Calf Blood Compound (CFC) and Homeopathic Drug Induce Differentiation of Primary Human Skeletal Muscle Cells.

The use of injections to treat structural muscle injuries is controversially discussed. In our controlled in vitro study, we investigated the biological impact of Actovegin and Traumeel alone and in combination on primary human skeletal muscle cells. Cells were characterized by immunofluorescence staining for myogenic factor 5 (Myf5) and MyoD, and cultured with or without Actovegin and / or Traumeel. The effects of these agents were assayed by cell viability and gene expression of the specific markers MyoD, Myf5, neural adhesion molecule (NCAM), and CD31. Myotube formation was determined by myosin staining. Neither Actovegin nor Traumeel showed toxic effects or influenced cell viability significantly. High volumes of Actovegin down-regulated gene expression of NCAM after 3 days but had no effect on MyoD, Myf5, and CD31 gene expression. High volumes of Traumeel inhibited MyoD gene expression after 3 days, whereas after 7 days MyoD expression was significantly up-regulated. The combination of both agents did not significantly influence cell viability or gene expression. This is the first study demonstrating that Actovegin and Traumeel potentially modulate human skeletal muscle cells. The relevance of these in vitro findings has to be highlighted in further in vivo studies.

TCDD disrupts posterior palatogenesis and causes cleft palate.

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion.

Effects of genistein and estrogen on the genioglossus in rats exposed to chronic intermittent hypoxia may be HIF-1α dependent.

OBJECTIVES: Chronic intermittent hypoxia (CIH) is a frequent feature of OSAHS. The present study was designed to evaluate the effects of genistein and estrogen on genioglossus contractile and regeneration properties in CIH rats and investigate the involvement of HIF-1α. METHODS: Ovariectomized female rats were exposed to CIH for 5 weeks. Genistein and estrogen were administered by intraperitoneal injection. The genioglossus myoblasts of rat were also isolated and cultured in vitro, and the HIF-1α shRNA lentivirus was used. RESULTS: Muscle fatigue resistance and myogenic regeneration were significantly decreased after CIH but were partially reversed by estrogen and genistein treatment. The effect of estrogen was more powerful than that of genistein. Compared with control group, RT-PCR and western blotting showed higher levels of HIF-1α mRNA and protein in the CIH group, but estrogen and genistein treatment reduced the levels of HIF-1α mRNA and protein in rats exposed to CIH. In genioglossus myoblasts, the expression of HIF-1α was up-regulated under hypoxia rather than normoxia and decreased over time under both hypoxia and normoxia during myogenic differentiation. HIF-1α knockdown relieved myogenesis inhibition under hypoxia. CONCLUSION: We concluded that genistein and estrogen may inhibit the overexpression of HIF-1α induced by CIH and improve the endurance and regeneration of the genioglossus muscle.

Cancer cachexia is regulated by selective targeting of skeletal muscle gene products.

Cachexia is a syndrome characterized by wasting of skeletal muscle and contributes to nearly one-third of all cancer deaths. Cytokines and tumor factors mediate wasting by suppressing muscle gene products, but exactly which products are targeted by these cachectic factors is not well understood. Because of their functional relevance to muscle architecture, such targets are presumed to represent myofibrillar proteins, but whether these proteins are regulated in a general or a selective manner is also unclear. Here we demonstrate, using in vitro and in vivo models of muscle wasting, that cachectic factors are remarkably selective in targeting myosin heavy chain. In myotubes and mouse muscles, TNF-alpha plus IFN-gamma strongly reduced myosin expression through an RNA-dependent mechanism. Likewise, colon-26 tumors in mice caused the selective reduction of this myofibrillar protein, and this reduction correlated with wasting. Under these conditions, however, loss of myosin was associated with the ubiquitin-dependent proteasome pathway, which suggests that mechanisms used to regulate the expression of muscle proteins may be cachectic factor specific. These results shed new light on cancer cachexia by revealing that wasting does not result from a general downregulation of muscle proteins but rather is highly selective as to which proteins are targeted during the wasting state.

Exogenous hepatocyte growth factor inhibits myoblast differentiation by inducing myf5 expression and suppressing myoD expression in an organ culture system of embryonic mouse tongue.

