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The ubiquitin-proteasome system is a major protein degradation pathway in the cell. Proteasomes produce several peptides that are rapidly degraded to free amino acids by intracellular aminopeptidases. Our previous studies reported that proteolysis via proteasomes and aminopeptidases is required for myoblast proliferation and differentiation. However, the role of intracellular aminopeptidases in myoblast proliferation and differentiation had not been clarified. In this study, we investigated the effects of puromycin-sensitive aminopeptidase (PSA) on C2C12 myoblast proliferation and differentiation by knocking down PSA. Aminopeptidase enzymatic activity was reduced in PSA-knockdown myoblasts. Knockdown of PSA induced impaired cell cycle progression in C2C12 myoblasts and accumulation of cells at the G2/M phase. Additionally, after the induction of myogenic differentiation in PSA-knockdown myoblasts, multinucleated circular-shaped myotubes with impaired cell polarity were frequently identified. Cell division cycle 42 (CDC42) knockdown in myoblasts resulted in a loss of cell polarity and the formation of multinucleated circular-shaped myotubes, which were similar to PSA-knockdown myoblasts. These data suggest that PSA is required for the proliferation of myoblasts in the growth phase and for the determination of cell polarity and elongation of myotubes in the differentiation phase., (© 2020 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals LLC.)

Recognized as a novel and important gasotransmitter, hydrogen sulfide (H 2 S) is widely present in various tissues and organs. Cystathionine gamma-lyase (CSE)-derived H 2 S has been shown to regulate oxidative stress and lipid metabolism. The aim of the present study is to examine the role of H 2 S in ferroptosis and lipid peroxidation in mouse myoblasts and skeletal muscles. Ferroptosis agonist RSL3 inhibited the expressions of Gpx4 and reduced CSE/H 2 S signaling, which lead to increased oxidative stress, lipid peroxidation, and ferroptotic cell death. In addition, ferroptosis antagonist ferrostatin-1 (Fer-1) up-regulated the expression of CSE, scavenged the generation of reactive oxygen species (ROS) and lipid peroxidation, and improved cell viability. Exogenously applied NaHS was also able to block RSL3-induced ferroptotic cell death. Neither RSL3 nor H 2 S affected cell apoptosis. Furthermore, H 2 S reversed RSL3-induced Drp1 expression and mitochondrial damage, which lead to abnormal lipid metabolism as evidenced by altered expressions of ACSL4, FAS, ACC and CPT1 as well as higher acetyl-CoA contents in both cytoplasm and mitochondria. RSL3 promoted the protein expression and acetylation of ALOX12, a key protein in initiating membrane phospholipid oxidation, while the addition of NaHS attenuated ALOX12 acetylation and protected from membrane lipid peroxidation. Moreover, we observed that CSE deficiency alters the expressions of ferroptosis and lipid peroxidation-related proteins and enhances global protein acetylation in mouse skeletal muscles under aging or injury conditions. These results indicate that downregulation of CSE/H 2 S signaling would contribute to mitochondrial damage, abnormal lipid metabolism, membrane lipid peroxidation, and ferroptotic cell death. CSE/H 2 S system can be a target for preventing ferroptosis in skeletal muscle., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD + ) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD + , mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H 2 O 2 -induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H 2 O 2 -triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD + -mediated mitochondrial-nuclear crosstalk., Competing Interests: Declaration of Interests The authors declare no financial interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A large number of intracellular proteins are degraded by the ubiquitin-proteasome system, one of the major protein degradation pathways. It produces peptides of several different sizes through protein degradation, and these peptides are rapidly degraded into free amino acids by various intracellular aminopeptidases. Previously, we reported that the activity of proteasomes and aminopeptidases in the proteolysis pathway are necessary for myoblast proliferation and differentiation. However, the detailed function of intracellular aminopeptidases in myoblast proliferation and differentiation has not yet been elucidated. In this study, we focused on alanine aminopeptidase (APN) and investigated the function of APN in C2C12 myoblast proliferation and differentiation. In myoblasts and myotubes, APN was mainly localized in the cell membrane as well as expressed at low levels in the cytoplasm and nucleus. The reduction of the APN enzymatic activity impaired the cell cycle progression in C2C12 myoblasts. In addition, apoptosis was induced after APN-knockdown. Finally, myogenic differentiation was also delayed in the APN-suppressed myoblasts. These findings indicate that APN is required for myoblast proliferation and differentiation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Msh homeobox (Msx) is a subclass of homeobox transcriptional regulators that control cell lineage development, including the early stage of vertebrate limb development, although the underlying mechanisms are not clear. Here, we demonstrate that Msx1 promotes the proliferation of myoblasts and mesenchymal stem cells (MSCs) by enhancing mitogen-activated protein kinase (MAPK) signaling. Msx1 directly binds to and upregulates the expression of fibroblast growth factor 9 (Fgf9) and Fgf18. Accordingly, knockdown or antibody neutralization of Fgf9/18 inhibits Msx1-activated extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. Mechanistically, we determined that the phosphorylation of Msx1 at Ser136 is critical for enhancing Fgf9 and Fgf18 expression and cell proliferation, and cyclin-dependent kinase 1 (CDK1) is apparently responsible for Ser136 phosphorylation. Furthermore, mesenchymal deletion of Msx1/2 results in decreased Fgf9 and Fgf18 expression and Erk1/2 phosphorylation, which leads to serious defects in limb development in mice. Collectively, our findings established an important function of the Msx1-Fgf-MAPK signaling axis in promoting cell proliferation, thus providing a new mechanistic insight into limb development., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent findings suggest a pathologic role of skeletal muscle in amyotrophic lateral sclerosis (ALS) onset and progression. However, the exact mechanism by which this occurs remains elusive due to limited human-based studies. To this end, phenotypic ALS skeletal muscle models were developed from induced pluripotent stem cells (iPSCs) derived from healthy individuals (WT) and ALS patients harboring mutations in the superoxide dismutase 1 (SOD1) gene. Although proliferative, SOD1 myoblasts demonstrated delayed and reduced fusion efficiency compared to WT. Additionally, SOD1 myotubes exhibited significantly reduced length and cross-section. Also, SOD1 myotubes had loosely arranged myosin heavy chain and reduced acetylcholine receptor expression per immunocytochemical analysis. Functional analysis indicated considerably reduced contractile force and synchrony in SOD1 myotubes. Mitochondrial assessment indicated reduced inner mitochondrial membrane potential (ΔΨm) and metabolic plasticity in the SOD1-iPSC derived myotubes. This work presents the first well-characterized in vitro iPSC-derived muscle model that demonstrates SOD1 toxicity effects on human muscle regeneration, contractility and metabolic function in ALS. Current findings align with previous ALS patient biopsy studies and suggest an active contribution of skeletal muscle in NMJ dysfunction. Further, the results validate this model as a human-relevant platform for ALS research and drug discovery studies.

