB), also known as soluble ActRIIB. After 48 h of treatment, MyHC and MyLC, important myogenic markers during differentiation, were measured by western blot. The results showed that neutralizing endogenous myostatin in primary culture significantly rescued MyHC protein levels in Smad3-null primary myotubes at 48 h post differentiation (Figure 5I and and5J).5J). The expression level of another marker, MyLC, was also improved considerably (Figure 5I and and5J).5J). This result further confirms the role of myostatin in muscle atrophy of Smad3-null mice.
Among the TGF-β superfamily members, TGF-β and myostatin rely on canonical Smad signaling, specifically Smad2/3, to elicit biological functions during myogenesis 11, 12, 26. Since myostatin is a negative regulator of myogenesis, one might expect that loss of Smad3 would enhance myogenesis due to interference with the myostatin-signaling pathway. However contrary to this, we show that lack of Smad3 results in dramatic muscular atrophy. Impaired SC function combined with increased proteasomal protein degradation appears to be the molecular mechanism behind the observed atrophy in Smad3-null muscles.
SCs are adult muscle precursor cells, and impaired function and/or number of SCs lead to reduced myogenesis and muscular atrophy 27. In Smad3-null mice, there is a significant reduction in the SC number (Figure 2E and and2F),2F), and data presented here suggest that this could be due to reduced self-renewal of SCs (Figure 2B and and2D).2D). SC self-renewal refers to a process whereby a pool of myoblasts (derived from activated SCs), which express high levels of Pax7, withdraws from proliferation to replenish the SC population 28, 29. Therefore, proliferation rate plays an important role in determining the pool of self-renewed SCs 8 during postnatal myogenesis. Cell cycle progression and proliferation of myoblasts are in turn regulated at G1/S and G2/M checkpoints, and perturbations at the checkpoints would lead to altered proliferation of myoblasts, which would in turn affect SC self-renewal. Our data here clearly demonstrate that in Smad3-inactivated myoblasts, there is increased cell cycle arrest in G1 and G2/M phases of the cell cycle (Table 1). Furthermore, there is significant reduction in cyclin-E and Cdk2 expression, which in turn leads to hypophosphorylation of Rb (Figure 1D) and cell cycle arrest, resulting in the reduced proliferation rate observed in Smad3-null myoblasts (Figure 1B and and1C).1C). This, we suspect, would lead to a diminished pool of myoblasts and reduced self-renewal of SCs.
Differentiated Smad3-null myotubes showed an atrophic phenotype (Supplementary information, Figure S3), with both reduced fusion index and reduced average myonuclei number (Figure 1F and and1G).1G). The fusion defects were further confirmed by reduced expression of three major fusion-related genes, caveolin3, β1D integrin and SHP-2 (Figure 1H and and1J).1J). Caveolin3 and β1D integrin are membrane-associated proteins, which play important roles in myoblast fusion. SHP-2 is a known upstream modulator of the NFATc2 pathway, which is important for fusion between nascent myotubes and myoblasts 30. Therefore, results presented here suggest that Smad3 is essential for proper temporal expression of differentiation genes.
Both neonatal (data not shown) and 6-week-old postnatal Smad3-null mice show severe muscle fiber atrophy (Figure 1A). Skeletal muscle atrophy could be due to reduced protein synthesis and/or increased protein catabolism due to targeted ubiquitination of muscle-specific proteins. Two muscle-specific E3 ligases, Atrogin1 and MuRF1, are up-regulated in skeletal muscle tissue in response to numerous atrophic signals 31, 32, 33. Western blot analysis confirmed that there is increased ubiquitination of intracellular proteins in skeletal muscle tissue from Smad3-null mice as compared to control mice (Figure 4A). Furthermore, real-time PCR and western blot analysis confirmed that MuRF1 was up-regulated in Smad3-null muscles (Figure 4A and and4B).4B). Previous studies showed that expression of MuRF1 could be regulated by either the FoxO1 or FoxO3 transcription factor during skeletal muscle atrophy 34. In this study, we did not detect any changes in the expression of FoxO1 at both total and phosphorylated protein level (Figure 4A). Therefore, we speculate that up-regulation of MuRF1 in Smad3-null mice might be due to an increase in FoxO3 levels or activity. Alternatively, this could be due to an increase in the activity of NF-κB transcription factors, which can be activated by inflammatory cytokines 14, 35. Consistent with increased MuRF1 levels, MyHC, a known target of MuRF1 25, was down-regulated (Figure 4A and and4B).4B). Thus, these results clearly suggest that the muscular atrophy seen in Smad3-null mice is much akin to cachectic-like muscle wasting. Although there was no significant change in terms of Akt phosphorylation, the impact of reduced protein synthesis cannot be ruled out at this stage, as increased levels of myostatin can also inhibit protein synthesis by down-regulating elongation factors (Lokireddy et al., unpublished data and see discussion below).
