Myogenic differentiation induces taurine transporter in association with taurine-mediated cytoprotection in skeletal muscles

Skeletal muscle homoeostasis is maintained by a variety of cytoprotective mechanisms. Since ablation of the TauT (taurine transporter) gene results in susceptibility to exercise-induced muscle weakness in vivo, it has been suggested that TauT is essential for skeletal muscle function. However, the regulatory mechanisms of TauT expression remain to be elucidated. In the present study, we demonstrated that TauT was up-regulated during myogenesis in C2C12 cells. Treatment with bFGF (basic fibroblast growth factor), which inhibited muscle differentiation, abrogated myogenic induction of TauT. The promoter activities of TauT were up-regulated during muscle differentiation in C2C12 cells. Database analyses identified an MEF2 (myocyte enhancer binding factor 2) consensus sequence at −844 in the rat TauT gene. Truncation of the promoter region containing the MEF2 site significantly reduced the promoter activity, demonstrating the functional importance of the MEF2 site. Electrophoretic mobility-shift assays confirmed that MEF2 bound to the MEF2 consensus sequence and that DNA–protein complex levels were increased during differentiation. Promoter analyses using mutated promoter-reporter plasmids demonstrated that this site was functional. Importantly, transfection with a MyoD expression vector markedly enhanced TauT promoter activity in the (non-myogenic) 10T1/2 cells. Moreover, co-transfection with an MEF2 expression vector augmented MyoD-induced TauT promoter activity, suggesting that MEF2 is required for full activation of TauT expression. Finally, we examined the effects of taurine on myotube atrophy to clarify the biological significance of the up-regulation of TauT, and demonstrated that taurine attenuated muscle atrophy induced by dexamethasone. TauT expression is regulated under the control of the myogenic programme, and we propose that this is the mechanism for taurine-mediated resistance to muscle atrophy.

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6-Bromoindirubin-3′-oxime intercepts GSK3 signaling to promote and enhance skeletal muscle differentiation affecting miR-206 expression in mice

Scientific Reports ◽

10.1038/s41598-019-54574-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Elvira Ragozzino ◽

Mariarita Brancaccio ◽

Antonella Di Costanzo ◽

Francesco Scalabrì ◽

Gennaro Andolfi ◽

...

Keyword(s):

Skeletal Muscle ◽

Myogenic Differentiation ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Skeletal Muscle Differentiation ◽

Myoblast Proliferation ◽

Enhanced Expression ◽

Pharmacologically Active

AbstractDystrophies are characterized by progressive skeletal muscle degeneration and weakness as consequence of their molecular abnormalities. Thus, new drugs for restoring skeletal muscle deterioration are critically needed. To identify new and alternative compounds with a functional role in skeletal muscle myogenesis, we screened a library of pharmacologically active compounds and selected the small molecule 6-bromoindirubin-3′-oxime (BIO) as an inhibitor of myoblast proliferation. Using C2C12 cells, we examined BIO’s effect during myoblast proliferation and differentiation showing that BIO treatment promotes transition from cell proliferation to myogenic differentiation through the arrest of cell cycle. Here, we show that BIO is able to promote myogenic differentiation in damaged myotubes in-vitro by enriching the population of newly formed skeletal muscle myotubes. Moreover, in-vivo experiments in CTX-damaged TA muscle confirmed the pro-differentiation capability of BIO as shown by the increasing of the percentage of myofibers with centralized nuclei as well as by the increasing of myofibers number. Additionally, we have identified a strong correlation of miR-206 with BIO treatment both in-vitro and in-vivo: the enhanced expression of miR-206 was observed in-vitro in BIO-treated proliferating myoblasts, miR-206 restored expression was observed in a forced miR-206 silencing conditions antagomiR-mediated upon BIO treatment, and in-vivo in CTX-injured muscles miR-206 enhanced expression was observed upon BIO treatment. Taken together, our results highlight the capacity of BIO to act as a positive modulator of skeletal muscle differentiation in-vitro and in-vivo opening up a new perspective for novel therapeutic targets to correct skeletal muscle defects.