Caspase-2, -9, and -3 are reported to control myoblast differentiation into myotubes. This had been previously explained by phosphatidylserine exposure on apoptotic myoblasts inducing differentiation in neighboring cells. Here we show for the first time that caspase-3 is activated in the myoblasts undergoing differentiation. Using RNAi, we also demonstrate that differentiation requires both cytochrome c and Apaf-1, and by using a new pharmacological approach, we show that apoptosome formation is required. We also show that Bid, whose cleavage links caspase-2 to the mitochondrial death pathway, was required for differentiation, and that the caspase cleavage product, tBid, was generated during differentiation. Taken together, these data suggest that myoblast differentiation requires caspase-2 activation of the mitochondrial death pathway, and that this occurs in the cells that differentiate. Our data also reveal a hierarchy of caspases in differentiation with caspase-2 upstream of apoptosome activation, and exerting a more profound control of differentiation, while caspases downstream of the apoptosome primarily control cell fusion.

The ubiquitin-proteasome pathway is essential for skeletal muscle growth and development. Proteasomes generate oligopeptides in the cytoplasm, and these peptides are considered to be rapidly degraded to amino acids by several intracellular aminopeptidases. However, the role of intracellular aminopeptidases in muscle growth remains unknown. In this study, therefore, we investigated the role of intracellular aminopeptidases in C2C12 myoblast proliferation and differentiation. Inhibition of intracellular aminopeptidases by Bestatin methyl ester (Bes-ME) decreased leucine and alanine aminopeptidase activity, and impaired proliferation and differentiation of C2C12 myoblasts. Furthermore, we observed that the inhibition of intracellular aminopeptidases reduced intracellular levels of amino acid and ATP level, and suppressed the phosphorylation of the mTOR pathway. These results suggested that intracellular aminopeptidases affect C2C12 myoblast proliferation and differentiation via mTOR pathway; however, further studies are required to clarify the role of aminopeptidase in skeletal muscle., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Whether the histone deacetylase (HDAC) and sirtuin families of protein deacetylases regulate insulin-stimulated glucose uptake, independent of their transcriptional effects, has not been studied. Our objective was to determine the nontranscriptional role of HDACs and sirtuins in regulation of skeletal muscle insulin action. Basal and insulin-stimulated glucose uptake and signaling and acetylation were assessed in L6 myotubes and skeletal muscle from C57BL/6J mice that were treated acutely (1 h) with HDAC (trichostatin A, panobinostat, TMP195) and sirtuin inhibitors (nicotinamide). Treatment of L6 myotubes with HDAC inhibitors or skeletal muscle with a combination of HDAC and sirtuin inhibitors increased tubulin and pan-protein acetylation, demonstrating effective impairment of HDAC and sirtuin deacetylase activities. Despite this, neither basal nor insulin-stimulated glucose uptake or insulin signaling was impacted. Acute reduction of the deacetylase activity of HDACs and/or sirtuins does not impact insulin action in skeletal muscle.

Adult skeletal muscle regeneration after injury depends on normal myoblast function. However, the intrinsic mechanisms for the control of myoblast behaviors are not well defined. Herein, we identified Pim1 kinase as a novel positive regulator of myoblast behaviors in vitro and muscle regeneration in vivo. Specifically, knockdown of Pim1 significantly restrains the proliferation and accelerates the apoptosis of myoblasts in vitro, indicating that Pim1 is critical for myoblast survival and amplification. Meanwhile, we found that Pim1 kinase is increased and translocated from cytoplasm into nucleus during myogenic differentiation. By using Pim1 kinase inhibitor, we proved that inhibition of Pim1 activity prevents myoblast differentiation and fusion, suggesting the necessity of Pim1 kinase activity for proper myogenesis. Mechanistic studies demonstrated that Pim1 kinase interacts with myogenic regulator MyoD and controls its transcriptional activity, inducing the expression of muscle-specific genes, which consequently promotes myogenic differentiation. Additionally, in skeletal muscle injury mouse model, deletion of Pim1 hinders the regeneration of muscle fibers and the recovery of muscle strength. Taken together, our study provides a potential target for the manipulation of myoblast behaviors in vitro and the myoblast-based therapeutics of skeletal muscle injury.

Objective: To apply hypoxia of different oxygen concentration on C2C12 cells to study the changes of Nrf2 antioxidant system under H 2 O 2 ., Methods: The perfect simulative effect time and concentration of H 2 O 2 were chosen. Cell vitality was tested after C2C12 cells cultured in 0.1 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 0.75 mmol/L, 1 mmol/L and 2 mmol/L H 2 O 2 for 1 or 2 h respectively. The C2C12 cells were divided into different oxygen concentration group: 21%O 2 , 12%O 2 , 8%O 2 , 5%O 2 respectively. And then cells were treated with H 2 O 2 for 1 h, and collected for determination. Immunofluorescence of Nrf2 and the protein expression of Nrf2 were detected. The expressions of antioxidant enzymes superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase(CAT), NADPH quinine oxidoreductase-1 (NQO-1), glutathione peroxidase-1 (GPX-1), Heme oxygenase-1 (HO-1) mRNA and cellular ROS levels were tested by high quality fluorescence assay., Results: 0.5 mmol/L H 2 O 2 for 1 h was selected as the conditions of H 2 O 2 stimulation. Compared with 21% O 2 group, the expressions of Nrf2 mRNA and protein, antioxidant enzymes SOD1, SOD2, CAT, HO-1, NQO-1, GPX-1 mRNA were increased significantly (P＜0.05 or P＜0.01), and ROS level was lower (P＜0.01) in 12%O 2 group cells; only the expression of GPX-1 mRNA was increased (P＜0.05) in 8%O 2 group; the expressions of Nrf2 mRNA and protein expression, antioxidant enzymes SOD1, SOD2, NQO-1, GPX-1 mRNA were decreased significantly(P＜0.05 or P＜0.01), and ROS level was higher (P＜0.01) in 5%O 2 group., Conclusion: Hypoxia can affect the Nrf2 antioxidant system, and the different oxygen concentrations have different impact. In addition, 12% O 2 for 12 h could promote the Nrf2 antioxidant system, and 5% extremely low oxygen may inhibit it.