We examined the effects of exogenous hepatocyte growth factor (HGF) on the differentiation and proliferation of tongue myoblasts by using an organ culture system of tongue obtained from mouse embryos at embryonic day (E) 13. Exogenous HGF induced reductions in the quantities of muscle creatine kinase and myogenin mRNAs and in the number of fast myosin heavy chain-positive myoblasts and myotubes, suggesting that HGF suppressed the differentiation of myoblasts in the cultured E13 tongues. Exogenous HGF induced no significant changes in the percentage of proliferating cell nuclear antigen (PCNA)-positive cell nuclei to total cell nuclei (labeling index) in the muscle portion of the cultured E13 tongue, suggesting that HGF did not affect the proliferation of myoblasts. Exogenous HGF induced the expression of myf5 mRNA but inhibited the expression of myoD mRNA. Since mouse tongue myoblasts are reported to complete proliferation by E13, it appears that exogenous HGF arrests myoblasts in the cell cycle and does not allow them to enter the differentiation process. This is achieved by controlling the expression of myf5 and myoD mRNAs, thus inhibiting the differentiation of tongue myoblasts.

Injection of FGF6 accelerates regeneration of the soleus muscle in adult mice.

FGF6, a member of the fibroblast growth factor (FGF) family, accumulated almost exclusively in the myogenic lineage, supporting the finding that FGF6 could specifically regulate myogenesis. Using FGF6 (-/-) mutant mice, important functions in muscle regeneration have been proposed for FGF6 but remain largely controversial. Here, we examined the effect of a single injection of recombinant FGF6 (rhFGF6) on the regeneration of mouse soleus subjected to cardiotoxin injection, specifically looking for molecular and morphological phenotypes. The injection of rhFGF6 has two effects. First, there is an up-regulation of cyclin D1 mRNA, accounting for the regulating role of a high FGF6 concentration on proliferation, and second, differentiation markers such as CdkIs and MHC I and Tn I increase and cellular differentiation is accelerated. We also show a down-regulation of endogenous FGF6, acceleration of FGFR1 receptor expression and deceleration of the FGFR4 receptor expression, possibly accounting for biphasic effects of exogenous FGF6 on muscle regeneration.

Calcineurin is a potent regulator for skeletal muscle regeneration by association with NFATc1 and GATA-2.

The molecular signaling pathways involved in regeneration after muscle damage have not been identified. In the present study, we tested the hypothesis that calcineurin, a calcium-regulated phosphatase recently implicated in the signaling of fiber-type conversion and muscle hypertrophy, is required to induce skeletal muscle remodeling. The amount of calcineurin and dephosphorylated nuclear factor of activated T cells c1 (NFATc1) proteins was markedly increased in the regenerating muscle of rats. The amount of calcineurin co-precipitating with NFATc1 and GATA-2, and NFATc1 co-precipitating with GATA-2 gradually increased in the tibialis anterior muscle after bupivacaine injection. Calcineurin protein was present in the proliferating satellite cells labeled with BrdU in the damaged muscle after 4 days. In contrast, calcineurin was not detected in the quiescent nonactivating satellite cells expressing Myf-5. At 4 days post injection, many macrophages detected in the damaged and regenerating area did not possess calcineurin protein. Calcineurin protein was abundant in many myoblasts and myotubes that expressed MyoD and myogenin at 4 and 6 days post injection. In the intact muscle, no immunoreactivity of calcineurin or BrdU was detected in the cell membrane, cytosol or the extracellular connective tissue. In mice, intraperitoneal injection of cyclosporin A, a potent inhibitor of calcineurin, induced extensive inflammation, marked fiber atrophy, the appearance of immature myotubes, and calcification in the regenerating muscle compared with phosphate-buffered saline-administered mice. Thus, calcineurin may have an important role in muscle regeneration in association with NFATc1 and GATA-2.

A myogenic differentiation checkpoint activated by genotoxic stress.

Cell-cycle checkpoints help to protect the genomes of proliferating cells under genotoxic stress. In multicellular organisms, cell proliferation is often directed toward differentiation during development and throughout adult homeostasis. To prevent the formation of differentiated cells with genetic instability, we hypothesized that genotoxic stress may trigger a differentiation checkpoint. Here we show that exposure to genotoxic agents causes a reversible inhibition of myogenic differentiation. Muscle-specific gene expression is suppressed by DNA-damaging agents if applied prior to differentiation induction but not after the differentiation program is established. The myogenic determination factor, MyoD (encoded by Myod1), is a target of the differentiation checkpoint in myoblasts. The inhibition of MyoD by DNA damage requires a functional c-Abl tyrosine kinase (encoded by Abl1), but occurs in cells deficient for p53 (transformation-related protein 53, encoded by Trp53) or c-Jun (encoded by the oncogene Jun). These results support the idea that genotoxic stress can regulate differentiation, and identify a new biological function for DNA damage-activated signaling network.