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TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake

Cell Death and Disease ◽

10.1038/s41419-021-04383-3 ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Barbara Toffoli ◽

Federica Tonon ◽

Veronica Tisato ◽

Giorgio Zauli ◽

Paola Secchiero ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucose Uptake ◽

Therapeutic Potential ◽

Myogenic Differentiation ◽

Body Weight Gain ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Acid Oxidation ◽

Skeletal Muscle Differentiation ◽

Akt Phosphorylation

AbstractTNF-related apoptosis-inducing ligand (TRAIL) is a protein that induces apoptosis in cancer cells but not in normal ones, where its effects remain to be fully understood. Previous studies have shown that in high-fat diet (HFD)-fed mice, TRAIL treatment reduced body weight gain, insulin resistance, and inflammation. TRAIL was also able to increase skeletal muscle free fatty acid oxidation. The aim of the present work was to evaluate TRAIL actions on skeletal muscle. Our in vitro data on C2C12 cells showed that TRAIL treatment significantly increased myogenin and MyHC and other hallmarks of myogenic differentiation, which were reduced by Dr5 (TRAIL receptor) silencing. In addition, TRAIL treatment significantly increased AKT phosphorylation, which was reduced by Dr5 silencing, as well as glucose uptake (alone and in combination with insulin). Our in vivo data showed that TRAIL increased myofiber size in HFD-fed mice as well as in db/db mice. This was associated with increased myogenin and PCG1α expression. In conclusion, TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake. These data shed light onto a pathway that might hold therapeutic potential not only for the metabolic disturbances but also for the muscle mass loss that are associated with diabetes.

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Coordinated Vascular Endothelial Growth Factor Expression and Signaling During Skeletal Myogenic Differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e07-09-0856 ◽

2008 ◽

Vol 19 (3) ◽

pp. 994-1006 ◽

Cited By ~ 76

Author(s):

Brad A. Bryan ◽

Tony E. Walshe ◽

Dianne C. Mitchell ◽

Josh S. Havumaki ◽

Magali Saint-Geniez ◽

...

Keyword(s):

Skeletal Muscle ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Myogenic Differentiation ◽

Es Cells ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Vascular Endothelial ◽

Skeletal Muscle Differentiation ◽

Endothelial Growth Factor

Angiogenesis is largely controlled by hypoxia-driven transcriptional up-regulation and secretion of vascular endothelial growth factor (VEGF) and its binding to the endothelial cell tyrosine receptor kinases, VEGFR1 and VEGFR2. Recent expression analysis suggests that VEGF is expressed in a cell-specific manner in normoxic adult tissue; however, the transcriptional regulation and role of VEGF in these tissues remains fundamentally unknown. In this report we demonstrate that VEGF is coordinately up-regulated during terminal skeletal muscle differentiation. We reveal that this regulation is mediated in part by MyoD homo- and hetero-dimeric transcriptional mechanisms. Serial deletions of the VEGF promoter elucidated a region containing three tandem CANNTG consensus MyoD sites serving as essential sites of direct interaction for MyoD-mediated up-regulation of VEGF transcription. VEGF-null embryonic stem (ES) cells exhibited reduced myogenic differentiation compared with wild-type ES cells, suggesting that VEGF may serve a role in skeletal muscle differentiation. We demonstrate that VEGFR1 and VEGFR2 are expressed at low levels in myogenic precursor cells and are robustly activated upon VEGF stimulation and that their expression is coordinately regulated during skeletal muscle differentiation. VEGF stimulation of differentiating C2C12 cells promoted myotube hypertrophy and increased myogenic differentiation, whereas addition of sFlt1, a VEGF inhibitor, resulted in myotube hypotrophy and inhibited myogenic differentiation. We further provide evidence indicating VEGF-mediated myogenic marker expression, mitogenic activity, migration, and prosurvival functions may contribute to increased myogenesis. These data suggest a novel mechanism whereby VEGF is coordinately regulated as part of the myogenic differentiation program and serves an autocrine function regulating skeletal myogenesis.

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Inositide-Dependent Phospholipase C Signaling Mimics Insulin in Skeletal Muscle Differentiation by Affecting Specific Regions of the Cyclin D3 Promoter

Endocrinology ◽

10.1210/en.2006-1003 ◽

2007 ◽

Vol 148 (3) ◽

pp. 1108-1117 ◽

Cited By ~ 42

Author(s):

Irene Faenza ◽

Giulia Ramazzotti ◽

Alberto Bavelloni ◽

Roberta Fiume ◽

Gian Carlo Gaboardi ◽

...