Neuronal nitric oxide synthase (nNOS) plays a crucial role in the maintenance of correct skeletal muscle function due, at least in part, to S-nitrosylation of specific protein targets. Similarly, we recently provided evidence for a muscular phenotype in mice lacking the denitrosylase S-nitrosoglutathione reductase (GSNOR). Here, we demonstrate that nNOS and GSNOR are concomitantly expressed during differentiation of C2C12. They colocalizes at the sarcolemma and co-immunoprecipitate in cells and in myofibers. We also provide evidence that GSNOR expression decreases in mouse models of muscular dystrophies and of muscle atrophy and wasting, i.e., aging and amyotrophic lateral sclerosis, suggesting a more general regulatory role of GSNOR in skeletal muscle homeostasis.

Mechanical overloading can lead to skeletal muscle damage instead of remodeling. This is attributed to the excessive apoptosis of myoblasts, mechanism of which remains to be elucidated. The present study aimed to investigate the involvement of endoplasmic reticulum stress (ERS) and caspase-12 in mediating the stretch-induced apoptosis of myoblasts. Myoblast apoptosis was evaluated by Hoechst staining, DNA fragmentation assay, Annexin V binding, and propidium iodide staining, as well as caspase-3 and poly-ADP-ribose polymerase 1 cleavage. First, our results showed that apoptosis was elevated in a time-dependent manner when myoblasts were subjected to cyclic mechanical stretch (CMS) for 12, 24, and 36 hr. Concomitantly, CMS triggered the ERS and caspase-12 cleavage; ERS inhibitor GSK 2606414 suppressed the CMS-induced cleavage of caspase-12 and myoblast apoptosis. Silencing caspase-12 attenuated the apoptosis of myoblasts under CMS. Furthermore, CMS-induced myoblast apoptosis was partially recovered by overexpressing wild-type caspase-12 in caspase-12-silenced myoblasts. In contrast, overexpressing mutant caspase-12 (D94N), which cannot be cleaved into the active caspase-12 fragments, failed to accomplish the same effect. Finally, C2C12 overexpressing truncated caspase-12 segment (TC-casp12-D94), which starts from Asp94 and ends at Asn419, underwent apoptosis under both static and stretched conditions. Interestingly, C2C12 myoblasts seemed to be resistant to stretch-induced apoptosis upon low-serum-induced differentiation. In conclusion, our study provided evidence that caspase-12 cleavage at Asp94, induced by ERS under mechanical stimuli, is the key molecule in initiating the stretch-triggered apoptosis of myoblasts., (© 2018 Wiley Periodicals, Inc.)

Bisphenol A (BPA), one of the most widespread endocrine disrupting chemicals, is known as an artificial estrogen, which interacts with estrogen receptor (ER). In this study, we investigated the effects of BPA and estradiol on myoblast differentiation and the underlying signaling mechanism. Exposure to BPA (0.01-1 μM) in mouse myoblast C2C12 cells attenuated myogenic differentiation via the reduced expression of muscle-specific genes, such as myosin heavy chain (MHC), MyoD, and Myogenin, without the alteration of cell proliferation and viability. BPA-exposed C2C12 myoblasts also showed a reduction of Akt phosphorylation ((37-61) %, p < 0.001), a key event for myogenesis. Similarly to BPA, estradiol (0.01-1 μM) reduced the expression of muscle-specific proteins and the formation of multinucleated myotubes, and attenuated the muscle differentiation-specific phosphorylation of Akt ((42-59) %, p < 0.001). We conclude that BPA and estradiol suppress myogenic differentiation through the inhibition of Akt signaling., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Myoblast proliferation is crucial to skeletal muscle hypertrophy and regeneration. Our previous study indicated that mechanical stretch altered the proliferation of C2C12 myoblasts, associated with insulin growth factor 1 (IGF-1)-mediated phosphoinositide 3-kinase (PI3K)/Akt (also known as protein kinase B) and mitogen-activated protein kinase (MAPK) pathways through IGF-1 receptor (IGF-1R). The purpose of this study was to explore the same stretches on the proliferation of L6 myoblasts and its association with IGF-1-regulated PI3K/Akt and MAPK activations. L6 myoblasts were divided into three groups: control, 15% stretch, and 20% stretch. Stretches were achieved using FlexCell Strain Unit. Cell proliferation and IGF-1 concentration were detected by CCK8 and ELISA, respectively. IGF-1R expression, and expressions and activities of PI3K, Akt, and MAPKs (including extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38) were determined by Western blot. We found that 15% stretch promoted, while 20% stretch inhibited L6 myoblast proliferation. A 15% stretch increased IGF-1R level, although had no effect on IGF-1 secretion of L6 myoblasts, and PI3K/Akt and ERK1/2 (not p38) inhibitors attenuated 15% stretch-induced pro-proliferation. Exogenous IGF-1 reversed 20% stretch-induced anti-proliferation, accompanied with increases in IGF-1R level as well as PI3K/Akt and MAPK (ERK1/2 and p38) activations. In conclusion, stretch regulated L6 myoblasts proliferation, which may be mediated by the changes in PI3K/Akt and MAPK activations regulated by IGF-1R, despite no detectable IGF-1 from stretched L6 myoblasts.

Background: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context., Methods: By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3'UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS., Results: TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3'UTR-luciferase assay, confirming their interaction with the NOS1-3'UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts., Conclusion: These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle.

