Resistance exercise alters MRF and IGF-I mRNA content in human skeletal muscle.

Increasing evidence suggests that the myogenic regulatory factors (MRFs) and IGF-I have important roles in the hypertrophy response observed after mechanical loading. We, therefore, hypothesized that a bout of heavy-resistance training would affect the MRF and IGF-I mRNA levels in human skeletal muscle. Six male subjects completed four sets of 6-12 repetitions on a leg press and knee extensor machine separated by 3 min. Myogenin, MRF4, MyoD, IGF-IEabc (isoforms a, b, and c) and IGF-IEbc (isoform b and c) mRNA levels were determined in the vastus lateralis muscle by RT-PCR before exercise, immediately after, and 1, 2, 6, 24, and 48 h postexercise. Myogenin, MyoD, and MRF4 mRNA levels were elevated (P < 0.005) by 100-400% 0-24 h postexercise. IGF-IEabc mRNA content decreased (P < 0.005) by approximately 44% after 1 and 6 h of recovery. The IGF-IEbc mRNA level was unaffected. The present study shows that myogenin, MyoD, and MRF4 mRNA levels are transiently elevated in human skeletal muscle after a single bout of heavy-resistance training, supporting the idea that the MRFs may be involved in regulating hypertrophy and/or fiber-type transitions. The results also suggest that IGF-IEa expression may be downregulated at the mRNA level during the initial part of recovery from resistance exercise.     

IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30 °C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30 °C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30 °C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.     

Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family

The muscle regulatory factors (MRF) gene family regulate muscle fibre development. Several hormones and drugs also affect muscle development. Glucocorticoids are the only drugs reported to have a beneficial effect on muscle degenerative disorders. We investigated the glucocorticoid-related effects on C2C12 myoblast proliferation rate, morphological differentiation, and subsequent mRNA expression patterns of the MRF genes. C2C12 cells were incubated with the glucocorticoids dexamethasone or alpha-methyl-prednisolone. Both glucocorticoids showed comparable effects. Glucocorticoid treatment of C2C12 cells during the proliferative phase reduced the proliferation rate of the cells dose dependently, especially during the third and fourth day of culture, increased MyoD1, myf-5, and MRF4 mRNA levels, and reduced myogenin mRNA level, compared to untreated control cells. Thus, the mRNA level of proliferation-specific MyoD1 and myf-5 expression does not seem to associate with C2C12 myoblast proliferation rate. Glucocorticoid treatment of C2C12 cells during differentiation reduced the differentiation capacity dose dependently, which is accompanied by a dose dependent reduction of myogenin mRNA level, and increased MyoD1, myf-5, and MRF4 mRNA levels compared to untreated control cells. Therefore, we conclude that glucocorticoid treatment reduces differentiation of C2C12 myoblasts probably through reduction of differentiation-specific myogenin mRNA level, while inducing higher mRNA levels of proliferation-associated MRF genes.     

Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.     

Possible Correlation between Selenoprotein W and Myogenic Regulatory Factors in Chicken Embryonic Myoblasts

The biological function of selenium (Se) is mainly elicited through Se-containing proteins. Selenoprotein W (SelW), one member of the selenoprotein family, is essential for the normal function of the skeletal muscle system. To investigate the possible relationship of Se in the process of differentiation in chicken myoblasts and the expression of SelW, the cultured chicken embryonic myoblasts were incubated with sodium selenite at different concentrations for 72?h, and then the mRNA levels of SelW and myogenic regulatory factors (MRFs) in myoblasts were determined at 12, 24, 48, and 72?h, respectively. Furthermore, the correlation between SelW mRNA expression and MRF mRNA expression was assessed. The results showed that the sodium selenite medium enhanced the mRNA expression of SelW, Myf-5, MRF4, and myogenin in chicken myoblasts. The mRNA expression levels of MRFs were significantly correlated with those of SelW at 24, 48, and 72?h. These data demonstrate that Se is involved in the differentiation of chicken embryonic myoblasts, and SelW showed correlation with MRFs.     