Keyword(s):

Skeletal Muscle ◽

Phospholipase C ◽

Myogenic Differentiation ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Cyclin D3 ◽

Expression Vectors ◽

Skeletal Muscle Differentiation ◽

Small Interfering Rnas

Our main goal in this study was to investigate the role of phospholipase C (PLC) β1 and PLCγ1 in skeletal muscle differentiation and the existence of potential downstream targets of their signaling activity. To examine whether PLC signaling can modulate the expression of cyclin D3, a target of PLCβ1 in erythroleukemia cells, we transfected C2C12 cells with expression vectors containing PLCβ1 or PLCγ1 cDNA and with small interfering RNAs from regions of the PLCβ1 or PLCγ1 gene and followed myogenic differentiation in this well-established cell system. Intriguingly, overexpressed PLCβ1 and PLCγ1 were able to mimic insulin induction of both cyclin D3 and muscle differentiation. By knocking down PLCβ1 or PLCγ1 expression, C2C12 cells almost completely lost the increase in cyclin D3, and the differentiation program was down-regulated. To explore the induction of the cyclin D3 gene promoter during this process, we used a series of 5′-deletions of the 1.68-kb promoter linked to a reporter gene and noted a 5-fold augmentation of promoter activity upon insulin stimulation. These constructs were also cotransfected with PLCβ1 or PLCγ1 cDNAs and small interfering RNAs, respectively. Our data indicate that PLCβ1 or PLCγ1 signaling is capable of acting like insulin in regard to both the myogenic differentiation program and cyclin D3 up-regulation. Taken together, this is the first study that hints at cyclin D3 as a target of PLCβ1 and PLCγ1 during myogenic differentiation in vitro and implies that up-regulation of these enzymes is sufficient to mimic the actions of insulin in this process.

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Interleukin-6 promotes myogenic differentiation of mouse skeletal muscle cells: role of the STAT3 pathway

AJP Cell Physiology ◽

10.1152/ajpcell.00025.2012 ◽

2013 ◽

Vol 304 (2) ◽

pp. C128-C136 ◽

Cited By ~ 55

Author(s):

Miriam Hoene ◽

Heike Runge ◽

Hans Ulrich Häring ◽

Erwin D. Schleicher ◽

Cora Weigert

Keyword(s):

Skeletal Muscle ◽

Mrna Expression ◽

Myogenic Differentiation ◽

Muscle Cells ◽

C2c12 Cells ◽

Skeletal Muscle Cells ◽

Wild Type ◽

C2c12 Myoblasts ◽

Sequence Of Events ◽

Primary Mouse

Myogenic differentiation of skeletal muscle cells is characterized by a sequence of events that include activation of signal transducer and activator of transcription 3 (STAT3) and enhanced expression of its target gene Socs3. Autocrine effects of IL-6 may contribute to the activation of the STAT3-Socs3 cascade and thus to myogenic differentiation. The importance of IL-6 and STAT3 for the differentiation process was studied in C2C12 cells and in primary mouse wild-type and IL-6−/− skeletal muscle cells. In differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA expression and protein secretion started after increased phosphorylation of STAT3 on tyrosine 705 and increased mRNA expression of Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in differentiating C2C12 myoblasts impaired the expression of the myogenic markers myogenin and MyHC IIb and subsequently myotube fusion. However, the knockdown of IL-6 did not prevent the induction of STAT3 tyrosine phosphorylation. The IL-6-independent activation of STAT3 was verified in differentiating primary IL-6−/− myoblasts. The phosphorylation of STAT3 and the expression levels of STAT3, Socs3, and myogenin during differentiation were comparable in the primary myoblasts independent of the genotype. However, IL-6−/− cells failed to induce MyHC IIb expression to the same level as in wild-type cells and showed reduced myotube formation. Supplementation of IL-6 could partially restore the fusion of IL-6−/− cells. These data demonstrate that IL-6 depletion during myogenic differentiation does not reduce the activation of the STAT3-Socs3 cascade, while IL-6 and STAT3 are both necessary to promote myotube fusion.

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AMP-activated protein kinase agonists increase mRNA content of the muscle-specific ubiquitin ligases MAFbx and MuRF1 in C2C12 cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00622.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. E1555-E1567 ◽

Cited By ~ 93

Author(s):