Interleukin-11 induces osteoblast differentiation and acts synergistically with bone morphogenetic protein-2 in C3H10T1/2 cells.

Interleukin-11 (IL-11) is a pleiotropic cytokine that supports various types of hematopoietic cell growth and is involved in bone resorption. We report here the involvement of recombinant human IL-11 (rHuIL-11) in osteoblast differentiation in mouse mesenchymal progenitor cells, C3H10T1/2. rHuIL-11 alone increased alkaline phosphatase (ALP) activity and upregulated expression levels of osteocalcin (OC), bone sialo protein (BSP), and parathyroid hormone receptor (PTHR) mRNA. rHuIL-11 had no effect on expression of type II collagen, peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2), adipocyte fatty acid-binding protein P2 (aP2), and myogenic MyoD protein (MyoD). Recombinant human bone morphogenetic protein (rHuBMP)-2 increased ALP activity and mRNA expression of these genes except for MyoD. The expression patterns of ALP activity and osteoblast-specific or chondrocyte-specific genes suggest that rHuIL-11 may be involved in early differentiation of osteoblasts at a step earlier than that which is affected by rHuBMP-2. In support of this hypothesis, combined treatment with rHuIL-11 and rHuBMP-2 synergistically increased ALP activity and mRNA expression of OC and type II collagen, rHuIL-11 also abrogated the increased levels of PPAR-gamma2, aP2 mRNA caused by rHuBMP-2. Our results suggest that rHuIL-11 alone and in combination with rHuBMP-2 can induce osteoblastic differentiation of progenitor cells and plays an important role in osteogenesis.

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.

The 2'-5' oligoadenylate/RNase L/RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation.

The 2'-5' oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.

Mutation of Thr115 in MyoD positively regulates function in murine fibroblasts and human rhabdomyosarcoma cells.

Committed skeletal muscle myoblasts undergo terminal differentiation when shifted from a high-mitogen medium to a low-mitogen medium. However, expression of the myogenic regulatory factor MyoD seems to be similar in proliferating and differentiating cells, suggesting that its function is attenuated in proliferating myoblasts. To further understand the potential mechanisms that may attenuate MyoD function, we have examined the effect of posttranslational modification. By analogy with myogenin, we have examined the role of phosphorylation in regulating the function of MyoD. MyoD contains two putative protein kinase C (PKC) phosphorylation sites (Thr115 and Ser200). The former site is analogous to Thr85 within the highly conserved basic domain of myogenin that has been demonstrated to negatively regulate the myogenic differentiation functions of myogenin. To test whether hyperphosphorylation of the same PKC site in MyoD would attenuate its function, we generated a mutant MyoD with a single amino acid substitution (Thr115-Ala) that disrupts the PKC phosphorylation site (Thr115) within the conserved basic domain. Wild-type and mutant MyoD were introduced into cells using an E1, E3-deleted adenoviral vector. In mouse C3H10T1/2 fibroblasts, both wild-type and mutant MyoD induced terminal myogenic differentiation when growth factors were withdrawn from the cell culture. Consistent with these results, nuclear extracts from infected cells, but not those from uninfected cells, demonstrated complex formation with an oligonucleotide containing an E-box consensus sequence. Growth arrest was associated with the up-regulation of p21cip1, cell fusion to multinucleated myotubes, and the expression of a muscle differentiation marker (myosin heavy chain). On the other hand, when infected cells were maintained under high mitogenic conditions (in the presence of 10% fetal bovine serum), the expression of wild-type or mutant MyoD slowed cell growth and induced p21cip1. Only mutant MyoD caused cell fusion, myosin heavy chain expression, and altered mobility of the E-box oligonucleotide in gel shift assays. Furthermore, after infection, MyoD was phosphorylated, and phosphothreonine was detected in wild-type MyoD immunoprecipitated only from C3H10T1/2 cells grown under high mitogenic conditions. These results suggest that Thr115 may play an important role in the regulation of MyoD function under conditions of high mitogenesis. MyoD was also phosphorylated in malignant rhabdomyosarcoma (RMS) cells in which MyoD function was attenuated. Phosphothreonine was also detected in MyoD immunoprecipitates. Rh30 alveolar RMS cells were infected with an adenovirus expressing either wild-type or mutant MyoD. In contrast to the results in fibroblasts, when overexpressed in malignant Rh30 RMS cells, mutant MyoD arrested cell growth without inducing p21cip1 and caused cell fusion. However, no muscle differentiation markers were detected, indicating that an overexpression of mutant MyoD lacking Thr115 caused Rh30 cells to become quiescent and recapitulate at least some aspects of myogenesis (cell fusion).

