Muscle-specific Transcription Factor Myogenin Research Articles

Myogenesis controlled by a long non-coding RNA 1700113A16RIK and post-transcriptional regulation

Long non-coding (lnc) RNA plays important roles in many cellular processes. The function of the vast majority of lncRNAs remains unknown. Here we identified that lncRNA-1700113A16RIK existed in skeletal muscle stem cells (MuSCs) and was significantly elevated during MuSC differentiation. Knockdown of 1700113A16RIK inhibits the differentiation of muscle stem cells. In contrast, overexpression of 1700113A16RIK promotes the differentiation of muscle stem cells. Further study shows the muscle specific transcription factor Myogenin (MyoG) positively regulates the expression of 1700113A16RIK by binding to the promoter region of 1700113A16RIK. Mechanistically, 1700113A16RIK may regulate the expression of myogenic genes by directly binding to 3’UTR of an important myogenic transcription factor MEF2D, which in turn promotes the translation of MEF2D. Taken together, our results defined 1700113A16RIK as a positive regulator of MuSC differentiation and elucidated a mechanism as to how 1700113A16RIK regulated MuSC differentiation.

Myogenin suppresses apoptosis induced by angiotensin II in human induced pluripotent stem cell-derived cardiomyocytes

BackgroundAngiotensin II (Ang II), an important component of the renin–angiotensin system (RAS), plays a critical role in the pathogenesis of cardiovascular disorders. In addition, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been considered as a promising platform for studying personalized medicine for heart diseases. However, whether Ang II can induce the apoptosis of hiPSC-CMs is not known. MethodsIn this study, we treated hiPSC-CMs with different concentrations of Ang II [0 nM (vehicle as a control), 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM] for various time periods (24 h, 48 h, 6 days, and 10 days) and analyzed the viability and apoptosis of hiPSC-CMs. ResultsWe found that treatment with 1 mM Ang II for 10 days reduced the viability of hiPSC-CMs by 41% (p = 2.073E-08) and increased apoptosis by 2.74-fold, compared to the control group (p = 6.248E-12). MYOG, which encodes the muscle-specific transcription factor myogenin, was also identified as an apoptosis-suppressor gene in Ang II-treated hiPSC-CMs. Ectopic MYOG expression decreased the apoptosis and increased the viability of Ang II-treated hiPSC-CMs. Further analysis of the RNA sequencing (RNA-seq) data illustrated that myogenin ameliorated Ang II-induced apoptosis of hiPSC-CMs by downregulating the expression of proinflammatory genes. ConclusionOur findings suggest that Ang II induces the apoptosis of hiPSC-CMs and that myogenin attenuates Ang II-induced apoptosis.

Myogenin gene expression is not altered in the developing diaphragm of nitrofen-induced congenital diaphragmatic hernia.

Pleuroperitoneal folds (PPFs) represent the only source of muscle precursors cells (MPCs) in the primordial diaphragm. However, the exact pathogenesis of malformed PPFs and congenital diaphragmatic hernia (CDH) remains unclear. The muscle-specific transcription factor myogenin plays a key role during development and muscularization of the fetal diaphragm. Although myogenin knockout mice lack skeletal muscle fibers, the diaphragmatic musculature is intact without any defects. It has further been demonstrated that proliferation and differentiation of MPCs in PPFs and developing diaphragms are normal in rodent CDH models. We hypothesized that myogenin gene expression is not altered in malformed PPFs, developing diaphragms and diaphragmatic musculature in the nitrofen-induced CDH model. Pregnant rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested during PPF formation (D13), diaphragmatic development (D14-15) and muscularization (D18-21). Fetal PPFs, developing diaphragms and diaphragmatic musculature were dissected and divided into nitrofen and control groups. Myogenin mRNA levels were analyzed by quantitative real-time polymerase chain reaction, while immunohistochemistry was performed to investigate myogenin protein expression and distribution. Relative mRNA expression of myogenin was not significant different in PPFs (0.30±0.09 vs. 0.48±0.09; P=0.37), developing diaphragms (1.25±0.29 vs. 1.60±0.32; P=0.53) and diaphragmatic musculature (1.08±0.24 vs. 1.59±0.20; P = 0.15) of nitrofen-exposed fetuses compared to controls. Myogenin immunoreactivity was not altered in the muscular components of malformed PPFs, developing diaphragms and diaphragmatic musculature of nitrofen-exposed fetuses compared to controls. Myogenin gene expression is not altered in PPFs, developing diaphragms and diaphragmatic musculature in the nitrofen-induced CDH model, thus suggesting that diaphragmatic defects in this model develop independent of myogenic processes.