Brian J. Krawiec ◽

Gerald J. Nystrom ◽

Robert A. Frost ◽

Leonard S. Jefferson ◽

Charles H. Lang

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Mrna Expression ◽

Muscle Atrophy ◽

Ubiquitin Ligases ◽

C2c12 Cells ◽

Skeletal Muscle Atrophy ◽

Compound C ◽

Mrna Content ◽

Amp Activated Protein Kinase

The hypothesis of the present study was that exposure of differentiated muscle cells to agonists of the AMP-activated protein kinase (AMPK) would increase the mRNA content of the muscle-specific ubiquitin ligases muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). C2C12 cells were incubated with incremental doses of 5-aminoimidazol-4-carboximide ribonucleoside (AICAR) or metformin for 24 h. Both MAFbx and MuRF1 mRNA increased dose dependently in response to these AMPK activators. AICAR, metformin, and 2-deoxy-d-glucose produced time-dependent alterations in ubiquitin ligase expression, typified by a biphasic pattern of expression marked by an acute repression followed by a sustained induction. AMPK-activating treatments in conjunction with dexamethasone produced a pronounced synergistic effect on ligase mRNA expression at later time points. This cooperative response occurred in the absence of a dexamethasone-dependent increase in AMPK expression or activity, as determined by immunoblotting for phosphorylation and expression of AMPKα and its downstream target acetyl-CoA carboxylase (ACC). These responses elicited by AMPK activation singly or in combination with dexamethasone did not extend to the mRNA expression of the UBR box family E3s UBR1/E3αI and UBR2/E3αII. Treatment with the AMPK inhibitor compound C prevented increases in MAFbx and MuRF1 mRNA in response to serum deprivation, as well as AICAR and dexamethasone treatment individually or jointly. Stimulation of AMPK activity in vivo via AICAR injection increased both MAFbx and MuRF1 mRNA in murine skeletal muscle. These data suggest that activation of AMPK in skeletal muscle results in a specific upregulation of MAFbx and MuRF1, responses that are reminiscent of the proposed atrophic transcriptional program executed under various conditions of skeletal muscle wasting. Therefore, AMPK may be a critical component of the intercalated network of signaling pathways governing skeletal muscle atrophy, where its input acts to modify anti- and proatrophic signals to influence gene expression in reaction to catabolic perturbations.

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Hydrogen sulfide alleviates hyperhomocysteinemia-mediated skeletal muscle atrophy via mitigation of oxidative and endoplasmic reticulum stress injury

AJP Cell Physiology ◽

10.1152/ajpcell.00147.2018 ◽

2018 ◽

Vol 315 (5) ◽

pp. C609-C622 ◽

Cited By ~ 17

Author(s):

Avisek Majumder ◽

Mahavir Singh ◽

Jyotirmaya Behera ◽

Nicholas T. Theilen ◽

Akash K. George ◽

...

Keyword(s):

Oxidative Stress ◽

Skeletal Muscle ◽

Endoplasmic Reticulum ◽

Hydrogen Sulfide ◽

Er Stress ◽

Muscle Atrophy ◽

C2c12 Cells ◽

Skeletal Muscle Atrophy ◽

Stress Injury ◽

Western Blots

Although hyperhomocysteinemia (HHcy) occurs because of the deficiency in cystathionine-β-synthase (CBS) causing skeletal muscle dysfunction, it is still unclear whether this effect is mediated through oxidative stress, endoplasmic reticulum (ER) stress, or both. Nevertheless, there is no treatment option available to improve HHcy-mediated muscle injury. Hydrogen sulfide (H2S) is an antioxidant compound, and patients with CBS mutation do not produce H2S. In this study, we hypothesized that H2S mitigates HHcy-induced redox imbalance/ER stress during skeletal muscle atrophy via JNK phosphorylation. We used CBS+/−mice to study HHcy-mediated muscle atrophy, and treated them with sodium hydrogen sulfide (NaHS; an H2S donor). Proteins and mRNAs were examined by Western blots and quantitative PCR. Proinflammatory cytokines were also measured. Muscle mass and strength were studied via fatigue susceptibility test. Our data revealed that HHcy was detrimental to skeletal mass, particularly gastrocnemius and quadriceps muscle weight. We noticed that oxidative stress was reversed by NaHS in homocysteine (Hcy)-treated C2C12 cells. Interestingly, ER stress markers (GRP78, ATF6, pIRE1α, and pJNK) were elevated in vivo and in vitro, and NaHS mitigated these effects. Additionally, we observed that JNK phosphorylation was upregulated in C2C12 after Hcy treatment, but NaHS could not reduce this effect. Furthermore, inflammatory cytokines IL-6 and TNF-α were higher in plasma from CBS as compared with wild-type mice. FOXO1-mediated Atrogin-1 and MuRF-1 upregulation were attenuated by NaHS. Functional studies revealed that NaHS administration improved muscle fatigability in CBS+/−mice. In conclusion, our work provides evidence that NaHS is beneficial in mitigating HHcy-mediated skeletal injury incited by oxidative/ER stress responses.