Fibrosis is a common feature of several chronic diseases and is characterized by exacerbated accumulation of ECM. An understanding of the cellular and molecular mechanisms involved in the development of this condition is crucial for designing efficient treatments for those pathologies. Connective tissue growth factor (CTGF/CCN2) is a pleiotropic protein with strong profibrotic activity. In this report, we present experimental evidence showing that ECM stimulates the synthesis of CTGF in response to lysophosphatidic acid (LPA).The integrin/focal adhesion kinase (FAK) signaling pathway mediates this effect, since CTGF expression is abolished by the use of the Arg-Gly-Asp-Ser peptide and also by an inhibitor of FAK autophosphorylation at tyrosine 397. Cilengitide, a specific inhibitor of αv integrins, inhibits the expression of CTGF mediated by LPA or transforming growth factor β1. We show that ECM obtained from decellularized myofibroblast cultures or derived from activated fibroblasts from muscles of the Duchenne muscular dystrophy mouse model ( mdx) induces the expression of CTGF. This effect is dependent on FAK phosphorylation in response to its activation by integrin. We also found that the fibrotic ECM inhibits skeletal muscle differentiation. This novel regulatory mechanism of CTGF expression could be acting as a positive profibrotic feedback between the ECM and CTGF, revealing a novel concept in the control of fibrosis under chronic damage.

Mammalian target of rapamycin (mTOR) signaling controls skeletal muscle cell differentiation, growth, and metabolism by sensing the intracellular energy status and nutrients. Recently, leucyl-tRNA synthetase (Lars) was identified as an intracellular sensor of leucine involved in the activation of mTOR signaling. However, there is still no evidence for the activation of mTOR signaling by Lars and its physiological roles in skeletal muscle cells. In this study, we determined the potential roles of Lars for the activation of mTOR signaling, skeletal muscle cell differentiation, hypertrophy, and metabolism using small interfering (si)-RNA knockdown. siRNA-mediated knockdown of Lars decreased phosphorylated p70 S6 kinase and inhibited the differentiation of C2C12 mouse myoblasts into myotubes, as evidenced by a decreased fusion index and decreased mRNA and protein expression levels of myogenic markers. Importantly, si-Lars decreased the level of Insulin-like growth factor 2 (Igf2) mRNA expression from the early stages of differentiation, indicating the possibility of an association between the mTOR-IGF2 axis and Lars. However, Lars knockdown did not decrease phosphorylated mTOR in differentiated myotubes, nor did it affect the hypertrophy of myotubes as evidenced by measuring their diameters and detecting the mRNA and protein expression of hypertrophy markers. Similarly, an extracellular flux analyzer showed that Lars knockdown did not affect the metabolism (glycolysis and mitochondrial respiration) of myotubes. These results demonstrate that Lars is required for skeletal muscle differentiation through the activation of mTOR signaling, but not for hypertrophy or metabolic alteration of myotubes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In Duchenne muscular dystrophy (DMD), lack of dystrophin leads to progressive muscle degeneration, with DMD patients suffering from cardiorespiratory failure. Cell therapy is an alternative to life-long corticoid therapy. Satellite cells, the stem cells of skeletal muscles, do not completely compensate for the muscle damage in dystrophic muscles. Elevated levels of proinflammatory and profibrotic factors, such as metalloproteinase 9 (MMP-9), impair muscle regeneration, leading to extensive fibrosis and poor results with myoblast transplantation therapies. Omega-3 is an anti-inflammatory drug that protects against muscle degeneration in the mdx mouse model of DMD. In the present study, we test our hypothesis that omega-3 affects MMP-9 and thereby benefits muscle regeneration and myoblast transplantation in the mdx mouse. We observe that omega-3 reduces MMP-9 gene expression and improves myoblast engraftment, satellite cell activation, and muscle regeneration by mechanisms involving, at least in part, the regulation of macrophages, as shown here with the fluorescence-activated cell sorting technique. The present study demonstrates the benefits of omega-3 on satellite cell survival and muscle regeneration, further supporting its use in clinical trials and cell therapies in DMD.

Objects: To detect the effects of androgen receptor (AR) on cyclic mechanical stretch-modulated proliferation of C2C12 myoblasts and its pathways: roles of IGF-1, PI3K and MAPK., Methods: C2C12 were randomly divided into five groups: un-stretched control, six or 8 h of fifteen percent stretch, and six or 8 h of twenty percent stretch. Cyclic mechanical stretch of C2C12 were completed using a computer-controlled FlexCell Strain Unit. Cell proliferation and IGF-1 concentration in medium were detected by CCK8 and ELISA, respectively. Expressions of AR and IGF-1R, and expressions and activities of PI3K, p38 and ERK1/2 in stretched C2C12 cells were determined by Western blot., Results: ①The proliferation of C2C12 cells, IGF-1 concentration in medium, expressions of AR and IGF-1R, and activities of PI3K, p38 and ERK1/2 were increased by 6 h of fifteen percent stretch, while decreased by twenty percent stretch for six or 8 h ②The fifteen percent stretch-increased proliferation of C2C12 cells was reversed by AR inhibitor, Flutamide. ③The increases of AR expression, activities of PI3K, p38 and ERK1/2 resulted from fifteen percent stretch were attenuated by IGF-1 neutralizing antibody, while twenty percent stretch-induced decreases of the above indicators were enhanced by recombinant IGF-1. ④Specific inhibitors of p38, ERK1/2 and PI3K all decreased the expression of AR in fifteen percent and twenty percent of stretched C2C12 cells., Conclusions: Cyclic mechanical stretch modulated the proliferation of C2C12 cells, which may be attributed to the alterations of AR via IGF-1-PI3K/Akt and IGF-1-MAPK (p38, ERK1/2) pathways in C2C12 cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Obstructive sleep apnea and hypopnea syndrome (OSAHS) is a clinical syndrome characterized by recurrent episodes of obstruction of the upper airway during sleep that leads to a hypoxic condition. Genioglossus, an important pharyngeal muscle, plays an important role in maintaining an open upper airway for effective breathing. Our previous study found that genistein (a kind of phytoestrogen) protects genioglossus muscle from hypoxia-induced oxidative injury. However, the underlying mechanism is still unknown. In the present study, we examined the effects of hypoxia on genioglossus myoblast proliferation, viability and apoptosis, and the protective effect of genistein and its relationship with the PI3K/Akt and ERK MAPK pathways. Cell viability and Bcl-2 were reduced under hypoxic condition, while ROS generation, caspase-3, MDA, and DNA damage were increased following a hypoxia exposure. However, the effects of hypoxia were partially reversed by genistein in an Akt- and ERK- (but not estrogen receptor) dependent manner. In conclusion, genistein protects genioglossus myoblasts against hypoxia-induced oxidative injury and apoptosis independent of estrogen receptor. The PI3K-Akt and ERK1/2 MAPK signaling pathways are involved in the antioxidant and anti-apoptosis effect of genistein on genioglossus myoblasts.

Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), berberine (100 μM), DNP (500 μM)], or directly activate AMPK [AICAR (250 μM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 μM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 μM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role., (Copyright © 2017 the American Physiological Society.)

S100 calcium-binding protein B (S100B), a multifunctional macromolecule mainly expressed in nerve tissues and adipocytes, has been suggested to contribute to the pathogenesis of obesity. To clarify the role of S100B in insulin action and glucose metabolism in peripheral tissues, we investigated the effect of S100B on glycolysis in myoblast and myotube cells. Rat myoblast L6 cells were treated with recombinant mouse S100B to examine glucose consumption, lactate production, glycogen accumulation, glycolytic metabolites and enzyme activity, insulin signaling, and poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Glycolytic metabolites were investigated by enzyme assays or metabolome analysis, and insulin signaling was assessed by Western blot analysis. Enzyme activity and poly(ADP-ribosyl)ation of GAPDH was evaluated by an enzyme assay and immunoprecipitation followed by dot blot with an anti-poly(ADP-ribose) antibody, respectively. S100B significantly decreased glucose consumption, glucose analog uptake, and lactate production in L6 cells, in either the presence or absence of insulin. In contrast, S100B had no effect on glycogen accumulation and insulin signaling. Metabolome analysis revealed that S100B increased the concentration of glycolytic intermediates upstream of GAPDH. S100B impaired GAPDH activity and increased poly(ADP-ribosyl)ated GAPDH proteins. The effects of S100B on glucose metabolism were mostly canceled by a poly(ADP-ribose) polymerase inhibitor. Similar results were obtained in C 2 C 12 myotube cells. We conclude that S100B as a humoral factor may impair glycolysis in muscle cells independent of insulin action, and the effect may be attributed to the inhibition of GAPDH activity from enhanced poly(ADP-ribosyl)ation of the enzyme., (Copyright © 2017 the American Physiological Society.)

The role of cullin E3-ubiquitin ligases for muscle homeostasis is best known during muscle atrophy, as the cullin-1 substrate adaptor atrogin-1 is among the most well-characterized muscle atrogins. We investigated whether cullin activity was also crucial during terminal myoblast differentiation and aggregation of acetylcholine receptors for the establishment of neuromuscular junctions in vitro. The activity of cullin E3-ligases is modulated through post-translational modification with the small ubiquitin-like modifier nedd8. Using either the Nae1 inhibitor MLN4924 (Pevonedistat) or siRNA against nedd8 in early or late stages of differentiation on C2C12 myoblasts, and primary satellite cells from mouse and human, we show that cullin E3-ligase activity is necessary for each step of the muscle cell differentiation program in vitro. We further investigate known transcriptional repressors for terminal muscle differentiation, namely ZBTB38, Bhlhe41, and Id1. Due to their identified roles for terminal muscle differentiation, we hypothesize that the accumulation of these potential cullin E3-ligase substrates may be partially responsible for the observed phenotype. MLN4924 is currently undergoing clinical trials in cancer patients, and our experiments highlight concerns on the homeostasis and regenerative capacity of muscles in these patients who often experience cachexia., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Reduced Na + -K + -ATPase activity has close relationship with cardiomyocyte death. Reactive oxygen species (ROS) also plays an important role in cardiac cell damage. It has been proved that Na + -K + -ATPase and ROS form a feed-forward amplifier. The aim of this study was to explore whether DRm217, a proved Na + /K + -ATPase's DR-region specific monoclonal antibody and direct activator, could disrupt Na + -K + -ATPase/ROS amplifier and protect cardiac cells from ROS-induced injury. We found that DRm217 protected myocardial cells against hydrogen peroxide (H 2 O 2 )-induced cardiac cell injury and mitochondrial dysfunction. DRm217 also alleviated the effect of H 2 O 2 on inhibition of Na + -K + -ATPase activity, Na + -K + -ATPase cell surface expression, and Src phosphorylation. H 2 O 2 -treatment increased intracellular ROS, mitochondrial ROS and induced intracellular Ca 2+ , mitochondrial Ca 2+ overload. DRm217 closed Na + -K + -ATPase/ROS amplifier, alleviated Ca 2+ accumulation and finally inhibited ROS and mitochondrial ROS generation. These novel results may help us to understand the important role of the Na + -K + -ATPase in oxidative stress and oxidative stress-related disease.