Our search for mechanism revealed increased expression of myostatin in Smad3-null mice (Figure 3A and and3C).3C). Myostatin is a secreted growth factor, which has been previously shown to be a potent negative regulator of myogenesis. Loss of myostatin results in increased muscle growth due to increased SC number and activation 8. Furthermore, myoblasts derived from myostatin-null SCs show increased proliferation and enhanced differentiation 12, 36. In contrast, increased levels of myostatin have been shown to inhibit myoblast proliferation, arresting cells at the G1 to S transition, further resulting in reduced differentiation 7. Furthermore, increased myostatin levels have also been shown to inhibit SC activation and self-renewal 8, 9, and also result in the development of severe cachectic-like muscle wasting, through activating components of the ubiquitin-proteasome pathway 37. The data presented in this manuscript, as well as previously published reports, strongly support the notion that the increased expression of myostatin observed in Smad3-null mice may be primarily responsible for the impaired SC function and increased protein catabolism.
Consistent with this hypothesis, myoblasts from DKO mice showed significantly increased proliferation when compared to Smad3-null primary myoblasts and moreover had a higher rate of proliferation than that of WT primary myoblasts (Figure 5F). Defects in fusion, during differentiation, were also partially recovered (Figure 5G, 5H and Supplementary information, Figure S6C). Both fusion index and average myonuclei number increased in DKO myotubes as compared to Smad3-null myotubes. Furthermore, loss of myostatin, at least in part, rescued the skeletal muscle atrophy phenotype observed in Smad3-null mice, as demonstrated by the increased musculature observed in DKO mice when compared to Smad3-null mice (Supplementary information, Table S1). Thus, the improved muscle mass and enhanced myogenesis in DKO mice provide evidence to support the role of myostatin in the progression of the atrophic phenotype in Smad3-null mice.
Myostatin is widely known to signal through canonical Smad signaling (mainly Smad3) to inhibit myoblast proliferation, differentiation, SC activation and self-renewal 12, 26, 38. However, other signaling pathways including Smad2 are also shown to be involved in myostatin signaling 9, 13, 39, 40, 41, 42. In this study, higher myostatin level was shown to be, in part, responsible for the muscle atrophy in Smad3-null mice. This further suggests that myostatin could signal independently of Smad3 via either Smad2 or other signaling pathways, such as p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and phosphatidylinositol 3-kinase (PI3K) pathways as shown previously 9, 39, 40, 41, 42. In the present manuscript, we have provided evidence to support the critical role of Smad3 in regulation of postnatal myogenesis and SC function in mice. Absence of Smad3 leads to impaired SC self-renewal, which is important for maintenance of the steady-state SC population during postnatal myogenesis. In addition, primary myoblasts derived from Smad3-null SCs exhibit defective proliferation and differentiation. Therefore, we propose that the dramatic atrophy observed in the absence of Smad3 may, in part, be due to impaired SC function. Smad3-null muscle also undergoes enhanced protein degradation through up-regulation of MuRF1 and ubiquitin-proteasome system. Furthermore, we also find increased myostatin expression and decreased IGF-1 expression in Smad3-null muscle and primary cultures. Since myostatin acts as a potent negative regulator in muscle growth, it is quite possible that elevated levels of myostatin may contribute to the muscle atrophy in Smad3-null mice. In addition, inactivation of myostatin in Smad3-null mice (DKO) partially rescues the loss of muscle mass, as well as improves the myogenic potential of SC-derived myoblasts in vitro. Nevertheless, inactivation of myostatin is unable to fully compensate for the muscle wasting observed in Smad3-null mice, suggesting the involvement of additional factors, as yet to be determined, which also contribute to the progression of the atrophic phenotype in Smad3-null mice.