Involvement of myogenic regulator factors during fusion in the cell line C2C12

The myogenic factors, MyoD, myogenin, Myf5 and MRF4, can activate skeletal muscle differentiation when overexpressed in non-muscular cells. Gene targeting experiments have provided much insight into the in vivo functions of MRF and have defined two functional groups of MRFs. MyoD and Myf5 may be necessary for myoblast determination while myogenin and MRF4 may be required later during differentiation. However, the specific role of these myogenic factors has not been clearly defined during one important stage of myogenesis: the fusion of myoblasts. Using cultured C2C12 mouse muscular cells, the time-course of these proteins was analyzed and a distinct expression pattern in fusing cells was revealed. In an attempt to clarify the role of each of these regulators during myoblast fusion, an antisense strategy using oligonucleotides with phosphorothioate backbone modification was adoped. The results showed that the inhibition of myogenin and Myf5 activity is capable of significantly preventing fusion. Furthermore, the inhibition of MyoD can wholly arrest the engaged fusion process in spite of high endogenous expression of both myogenin and Myf5. Consequently, each MRF seems to have, at this defined step of myogenesis, a specific set of functions that can not be substituted for by the others and therefore may regulate a distinct subset of muscle-specific genes at the onset of fusion.     

Involvement of p38 MAPK-mediated signaling in the calpeptin-mediated suppression of myogenic differentiation and fusion in C2C12 cells

Calpeptin inhibits myoblast fusion by inhibiting the activity of calpain. However, the mechanism by which calpeptin inhibits myogenesis is not completely understood. This study examined how calpeptin affects the expression of the myogenic regulatory factors (MRFs) and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in differentiating C2C12 myoblasts. Consistent with previous reports, calpeptin inhibited the induction of μ-calpain and the formation of myotubes in these cells. In particular, calpeptin inhibited the expression of the early and mid differentiation markers including MyoD, Myf5, myogenin, and MRF4 as well as the expression of the late markers such as troponin T and myosin heavy chain (MyHC). Calpeptin also suppressed the phosphorylation of p38 MAPK in C2C12 cells. SB203580, a specific p38 inhibitor, prevented the expression of the muscle-specific markers and their fusion into myotubes in these cells, which was further accelerated in the presence of calpeptin. These findings suggest that calpeptin inhibits the myogenesis of skeletal muscle cells by down-regulating the MRFs and involving p38 MAPK signaling.     

Insulin-like growth factor-I induces androgen receptor activation in differentiating C2C12 skeletal muscle cells

The modulating effect of IGF-I on the regulation of AR gene expression and activation in skeletal muscle cells remains poorly understood. In this study, the effects of IGF-I treatment on AR induction and activation in the absence of AR ligands were examined. Differentiating C2C12 cells were treated with different concentrations (0–250 ng/ml) of IGF-I or for various periods of time (0–60 min) of 250 ng/ml IGF-I. Treatment of C2C12 cells with IGF-I resulted in a dose- and time-dependent increase in total AR and phosphorylated AR (Ser 213). IGF-I treatment also led to significantly increased AR mRNA expression when compared with the control. The levels of skeletal α-actin and myogenin mRNA, known target genes of AR, were also significantly upregulated after 5 or 10 min of treatment with IGF-I. Confocal images revealed that IGF-I stimulated nuclear localization of AR in the absence of ligands. In addition, an electrophoretic mobility shift assay indicated that IGF-I stimulated the AR DNA binding activity in a time-dependent manner. The present results suggest that IGF-I stimulates the expression and activation of AR by ligand-independent mechanism in differentiating C2C12 mouse skeletal muscle cells.     

Stretch-induced myogenin, MyoD, and MRF4 expression and acute hypertrophy in quail slow-tonic muscle are not dependent upon satellite cell proliferation

 The objectives of these studies were to determine if (1) hypertrophy-stimulated myogenic regulatory factor (MRF) mRNA increases occur in the absense of proliferating satellite cells, and (2) acute hypertrophy occurs without satellite cell proliferation. Adult and aged quails were exposed to 0 or 2500 Rads gamma irradiation, and then wing muscles were stretch-overloaded for 3 or 7 days. MRF mRNA levels in stretch-overloaded and contralateral anterior latissimus dorsi (ALD) muscles were determined after 3 days; hypertrophy was determined after 7 days. The elimination of proliferating cells in irradiated muscles was verified histologically by bromodeoxyuridine incorporation. Relative levels of MRF4, MyoD, and myogenin mRNA were elevated 100%–400% in stretch-overloaded ALD muscles from irradiated adult quails indicating that satellite cell proliferation was not a prerequisite for MRF mRNA increases. Myogenin was the only MRF that exhibited mRNA increases that were lowered by irradiation. This suggests that satellite cells contribute only to myogenin mRNA increases in non-irradiated adult muscles following 3 days of stretch-overload. Stretch-overloaded ALD muscles from aged quails had a relative increase in myogenin mRNA of ∼150%. The myogenin increase was the same in non-irradiated and irradiated aged animals and also the same as that in stretch-overloaded muscles from irradiated adult quails. Together, these data indicate that attenuated increases in MRF expression in muscles from aged animals are attributable to lower satellite cell MRF expression. ALD muscle masses and protein contents in adult irradiated quails approximately doubled after 7 days of stretch-overload demonstrating hypertrophy despite the elimination of satellite cell proliferation. Received: 5 June 1998 / Accepted: 19 November 1998     

Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133

The expression of three microRNAs, miR-1, miR-206 and miR-133 is restricted to skeletal myoblasts and cardiac tissue during embryo development and muscle cell differentiation, which suggests a regulation by muscle regulatory factors (MRFs). Here we show that inhibition of C2C12 muscle cell differentiation by FGFs, which interferes with the activity of MRFs, suppressed the expression of miR-1, miR-206 and miR-133. To further investigate the role of myogenic regulators (MRFs), Myf5, MyoD, Myogenin and MRF4 in the regulation of muscle specific microRNAs we performed gain and loss-of-function experiments in vivo, in chicken and mouse embryos. We found that directed expression of MRFs in the neural tube of chicken embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in a loss of miR-1 and miR-206 expression. Taken together our results demonstrate differential requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis.     

Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression.

Stimulation of myogenic differentiation by the insulin-like growth factors (IGFs) has been established for many years, but our attempts to elucidate the mechanism of that stimulation have been successful only in eliminating some likely possibilities. The recent discovery of a family of muscle determination genes has opened a new approach to this question, allowing specific focus on those genes that might play central roles in controlling myogenesis. We now report that IGF-I stimulates terminal myogenic differentiation in L6A1 cells by inducing a large increase in expression of the myogenin gene. This conclusion is supported by the following observations. 1) Myogenin mRNA is elevated by IGF-I, with a concentration dependency that parallels the stimulation of differentiation, including a decrease in stimulation at higher concentrations. 2) The time course of elevation of myogenin mRNA is consistent with its acting as an intermediate in the signalling pathway between occupancy of the IGF-I receptor and induction of expression of muscle-specific genes. 3) Inhibitors of myogenesis also inhibit elevation of myogenin mRNA in response to IGF-I. 4) An antisense oligonucleotide to the N-terminus of myogenin prevents the stimulation of differentiation by IGF-I and IGF-II, but has no effect on other actions of IGF-I on myoblasts. MyoD has been reported not to be expressed in L6 cells, and the expression of myf-5 and herculin/myf-6/MRF4 is reportedly low or undetectable. Thus, the stimulation of differentiation by IGF-I can be attributed largely, if not entirely, to increased expression of the myogenin gene. However, the relatively long time period between addition of the IGFs and elevation of myogenin mRNA as well as the inhibition of this process by several inhibitors indicate that increased myogenin mRNA levels are not a simple direct result of occupation of the IGF-I receptor.     

Histone deacetylase inhibitor trichostatin A enhances myogenesis by coordinating muscle regulatory factors and myogenic repressors

Histone deacetylase inhibitors (HDACIs) are known to promote skeletal muscle formation. However, their mechanisms that include effects on the expression of major muscle components such as the dystrophin-associated proteins complex (DAPC) or myogenic regulatory factors (MRFs) remain unknown. In this study, we investigated the effects of HDACIs on skeletal muscle formation using the C2C12 cell culture system. C2C12 myoblasts were exposed to trichostatin A (TSA), one of the most potent HDACIs, and differentiation was subsequently induced. We found that TSA enhances the expression of myosin heavy chain without affecting DAPC expression. In addition, TSA increases the expression of the early MRFs, Myf5 and MEF2, whereas it suppresses the expression of the late MRF, myogenin. Interestingly, TSA also enhances the expression of Id1, Id2, and Id3 (Ids). Ids are myogenic repressors that inhibit myogenic differentiation. These findings suggest that TSA promotes gene expression in proliferation and suppresses it in the differentiation stage of muscle formation. Taken together, our data demonstrate that TSA enhances myogenesis by coordinating the expression of MRFs and myogenic repressors.