Background: Prolylcarboxypeptidase (PRCP, EC:3.4.16.2) is a cardioprotective protease. Plasma PRCP levels are elevated in type 2 diabetes (T2D) mellitus and cardiovascular diseases., Objective: Since diabetic cardiomyopathy is a late complication of uncontrolled diabetes, we tested the hypothesis that glucose and free fatty acid related risk factors for T2D mellitus and cardiovascular disease may reduce the cardioprotective property of PRCP., Method: We examined the effects of glucose, saturated fatty acids, and unsaturated fatty acids on PRCP expression in cultured H9c2 cells as an in-vitro model for pharmacological studies. Selective inhibitors, known cardioprotective agents and saturating amounts of neutralizing antibodies were used to validate the effect of free fatty acids on the expression and function of PRCP., Results: The palmitate-mediated reduction of PRCP was concentration and time-dependent. Next, we explored the cardioprotective potential of thyroxin (T4) and insulin. Both T4 and insulin were able to prevent the palmitate-mediated reduction of PRCP expression in H9c2 cells. Inhibition of NF-kB with its specific inhibitor Bay 11-7082 or blockade of palmitate with polyunsaturated fatty acids was ineffective in preventing palmitate-mediated decreases in PRCP expression., Conclusion: Our data indicate that elevated palmitate inhibits PRCP expression in rat cardiomyocyte. From this inference PRCP level should be monitored in obese or diabetic patients because this simple measure could identify individuals at high risk of developing health problems, such as heart failure., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Differentiation of myoblasts into myotubes is essential for skeletal muscle development and regeneration. Caspase-3 and caspase-9 are required for efficient myoblast differentiation. The caspase-activated endonuclease activity, CAD, and the DNA-damage repair protein XRCC1 have also been shown to be required to complete differentiation. DNA-damage associated with differentiation is accompanied by phosphorylation of Histone 2AX, an event normally catalysed by kinases ATR, ATM or DNA-PK. However, the kinase responsible for phosphorylation during differentiation is not known. Here we show that inhibition of DNA-PK, but not of ATR or ATM, blocked histone phosphorylation during differentiation. We also show that DNA-PK inhibition and siRNA-mediated DNA-PK knockdown blocked cell fusion. These data implicate a new role for DNA-PK in myogenic differentiation., (© 2016 Federation of European Biochemical Societies.)

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor of intracellular energy. Activation of AMPK is associated with increased phosphorylation of the α-subunit at threonine 172 (T172) and decreased phosphorylation at serine 485 in AMPKα1 and serine 491 in AMPKα2 (S485/491). One potential mediator of AMPK phosphorylation is phosphatidylinositol 3-kinase (PI3K); however, the mechanism and the identities of the specific PI3K isoforms that regulate AMPK activation are not known. To determine whether PI3K p110α regulated AMPK activation in muscle cells, C2C12 myoblasts were subjected to pharmacological inhibition of p110α, siRNA directed against p110α, or overexpression of constitutively-active or dominant negative p110α. Chemical inhibition, siRNA, and expression of dominant-negative p110α were all associated with increased AMPK T172 phosphorylation, whereas expression of constitutively-active p110α reduced T172 phosphorylation. Conversely, pharmacological inhibition of p110α reduced AMPK S485/491 phosphorylation, while constitutively-active p110α increased AMPK S485/491 phosphorylation. This p110α-mediated increase in AMPK S485/491 phosphorylation was eliminated in the presence of the Akt inhibitor MK2206, suggesting that p110α-mediated phosphorylation of AMPKα at S485/491 is Akt-dependent. In response to oligomycin or serum-starvation, AMPK T172 phosphorylation was elevated in p110α-deficient myoblasts compared to control myoblasts. Overall, our findings identify PI3K p110α as a mediator of AMPK phosphorylation in myoblasts., (Published by Elsevier Inc.)

Chronic inflammation augments the deleterious effects of several diseases, particularly diabetes, cancer, and sepsis. It is also involved in the process of metabolic shift from glucose oxidation to lactate production. Although several studies suggest that the change in activity of the pyruvate dehydrogenase complex (PDC) is a major factor causing this metabolic change, the exact mechanism of the inflammatory state remains unclear. In this study, we investigated the effect of lipopolysaccharide (LPS) on the expression of pyruvate dehydrogenase kinase 4 (PDK4), which is strongly associated with inactivation of the PDC in C2C12 myoblasts. In C2C12 myoblasts, LPS exposure led to increased PDK4 mRNA and protein expression levels as well as lactate production in culture medium. However, the expression levels of other PDK isoenzymes (PDK1 - 3) remained unchanged. Additionally, we observed that LPS treatment induced phosphorylation of Jun N-Terminal Kinases (JNK). To confirm the role of JNK, we inhibited the JNK pathway and observed that PDK4 expression and lactate production were decreased, but p38 and ERK were not significantly changed. Taken together, our results suggest that LPS induces PDK4 expression and alters glucose metabolism via the JNK pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

PI-PLCbeta1 plays an important role in cell differentiation, and particularly in myogenesis, osteogenesis and hematopoiesis. Indeed, the increase of PI-PLCbeta1, along with Cyclin D3, has been detected in C2C12 mouse myoblasts induced to differentiate, as well as in human cells obtained from myotonic dystrophy. Also in the case of osteogenic differentiation there is a specific induction of PI-PLCbeta1, but in this case the role of PI-PLCbeta1 seems to be independent from Cyclin D3, so that a different mechanism could be involved. As for the hematopoietic system, PI-PLCbeta1 has a peculiar behavior: it increases during myeloid differentiation and decreases during erythroid differentiation, thus confirming the role of PI-PLCbeta1 as a modulator of hematopoiesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: The purpose of this study is to explore whether antioxidant DJ-1 protein affects the atrophy of skeletal muscle cell induced by undernutrition., Main Methods: To determine cell atrophic responses, L6 cell line and skeletal primary cells from mouse hind limbs were cultivated under condition of FBS-free and low glucose. Changes of protein expression were analyzed using Western blot. Overexpression and knockdown of DJ-1 was performed in cells to assess its influence on cell atrophic responses., Key Findings: Undernutrition decreased cell size and increased the abundance of oxidized form and total form of DJ-1 protein in L6 myoblasts. The undernourished cells revealed an elevation in the expression of muscle-specific RING finger-1 (MuRF-1) and atrogin-1, and in the phosphorylations of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase compared with control groups. Moreover, DJ-1-knockout mice showed a decrease in cell size and an enhancement in the expression of MuRF-1 and atrogin-1, as well as in the phosphorylation of MAPKs in gastrocnemius muscles; these changes were also observed in L6 cells transfected with siRNA of DJ-1. On the other hand, L6 cells overexpressing full-length DJ-1 did not exhibit the alterations in cell size and ubiquitin ligases seen after undernourished states of control cells. Myotubes differentiated from L6 cells also showed elevated expression of MuRF-1 and atrogin-1 in response to undernutrition., Significance: These results suggest that DJ-1 protein may contribute to undernutrition-induced atrophy via MAPKs/ubiquitin ligase pathway in skeletal muscle cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Brg1 (Brahma-related gene 1) is a catalytic component of the evolutionarily conserved mammalian SWI/SNF ATP-dependent chromatin remodeling enzymes that disrupt histone-DNA contacts on the nucleosome. While the requirement for the SWI/SNF enzymes in cell differentiation has been extensively studied, its role in precursor cell proliferation and survival is not as well defined. Muscle satellite cells constitute the stem cell pool that sustains and regenerates myofibers in adult skeletal muscle. Here, we show that deletion of Brg1 in primary mouse myoblasts derived from muscle satellite cells cultured ex vivo leads to a cell proliferation defect and apoptosis. We determined that Brg1 regulates cell proliferation and survival by controlling chromatin remodeling and activating transcription at the Pax7 promoter, which is expressed during somite development and is required for controlling viability of the satellite cell population. Reintroduction of catalytically active Brg1 or of Pax7 into Brg1-deficient satellite cells rescued the apoptotic phenotype and restored proliferation. These data demonstrate that Brg1 functions as a positive regulator for cellular proliferation and survival of primary myoblasts. Therefore, the regulation of gene expression through Brg1-mediated chromatin remodeling is critical not just for skeletal muscle differentiation but for maintaining the myoblast population as well., (© 2015 Wiley Periodicals, Inc.)