Heterozygote Smad3-null mice were kindly gifted by Prof Walter Wahli (University of Lausanne, Lausanne, Switzerland), which were bred and genotyped according to the previously published protocol 14. Heterozygote myostatin-null mice were a gift from Prof Se-Jin Lee (The Johns Hopkins University, USA) and were genotyped according to previously published reports 3, 15. Mice were maintained on standard chow diet at a constant temperature (20 °C) under a 12 h/12 h artificial light/dark cycle with unlimited access to water. All experiments were performed according to the approved protocols of the institute animal ethics committee (IACUC), Singapore.
C2C12 myoblasts were cultured in proliferation medium, which consisted of Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS; Invitrogen). For differentiation, C2C12 cells were plated at a density of 25 000 cells/cm2. After an overnight attachment period, C2C12 cells were induced to differentiate in low-serum media (DMEM with 2% Horse Serum (HS; Invitrogen)). Primary myoblasts were cultured from hind-limb muscles using a modified method of Partridge TA 16. Briefly, hind-limb muscles were excised, minced and then digested in 0.2% collagenase type 1A for 90 min. Fibroblasts were removed by preplating the cells on uncoated plates for 3 h. Adhered myoblasts were cultured on 10% Matrigel-coated plates (BD Biosciences) and were maintained in the proliferation medium, which consists of DMEM with 20% FBS, 10% HS and 1% chicken embryo extract (CEE) at 37 °C/5% CO2. Primary myoblast differentiation was induced as described above. For the neutralization of myostatin in primary culture, the isolated primary myoblasts were seeded at the density of 25 000 cells/cm2 in growth media on 10% Matrigel-coated six-well plates. After overnight attachment, primary myoblasts were induced to differentiate with (0 h + 3 μg/ml soluble ActRIIB) or without soluble ActRIIB (0 h + 0 μg/ml soluble ActRIIB) for 48 h. For the treatment of proteasome inhibitor, primary myotubes (48 h) were incubated for 10 h with epoxomicin (48 h + 100 nM epox; Sigma, USA) or without epoxomicin (48 h + 0 nM epox).
Myoblast proliferation was assessed as described previously 7. Briefly, primary myoblasts or C2C12 cells were seeded on 96-well plates at a density of 1 000 cells per well in proliferation medium. Myoblasts were subsequently grown and fixed at regular 12 h intervals. Proliferation was assessed using the methylene blue photometric end-point assay as previously described 7.
Single muscle fibers from M. gastrocnemius muscle were isolated by the method of Rosenblatt et al. 17. Isolated muscle fibers were placed in four-well Lab-Tek chamber slides (Nunc), fixed in 4% paraformaldehyde for 10 min and rinsed in PBS. The fibers were then permeabilized in 0.5% TritonX-100 in PBS and blocked with 10% normal goat serum (NGS) and 0.35% carrageenan-λ (cλ) in PBS for 1 h. Fibers were incubated with mouse anti-Pax7 antibody (1:100; Developmental Studies Hybridoma Bank; DSHB) in 5% NGS and 0.35% cλ in PBS overnight, followed by incubation with the biotinylated sheep anti-mouse IgG (1:300; GE Healthcare Life Sciences) in 5% NGS and 0.35% cλ in PBS for 1 h. After 3× TBS-T washes, fibers were incubated with the streptavidin-conjugated Alexa Fluor 488 (1:400; Molecular Probes) in 5% NGS and 0.35% cλ in PBS for 1 h, counterstained with DAPI (1:1 000; Molecular Probes) and mounted with ProLong Gold antifade reagent (Invitrogen). Immunofluorescence images were captured using the Carl Zeiss LSM 510 META laser-scanning confocal microscope and LSM 510 META software, with a Zeiss Plan-NeoFluar 10×/0.30× objective (Carl Zeiss MicroImaging, Inc.).