Regulation of dorsal somitic cell fates: BMPs and Noggin control the timing and pattern of myogenic regulator expression.

Previous work has indicated that signals from the neural tube, notochord, and surface ectoderm promote somitic myogenesis. Here, we show that somitic myogenesis is under negative regulation as well; BMP signaling serves to inhibit the activation of MyoD and Myf5 in Pax3-expressing cells. Furthermore, we show that the BMP antagonist Noggin is expressed within the dorsomedial lip of the dermomyotome, where Pax3-expressing cells first initiate the expression of MyoD and Myf5 to give rise to myotomal cells in the medial somite. Consistent with the expression of Noggin in dorsomedial dermomyotomal cells that lie adjacent to the dorsal neural tube, we have found that coculture of somites with fibroblasts programmed to secrete Wnt1, which is expressed in dorsal neural tube, can induce somitic Noggin expression. Ectopic expression of Noggin lateral to the somite dramatically expands MyoD expression into the lateral regions of the somite, represses Pax3 expression in this tissue, and induces formation of a lateral myotome. Together, our findings indicate that the timing and location of myogenesis within the somite is controlled by relative levels of BMP activity and localized expression of a BMP antagonist.

E2F1 inhibition of transcription activation by myogenic basic helix-loop-helix regulators.

Cellular transcription factor E2F1 is thought to regulate the expression of genes important for cell cycle progression and cell proliferation. Deregulated E2F1 expression induces S-phase entry in quiescent cells and inhibits myogenic differentiation. We show here that E2F1 inhibits the activation of gene transcription by myogenic basic helix-loop-helix proteins myoD and myogenin. Transfection assay using different deletion constructs indicates that both the DNA binding and the transactivation domains of E2F1 are required for its inhibition of myoD transcription activation. However, the retinoblastoma protein (RB) binding domain is not required. Furthermore, co-transfection with the RB, which inhibits the transcription activity of E2F1, can also repress E2F1 inhibition of myoD transactivation. These results suggest an essential role of E2F1-mediated transcription in its inhibition of myogenesis.

Doxorubicin represses the function of the myogenic helix-loop-helix transcription factor MyoD. Involvement of Id gene induction.

We have shown recently that Doxorubicin (Dox, Adriamycin), a potent broad spectrum chemotherapeutic agent with a major side effect of cardiomyopathy, completely prevents myoblast fusion and accumulation of muscle-specific transcripts in the mouse C2 skeletal muscle cell line. Here we use mouse embryonic fibroblast 10T1/2 cells to demonstrate that Dox represses muscle-specific gene expression by interfering with MyoD activity. As assayed by transient cotransfection, Dox inhibits the ability of MyoD to trans-activate muscle-specific reporter genes. A stable cell system was developed in which MyoD is constitutively expressed in 10T1/2 cells (M10 cells). Dox-treated M10 cells express MyoD from a long terminal repeat-driven vector but fail to activate endogenous MyoD and myogenin loci. Dox did not effect E2A gene transcript levels, but Id mRNA levels are significantly increased in Dox-treated M10 cells. Interestingly, overexpression of E2-5, which forms inactive heterodimers with Id, can overcome the Dox-induced suppression of the trans-activation function of MyoD in 10T1/2 cells. Furthermore, we demonstrate that the 5'-flanking region of the Id2 gene mediates its Dox-inducible transcriptional expression. These findings support a model in which Dox inhibits muscle-specific gene expression by interfering with the function of MyoD protein through, at least in part, induction of Id gene expression. The implications of our results for the molecular mechanisms underlying the myofibrillar loss observed in Dox-induced cardiomyopathy are discussed.