Low-level laser irradiation (LLLI) is increasingly used to treat musculoskeletal disorders, with satisfactory results described in the literature. Skeletal muscle satellite cells play a key role in muscle regeneration. The aim of the present study was to evaluate the effect of LLLI on cell viability, creatine kinase (CK) activity, and the expression of myogenic regulatory factors in C2C12 myoblasts during the differentiation process. C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum and submitted to irradiation with GaAlAs diode laser (wavelength, 780 nm; output power, 10 mW; energy density, 5 J/cm2). Cell viability and the expression of myogenic regulatory factors were assessed 24, 48, and 72 h after irradiation by 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. CK activity was analyzed at 24 and 72 h. An increase in cell viability was found in the laser group in comparison to the control group at all evaluation times. CK activity was significantly increased in the laser group at 72 h. Myogenin messenger RNA (mRNA) demonstrated a tendency toward an increase in the laser group, but the difference in comparison to the control group was non-significant. In conclusion, LLLI was able to modulate cell viability and CK activity in C2C12 myoblasts during the differentiation process.

Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I-V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists.

The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Poly(ADP-ribose) polymerase 1 (PARP-1), the major isoform of the poly (ADP-ribose) polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose) (PAR) groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12) and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor) exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6). Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant-induced injury. Taken together, our data indicate that the reduction of PARP-1 expression during the process of the skeletal muscle differentiation serves as a protective mechanism to maintain the cellular functions of skeletal muscle during oxidative stress.

While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with KrasG12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic KrasG12D expression. KrasG12D overexpression transformed p53-/- myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53-/- myoblasts transformed by KrasG12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum.

Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis., (Copyright © 2015 the American Physiological Society.)

Toona Sinensis leaf (TSL) extract with a beneficial effect for managing hyperglycemia has been reported, however the underlying mechanism by which TSL extract acts as an insulin sensitizer remains uncertain, especially in peripheral tissues. TSL 95% ethanol extract exhibited the highest transactivity of PPARγ and contained the highest amounts of natural PPARγ ligands including palmitic acid, linoleic acid, and α-linolenic acid among different TSL ethanol extracts (0, 10, 50, 70, and 95%). The efficacy and the mechanism of TSL ethanol extract (95%) mediated anti-diabetic effects were examined by both in vivo and in vitro models in this study. An improved whole-body insulin sensitivity was observed in high-fat diet-fed (HFD) mice after 14 weeks of TSL treatment, as evidenced by a faster rate of plasma glucose clearing. The improved insulin sensitivity was through direct stimulation of PPARγ and adiponectin expression in adipose tissues of HFD mice. In addition to the PPARγ pathway, TSL stimulated glucose uptake via directly inducing AMPKα but not AS160 activation in C2C12 myotubes under palmitate-induced insulin resistance. TSL successfully induced sirtuin 1 and restored PGC1α, but failed to restore mitochondrial electron transport complexes I, III, IV and V in mRNA levels. Loss of the mitochondrial membrane potential coupled with AMPK activation suggests that TSL acts as a mitochondrial inhibitor to stimulate AMPK-mediated glucose uptake. This study demonstrated that TSL stimulated glucose uptake via AMPK activation in skeletal muscles and promoted PPARγ and normalized adiponectin expression in adipose tissues, thereby ameliorating insulin resistance.

Irisin is a novel myokine produced by skeletal muscle. However, its metabolic role is poorly understood. In the present study, irisin induced glucose uptake in differentiated skeletal muscle cells. It increased AMP-activated protein kinase (AMPK) phosphorylation and the inhibition of AMPK blocked glucose uptake. It also increased reactive oxygen species (ROS) generation. N-acetyl cysteine, a ROS scavenger, blocked irisin-induced AMPK phosphorylation. Moreover, irisin activated p38 MAPK in an AMPK-dependent manner. The inhibition and knockdown of p38 MAPK blocked irisin-induced glucose uptake. A colorimetric absorbance assay showed that irisin stimulated the translocation of glucose transporter type 4 to the plasma membrane and that this effect was suppressed in cells pretreated with a p38 MAPK inhibitor or p38 MAPK small interfering RNA. In primary cultured myoblast cells, irisin increased the concentration of intracellular calcium. STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, blocked irisin-induced AMPK phosphorylation, implying that calcium is involved in irisin-mediated signaling. Our results suggest that irisin plays an important role in glucose metabolism via the ROS-mediated AMPK pathway in skeletal muscle cells.

Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32's substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2., (© 2015. Published by The Company of Biologists Ltd.)

The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na+/K+-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na+/K+-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM) and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na+/K+-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na+/K+-ATPase.