New clues for organogenesis

Organogenesis is a conventional research area in developmental biology. Recent research progresses on stem cell, epigenetics, no-coding RNA and other newpropertiesofmulticellularorganismshavetransformed the organogenesis research as a frontier of life science. The collection of six research papers in this issue aims to cover timely developments in the research area of organogenesis from basic mechanism analysis to applied medical potential. In the first article, Dr Wu’s group reported that knockdown of the members of the cadherin cell adhesion superfamily proteins, protocadherin (Pcdh) clusters, resulted in dendritic simplification and spine defects in vivo and in cultured primary hippocampal neurons in vitro. Furthermore, the knockdown of the Pcdha cluster resulted in the activation of two cell-adhesion kinases (Pyk2 and Fak) and inhibition of Rho GTPases that is essential for neuronal morphogenesis and synaptic plasticity. The authors propose that Pcdh clusters and CAKs play important roles in dendritic development and spine elaboration. In the second article, Dr Rottier’s group analyzed the function of a transcription factor (TF) Sox2 in regulating the initial patterning of mouse primitive gut. The authors showed that ectopic expression of Sox2 in the posterior region of the primitive gut caused anteriorization of the gut toward a gastriclike phenotype. The authors further showed that Sox2 activated the foregut transcriptional program even in the condition of sustained co-expression of an endogenous TF Cdx2, which is recognized as a suppressor of anteriorization. They conclude that the balance between Sox2 and Cdx2 function is essential for proper specification of the primitive gut, and that Sox2 could reprogram the initial patterning of the primitive gut. It is known that a muscle-specific TF myogenin (Myog) and an MAP kinase (p38a) are required during the process of myogenesis. By comparing the expression profiling of Myog-activated alone with that of p38a activated alone in myogenesis with microarray approach, Dr Dilworth’s group showed that Myog and p38a played different roles in muscle differentiation. The authors found an un

Comparative expression profiling identifies differential roles for Myogenin and p38α MAPK signaling in myogenesis

Skeletal muscle differentiation is mediated by a complex gene expression program requiring both the muscle-specific transcription factor Myogenin (Myog) and p38α MAPK (p38α) signaling. However, the relative contribution of Myog and p38α to the formation of mature myotubes remains unknown. Here, we have uncoupled the activity of Myog from that of p38α to gain insight into the individual roles of these proteins in myogenesis. Comparative expression profiling confirmed that Myog activates the expression of genes involved in muscle function. Furthermore, we found that in the absence of p38α signaling, Myog expression leads to the down-regulation of genes involved in cell cycle progression. Consistent with this, the expression of Myog is sufficient to induce cell cycle exit. Interestingly, p38α-defective, Myog-expressing myoblasts fail to form multinucleated myotubes, suggesting an important role for p38α in cell fusion. Through the analysis of p38α up-regulated genes, the tetraspanin CD53 was identified as a candidate fusion protein, a role confirmed both ex vivo in primary myoblasts, and in vivo during myofiber regeneration in mice. Thus, our study has revealed an unexpected role for Myog in mediating cell cycle exit and has identified an essential role for p38α in cell fusion through the up-regulation of CD53.

Sodium arsenite delays the differentiation of C2C12 mouse myoblast cells and alters methylation patterns on the transcription factor myogenin

Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2), was investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20 nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (−473 to + 90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at −236 and −126 bp, whereas hypomethylated CpGs were found at −207 bp in arsenic-exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay in muscle differentiation may lead to developmental abnormalities.

The role of CMKLR1a in the differentiation and function of C2C12 myoblasts

Chemerin is a novel adipokine that exerts its effects via the G‐protein coupled receptor CMKLR1. Recent studies have shown that CMKLR1 plays an important role in the differentiation and function of cultured adipocytes and osteoblasts. We hypothesize that CMKLR1 plays a functional role in the differentiation of myoblasts due to their common mesenchymal ancestry with adipocytes and osteoblasts. In the present study we show that CMKLR1 mRNA is expressed in mouse skeletal muscle. In C2C12 mouse myoblasts, the expression of CMKLR1 increases 3‐fold with the differentiation of those cells into multinucleated myotubules. Consistent with the mRNA expression immunodetectable CMKLR1 is present in the myotubules. Abolishing CMKLR1 expression using adenoviral‐delivered shRNA: impairs the differentiation of C2C12 myoblasts into mature myotubules; inhibits the expression of muscle specific transcription factors myogenin and myoD, and reduces the expression of glucose transporter‐4 and the insulin receptor. We conclude that CMKLR1 signaling is active in skeletal muscle cells and that this pathway acts as a regulator of myogenesis. This work was supported by operating grants from CIHR, NSHRF, PEF and DMRF.