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Role of SIRT2 in regulating the dexamethasone-activated autophagy pathway in skeletal muscle atrophy

Biochemistry and Cell Biology ◽

10.1139/bcb-2020-0445 ◽

2021 ◽

Author(s):

Ziqiu HAN ◽

Cen CHANG ◽

Weiyi ZHU ◽

Yanlei ZHANG ◽

Jing ZHENG ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Skeletal Muscles ◽

Weight Ratio ◽

Beclin 1 ◽

C2c12 Cells ◽

Skeletal Muscle Atrophy ◽

Time To Exhaustion ◽

Endurance Capacity ◽

Polymerase Chain Reaction Inhibition

The proteolytic autophagy system is involved in a major regulatory pathway in dexamethasone (Dex)-induced muscle atrophy. Sirtuin 2 (SIRT2) is known to participate in modulating autophagy signaling, exerting effects in skeletal muscle atrophy. We aimed to determine the effects of SIRT2 on autophagy in Dex-induced myoatrophy. Mice were randomly divided into the normal, Dex, and sirtinol groups. C2C12 cells were differentiated into myotubes and transfected with short hairpin (sh)-Sirt2-green fluorescent protein (GFP) or Sirt2-GFP lentivirus. To evaluate the mass and function of skeletal muscles, we measured the myofiber cross-sectional area, myotube size, gastrocnemius muscle wet weight/body weight ratio (%), and time-to-exhaustion. The SIRT2, myosin heavy chain (MyHC), LC3, and Beclin-1 expression levels were detected by western blotting and quantitative reverse transcription-polymerase chain reaction. Inhibition of SIRT2 markedly attenuated the muscle mass and endurance capacity. The same phenotype was observed in Sirt2-shRNA-treated myotubes, as evidenced by their decreased size. Conversely, SIRT2 overexpression alleviated Dex-induced myoatrophy in vitro. Moreover, SIRT2 negatively regulated the expression of the LC3b and Beclin-1 in skeletal muscles. These findings suggested that SIRT2 activation protects myotubes against Dex-induced atrophy through the inhibition of the autophagy system; this phenomenon may potentially serve as a target for treating glucocorticoid-induced myopathy.

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Zfp422 promotes skeletal muscle differentiation by regulating EphA7 to induce appropriate myoblast apoptosis

Cell Death and Differentiation ◽

10.1038/s41418-019-0448-9 ◽

2019 ◽

Vol 27 (5) ◽

pp. 1644-1659 ◽

Cited By ~ 1

Author(s):

Yaping Nie ◽

Shufang Cai ◽

Renqiang Yuan ◽

Suying Ding ◽

Xumeng Zhang ◽

...

Keyword(s):

Skeletal Muscle ◽

Zinc Finger ◽

Muscle Development ◽

Zinc Finger Protein ◽

Critical Role ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Myoblast Differentiation ◽

Skeletal Muscle Differentiation ◽

Finger Protein

Abstract Zinc finger protein 422 (Zfp422) is a widely expressed zinc finger protein that serves as a transcriptional factor to regulate downstream gene expression, but until now, little is known about its roles in myogenesis. We found here that Zfp422 plays a critical role in skeletal muscle development and regeneration. It highly expresses in mouse skeletal muscle during embryonic development. Specific knockout of Zfp422 in skeletal muscle impaired embryonic muscle formation. Satellite cell-specific Zfp422 deletion severely inhibited muscle regeneration. Myoblast differentiation and myotube formation were suppressed in Zfp422-deleted C2C12 cells, isolated primary myoblasts, and satellite cells. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) revealed that Zfp422 regulated ephrin type-A receptor 7 (EphA7) expression by binding an upstream 169-bp DNA sequence, which was proved to be an enhancer of EphA7. Knocking EphA7 down in C2C12 cells or deleting Zfp422 in myoblasts will inhibit cell apoptosis which is required for myoblast differentiation. These results indicate that Zfp422 is essential for skeletal muscle differentiation and fusion, through regulating EphA7 expression to maintain proper apoptosis.

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Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice

eLife ◽

10.7554/elife.17462 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 22

Author(s):

Shinichiro Hayashi ◽

Ichiro Manabe ◽

Yumi Suzuki ◽

Frédéric Relaix ◽

Yumiko Oishi

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Myogenic Differentiation ◽

Muscle Differentiation ◽

Skeletal Muscle Regeneration ◽

Biological Processes ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

Acting In Concert

Krüppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2.

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