Primary myoblasts were fixed with 70% ethanol: formaldehyde:acetic acid (20:2:1) for 30 s and rinsed three times with PBS. Cells were permeabilized in 0.1% TritonX-100 in PBS and washed with PBS. Cells were blocked with 5% NGS, 5% normal sheep serum (NSS) and 0.35% carrageenan-λ (cλ) in PBS and incubated with mouse anti-Pax7 antibody (1:100; DSHB) and rabbit anti-MyoD antibody (1:100; Santa Cruz Biotechnology) in 5% NGS and 0.35% cλ in PBS overnight at 4 °C. After washing with TBS-T for three times, myoblasts were incubated for 1 h with biotinylated sheep anti-mouse IgG (1:300; GE Healthcare Life Sciences) in PBS containing 5% NGS and 0.35% cλ and then for 1h in streptavidin-conjugated Alexa Fluor 488 (1:400) and Alexa Fluor 546 conjugated goat anti-rabbit IgG (IgG; 1:300; Molecular Probes) in 5% NGS, 5% NSS and 0.35% cλ in PBS. After washing in TBS-T twice, myoblasts were stained with DAPI (1:1 000) and mounted with ProLong Gold antifade reagent. Immunofluorescence images were captured using the Carl Zeiss LSM 510 META laser-scanning confocal microscope and LSM 510 META software, with a Zeiss Plan-NeoFluar 10× and 20× objectives (Carl Zeiss MicroImaging, Inc.). For single muscle fibers, isolated muscle fibers were cultured in four-well Lab-Tek Permanox chamber slides (Nunc) coated with 10% Matrigel (BD Biosciences). After 144 h of culturing, primary myoblasts, which migrated from single muscle fibers, were fixed and immunostained with Pax7 and MyoD antibodies as described above.
H & E staining on cell cultures was performed according to McFarlane et al. 9. Primary cultures were grown on Thermanox coverslips with 10% Matrigel coating. Myotubes were fixed at 24-h intervals and stained with Gill's hematoxylin, followed by 1% eosin (Merck). For H & E staining on skeletal muscle sections, M. tibialis anterior (TA) muscles were covered with OCT compound and then frozen in isopentane cooled with liquid nitrogen. 10 μm of transverse sections were cut and mounted on slides for H & E staining. Images were captured using the Leica CTR 6500 microscope, equipped with the Leica DFC 310 FX camera and Image Pro Plus software (Media Cybernetics, USA).
Protein extracts from myoblasts were made by lysing myoblasts in protein lysis buffer (50 mM Tris, pH 7.6, 250 mM NaCl, 5 mM EDTA, 0.1% P-40, complete protease inhibitor cocktail from Roche Molecular Biochemicals). Skeletal muscle protein lysates were prepared by homogenizing muscles in RIPA buffer with EDTA, PMSF, aprotinin, NaF and NaVO4. Equal amount of proteins was resolved by SDS-PAGE (4-12% gradient; Invitrogen) and transferred to a nitrocellulose membrane. The membranes were blocked in 5% milk in TBS-T, and then incubated with primary and secondary antibodies in 5% milk in TBS-T. Immunoreactivity was detected using Western Lightning™ Chemiluminescence Reagent Plus (PerkinElmer). Band density was analyzed using the Quantity One imaging software (Bio-Rad). For Rb phosphorylation analysis, proteins were separated by 6% SDS-PAGE. Details of the antibody sources and dilutions used in this manuscript are provided in Supplementary information, Table S2.
Total RNA was isolated using TRIzol reagent (Invitrogen), and RNA integrity was monitored by electrophoresis. cDNA was synthesized according to iScript cDNA Synthesis kit (Bio-Rad). Quantitative real-time PCR reactions were carried out in triplicate using SsoFast EvaGreen supermix (Bio-Rad) and the CFX96 Real-Time System (Bio-Rad). Gene expression levels were normalized against that of GAPDH. Fold change of gene expression was calculated using the delta-delta Ct method. The sequences of primers used in this manuscript are provided in Supplementary information, Table S3.