S100B stimulates myoblast proliferation and inhibits myoblast differentiation by independently stimulating ERK1/2 and inhibiting p38 MAPK

The Ca2+-modulated protein of the EF-hand type, S100B, was shown to inhibit rat L6 myoblast differentiation and myotube formation by interacting with a high affinity with an unidentified receptor (Sorci et al., 2003). We show here that S100B independently inhibits the MKK6-p38 MAPK pathway and stimulates the Ras-MEK-ERK1/2 pathway. The inhibitory effect of S100B on p38 MAPK translates into a defective induction of the muscle-specific transcription factor myogenin and the antiproliferative factor p21(WAF1), while S100B's stimulatory effect on ERK1/2 results in stimulation of myoblast proliferation via cyclin D1 induction and Rb phosphorylation and protection against apoptosis via activation of NF-kappaB transcriptional activity. Also, the S100B's effects that are mediated by the Ras-MEK-ERK1/2 pathway that is, stimulation of proliferation and protection against apoptosis, depend on reactive oxygen species production, being inhibited by antioxidants, while the S100B inhibitory effect on the MKK6-p38 MAPK pathway is not. We propose that S100B might participate in the regulation of myoblast differentiation by stimulating myoblast proliferation, protecting myoblasts against apoptosis, and modulating myotube formation.

Pulmonary hypoplasia in the myogenin null mouse embryo.

Although fetal breathing movements are required for normal lung development, there is uncertainty concerning the specific effect of absent fetal breathing movements on pulmonary cell maturation. We set out to evaluate pulmonary development in a genetically defined mouse model, the myogenin null mouse, in which there is a lack of normal skeletal muscle fibers and thus skeletal muscle movements are absent in utero. Significant decreases were observed in lung:body weight ratio and lung total DNA at embryonic days (E)14, E17, and E20. Reverse transcriptase/polymerase chain reaction, in situ immunofluorescence, and electron microscopy revealed early lung cell differentiation in both null and wild-type lungs as early as E14. However at E14, myogenin null lungs had decreased 5'-bromo-2-deoxyuridine incorporation compared with that of wild-type littermates, whereas at E17 and E20, increased Bax immunolabeling and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining were detected in the myogenin null mice but not in the wild-type littermates. These observations highlight the importance of skeletal muscle contractile activity in utero for normal lung organogenesis. Null mice lacking the muscle-specific transcription factor myogenin exhibit a secondary effect on lung development such that decreased lung cell proliferation and increased programmed cell death are associated with lung hypoplasia.

Involvement of type 4 cAMP-phosphodiesterase in the myogenic differentiation of L6 cells.

Myogenic cell differentiation is induced by Arg(8)-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin.

Cloning of Novel Injury-regulated Genes: IMPLICATIONS FOR AN IMPORTANT ROLE OF THE MUSCLE-SPECIFIC PROTEIN skNAC IN MUSCLE REPAIR

To gain insight into the molecular mechanisms underlying the wound repair process, we searched for genes that are regulated by skin injury. Using the differential display reverse transcription-polymerase chain reaction technique, we identified a gene that was strongly induced as early as 12 h after wounding. Sequence analysis revealed the identity of the corresponding protein with skeletal muscle nascent polypeptide-associated complex (skNAC), a recently identified muscle-specific transcription factor. By in situ hybridization and immunohistochemistry, we demonstrated the specific expression of skNAC in skeletal muscle cells of the panniculus carnosus at the wound edge. Furthermore, in vitro studies with cultured myoblasts revealed expression of skNAC in differentiating and differentiated, but not in proliferating, nondifferentiated cells. Differentiation of cultured myoblasts was accompanied by simultaneous expression of skNAC and the muscle-specific transcription factor myogenin. Our results provide the first evidence for a role of skNAC in muscle repair processes. Furthermore, they demonstrate the usefulness of our approach in identifying new players in wound repair.

Early Skeletal Muscle Development Proceeds Normally in Parthenogenetic Mouse Embryos

In mouse chimeras with parthenogenetic cell contribution, the skeletal musculature appears to be largely devoid of parthenogenetically derived cells. To analyze the appearance and early distribution of mytomal cells in parthenotes, we determined the expression of the muscle-specific transcription factors myogenin, MYF-5, and MYF-6 by in situ hybridization in somites of Day 10 and 11 embryos. Here, we report that these myogenic regulatory proteins are expressed in parthenogenetic animals together with desmin, one of the early muscle-specific structural proteins. We also show that parthenogenetic cells contribute equally to dermatome, sclerotome, and myotome in Day 10 and 11 chimeras. These results suggest that early myotomal cells expressing the myogenic control proteins develop and allocate normally in parthenogenetic embryos and in parthenogenetic ↔ normal chimeras. The underrepresentation in older chimeras may therefore be due to selective elimination. These data also argue against imprinting of the myogenic factor genes myogenin, Myf-5, and Myf-6.