Enzymes that mediate posttranslational modifications of histone and nonhistone proteins have been implicated in regulation of skeletal muscle differentiation. However, functions of histone demethylases that could counter the actions of H3-K9 specific histone methyltransferases remain still obscure. Here we present evidences that KDM4B histone demethylase regulates expression of myogenic regulators such as MyoD and thereby controls myogenic differentiation of C2C12 myoblast cells. We demonstrate that expression of KDM4B gradually increases during myogenic differentiation and depletion of KDM4B using shRNA results in inhibition of differentiation in C2C12 myoblast cells, which is correlated with decreased expression of MyoD and myogenin. In addition, we find that KDM4B shRNA represses expression of MyoD promoter-driven luciferase reporter and exogenous expression of MyoD rescues myogenic potential in KDM4B-depleted myoblast cells. We further show that KDM4B interacts with MyoD, binds to MyoD and myogenin promoters in vivo, and finally, is involved in demethylation of tri-methylated H3-K9 on promoters of MyoD and myogenin. Taken together, our data suggest that KDM4B plays key roles in myogenic differentiation of C2C12 cells, presumably by its function as a H3-K9 specific histone demethylase., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rhadomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS cells resemble fetal myoblasts but are unable to complete myogenic differentiation. In previous work we showed that miR-206, which is low in RMS, when induced in RMS cells promotes the resumption of differentiation by modulating more than 700 genes. To better define the pathways involved in the conversion of RMS cells into their differentiated counterpart, we focused on 2 miR-206 effectors emerged from the microarray analysis, SMYD1 and G6PD. SMYD1, one of the most highly upregulated genes, is a H3K4 histone methyltransferase. Here we show that SMYD1 silencing does not interfere with the proliferative block or with the loss anchorage independence imposed by miR-206, but severely impairs differentiation of ERMS, ARMS, and myogenic cells. Thus SMYD1 is essential for the activation of muscle genes. Conversely, among the downregulated genes, we found G6PD, the enzyme catalyzing the rate-limiting step of the pentose phosphate shunt. In this work, we confirmed that G6PD is a direct target of miR-206. Moreover, we showed that G6PD silencing in ERMS cells impairs proliferation and soft agar growth. However, G6PD overexpression does not interfere with the pro-differentiating effect of miR-206, suggesting that G6PD downmodulation contributes to - but is not an absolute requirement for - the tumor suppressive potential of miR-206. Targeting cancer metabolism may enhance differentiation. However, therapeutic inhibition of G6PD is encumbered by side effects. As an alternative, we used DCA in combination with miR-206 to increase the flux of pyruvate into the mitochondrion by reactivating PDH. DCA enhanced the inhibition of RMS cell growth induced by miR-206, and sustained it upon miR-206 de-induction. Altogether these results link miR-206 to epigenetic and metabolic reprogramming, and suggest that it may be worth combining differentiation-inducing with metabolism-directed approaches.

In muscle and other mechanically active tissue, cell membranes are constantly injured, and their repair depends on the injury-induced increase in cytosolic calcium. Here, we show that injury-triggered Ca(2+) increase results in assembly of ESCRT III and accessory proteins at the site of repair. This process is initiated by the calcium-binding protein-apoptosis-linked gene (ALG)-2. ALG-2 facilitates accumulation of ALG-2-interacting protein X (ALIX), ESCRT III and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane. Lack of ALG-2, ALIX or Vps4B each prevents shedding, and repair of the injured cell membrane. These results demonstrate Ca(2+)-dependent accumulation of ESCRT III-Vps4 complex following large focal injury to the cell membrane and identify the role of ALG-2 as the initiator of sequential ESCRT III-Vps4 complex assembly that facilitates scission and repair of the injured cell membrane.

This study was designed to confirm the protective effect of Schisandrae Fructus, which are the dried fruits of Schisandra chinensis (Turcz.) Baill, against oxidative stress-induced cellular damage and to elucidate the underlying mechanisms in C2C12 myoblasts. Preincubating C2C12 cells with a Schisandrae Fructus ethanol extract (SFEE) significantly attenuated hydrogen peroxide (H2O2)-induced inhibition of growth and induced scavenging activity against intracellular reactive oxygen species (ROS) induced by H2O2. SFEE also inhibited comet tail formation and phospho-histone γH2A.X expression, suggesting that it prevents H2O2-induced cellular DNA damage. Furthermore, treating C2C12 cells with SFEE significantly induced heme oxygenase-1 (HO-1) and phosphorylation of nuclear factor-erythroid 2 related factor 2 (Nrf2). However, zinc protoporphyrin IX, a potent inhibitor of HO-1 activity, significantly reversed the protective effects of SFEE against H2O2-induced growth inhibition and ROS generation in C2C12 cells. Additional experiments revealed that the potential of the SFEE to induce HO-1 expression and protect against H2O2-mediated cellular damage was abrogated by transient transfection with Nrf2-specific small interfering RNA, suggesting that the SFEE protected C2C12 cells against oxidative stress-induced injury through the Nrf2/HO-1 pathway.

Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model., (Copyright © 2014 the American Physiological Society.)

Although mitochondrial dysfunction and oxidative stress have been proposed to play a crucial role in several types of muscular dystrophy (MD), whether a causal link between these two alterations exists remains an open question. We have documented that mitochondrial dysfunction through opening of the permeability transition pore plays a key role in myoblasts from patients as well as in mouse models of MD, and that oxidative stress caused by monoamine oxidases (MAO) is involved in myofiber damage. In the present study we have tested whether MAO-dependent oxidative stress is a causal determinant of mitochondrial dysfunction and apoptosis in myoblasts from patients affected by collagen VI myopathies. We find that upon incubation with hydrogen peroxide or the MAO substrate tyramine myoblasts from patients upregulate MAO-B expression and display a significant rise in reactive oxygen species (ROS) levels, with concomitant mitochondrial depolarization. MAO inhibition by pargyline significantly reduced both ROS accumulation and mitochondrial dysfunction, and normalized the increased incidence of apoptosis in myoblasts from patients. Thus, MAO-dependent oxidative stress is causally related to mitochondrial dysfunction and cell death in myoblasts from patients affected by collagen VI myopathies, and inhibition of MAO should be explored as a potential treatment for these diseases., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)