Two different sets of shRNA sequences were designed to specifically target the mouse Smad3 gene using the online software, RNAi finder and RNAi converter, available at the Applied Biosystems website (http://www.ambion.com/techlib/misc/siRNA_finder.html and http://www.ambion.com/techlib/misc/psilencer_converter.html): 5′-GATCCACTTTCTACTGCCACTTGGTTCAAGAGACCAAGTGGCAGTAGAAAGTTTA-3′ (sense) and 5′-AGCTTAAACTTTCTACTGCCACTTGGTCTCTTGAACCAAGTGGCAGTAGAAAGTG-3′ (anti-sense) for shSmad3-1 and 5′-GATCCGTTCTCCAGAGTTAAAAGCTTCAAGAGAGCTTTTAACTCTGGAGAACTTA-3′ (sense) and 5′-AGCTTAAGTTCTCCAGAGTTAAAAGCTCTCTTGAAGCTTTTAACTCTGGAGAACG-3′ (anti-sense) for shSmad3-2. The oligos were annealed and ligated into the pSilencer vector according to protocol recommended by the manufacturer (Applied Biosystems, USA). Sequence-verified constructs were selected and transfected into C2C12 myoblasts using Lipofectamine 2000 (Invitrogen). The stable cell lines were selected by continued puromycin treatment for a period of 20 days. A negative control pSilencer vector that expresses a hairpin siRNA with limited homology to any known sequences in the human, mouse and rat genomes databases was used. Negative control cells were maintained simultaneously, under the same conditions as described above.
Control and shSmad3 C2C12 myoblasts were cultured in proliferation medium and harvested. In all, 1-3 million cells were counted and centrifuged at 1 000× g for 5 min. After washing with cold PBS, cells were transferred to 10-ml glass tubes and washed again. Supernatant was carefully aspirated and 1 ml of ice-cold 70% ethanol was slowly added while vortexing at low speed. Fixed cells were stored at −20 °C until staining. Cells were centrifuged at 1 000× g for 5 min and 70% ethanol was carefully aspirated. While cells were vortexed at low speed, 1 ml of staining mixture (0.1 mg/ml propidium iodide and 0.05 mg/ml ribonuclease) was added slowly. After 45 min of incubation at 4 °C, cells were washed twice with cold PBS. Then, 1 ml of cold PBS was added and cells were transferred to FACS tubes and examined for propidium iodide fluorescence using a flow cytometer (FACSCalibur, Becton-Dickinson) and analyzed using ModFit software (Becton-Dickinson).
The extracellular domain of mouse ActRIIB cDNA was amplified by PCR and cloned in the pET-16b protein expression system (Novagen) according to the manufacturer's protocol (Novagen). The construct was transformed into BL21 bacterial strain. The soluble ActRIIB protein was expressed and purified according to Sharma et al. 18. The biological activity of the recombinant soluble ActRIIB was measured by myoblast proliferation assay.
Statistical significance was performed using the one-way ANOVA and the two-tailed Student's t-test, and results were considered significant at P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***). Results were expressed as mean ± SEM (standard error of mean).
We would like to thank Prof Se-Jin Lee (The Johns Hopkins University, USA) for the gift of the myostatin-null mice, and Esther Latres (Regeneron Pharmaceuticals, USA) for providing the Atrogin1 and MuRF1 antibodies used in this study. We would like to thank Developmental Studies Hybridoma Bank for some of the antibodies used in this study. We are indebted to Biomedical Research Council (BMRC), Singapore, for financial support.
(Supplementary information is linked to the online version of the paper on the Cell Research website.)
Analysis of Smad3-null muscles.
Relative Smad3 mRNA expression levels in shSmad3 and shControl C2C12 myoblasts, as measured by quantitative real time PCR.
Representative images of differentiating primary myoblasts from wild-type and Smad3-null mice at 0 h, 24 h, 48 h and 72 h of differentiation; cells were stained with Hematoxylin and Eosin (H & E).
Densitometric analysis of myogenic gene (Myf5, p21, MyoD, Myogenin and MyHC) expression in primary myotubes isolated from wild-type and Smad3-null mice, normalized to tubulin expression.
Representative images of Pax7 ICC on single muscle fibers (the arrows indicate the location of SCs).
Improved myogenic potential of double knockout (DKO) myoblasts.
The genotypes of the wild-type, Smad3-null, and DKO mice were confirmed by RT-PCR analysis.
Proportion of muscle weight to body weight (%).
Antibodies used in this manuscript.
Sequence of primers used in this manuscript.