Generating human skeletal myoblast spheroids for vascular myogenic tissue engineering.

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such asin vitrodrug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool forin vitrodrug-testing and human disease modeling.

HIF-1α/MMP-9 Axis Is Required in the Early Phases of Skeletal Myoblast Differentiation under Normoxia Condition In Vitro.

Hypoxia-inducible factor (HIF)-1α represents an oxygen-sensitive subunit of HIF transcriptional factor, which is usually degraded in normoxia and stabilized in hypoxia to regulate several target gene expressions. Nevertheless, in the skeletal muscle satellite stem cells (SCs), an oxygen level-independent regulation of HIF-1α has been observed. Although HIF-1α has been highlighted as a SC function regulator, its spatio-temporal expression and role during myogenic progression remain controversial. Herein, using biomolecular, biochemical, morphological and electrophysiological analyses, we analyzed HIF-1α expression, localization and role in differentiating murine C2C12 myoblasts and SCs under normoxia. In addition, we evaluated the role of matrix metalloproteinase (MMP)-9 as an HIF-1α effector, considering that MMP-9 is involved in myogenesis and is an HIF-1α target in different cell types. HIF-1α expression increased after 24/48 h of differentiating culture and tended to decline after 72 h/5 days. Committed and proliferating mononuclear myoblasts exhibited nuclear HIF-1α expression. Differently, the more differentiated elongated and parallel-aligned cells, which are likely ready to fuse with each other, show a mainly cytoplasmic localization of the factor. Multinucleated myotubes displayed both nuclear and cytoplasmic HIF-1α expression. The MMP-9 and MyoD (myogenic activation marker) expression synchronized with that of HIF-1α, increasing after 24 h of differentiation. By means of silencing HIF-1α and MMP-9 by short-interfering RNA and MMP-9 pharmacological inhibition, this study unraveled MMP-9's role as an HIF-1α downstream effector and the fact that the HIF-1α/MMP-9 axis is essential in morpho-functional cell myogenic commitment.

Establishment and characterization of a skeletal myoblast cell line of grass carp (Ctenopharyngodon idellus).

Skeletal muscle myoblastic cell lines can provide a valuable new in vitro model for the exploration of the mechanisms that control skeletal muscle development and its associated molecular regulation. In this study, the skeletal muscle tissues of grass carp were digested with trypsin and collagenase I to obtain the primary myoblast cell culture. Myoblast cells were obtained by differential adherence purification and further analyzed by cryopreservation and resuscitation, chromosome analysis, immunohistochemistry, and immunofluorescence. A continuous grass carp myoblast cell line (named CIM) was established from grass carp (Ctenopharyngodon idellus) muscle and has been subcultured > 100 passages in a year and more. The CIM cells revived at 79.78-95.06% viability after 1-6 months of cryopreservation, and shared a population doubling time of 27.24 h. The number of modal chromosomes of CIM cells was 48, and the mitochondrial 12S rRNA sequence of the CIM cell line shared 99% identity with those of grass carp registered in GenBank. No microorganisms (bacteria, fungi, or mycoplasma) were detected during the whole study. The cell type of CIM cells was proven to be myoblast by immunohistochemistry of specific myogenic protein markers, including CD34, desmin, MyoD, and MyHC, as well as relative expression of key genes. And the myogenic rate and fusion index of this cell line after 10 days of induced differentiation were 8.96 ~ 9.42% and 3-24%, respectively. The telomerase activity and transfection efficiency of CIM cell line were 0.027 IU/mgprot and 23 ~ 24%, respectively. These results suggest that a myoblast cell line named CIM with normal biological function has been successfully established, which may provide a valuable tool for related in vitro studies.

Vindoline Exhibits Anti-Diabetic Potential in Insulin-Resistant 3T3-L1 Adipocytes and L6 Skeletal Myoblasts.

Type 2 diabetes mellitus (T2DM) emerged as a major health care concern in modern society, primarily due to lifestyle changes and dietary habits. Obesity-induced insulin resistance is considered as the major pathogenic factor in T2DM. In this study, we investigated the effect of vindoline, an indole alkaloid of Catharanthus roseus on insulin resistance (IR), oxidative stress and inflammatory responses in dexamethasone (IR inducer)-induced dysfunctional 3T3-L1 adipocytes and high-glucose-induced insulin-resistant L6-myoblast cells. Results showed that dexamethasone-induced dysfunctional 3T3-L1 adipocytes treated with different concentrations of vindoline significantly enhanced basal glucose consumption, accompanied by increased expression of GLUT-4, IRS-1 and adiponectin. Similarly, vindoline-treated insulin-resistant L6 myoblasts exhibited significantly enhanced glycogen content accompanied with upregulation of IRS-1 and GLUT-4. Thus, in vitro studies of vindoline in insulin resistant skeleton muscle and dysfunctional adipocytes confirmed that vindoline treatment significantly mitigated insulin resistance in myotubes and improved functional status of adipocytes. These results demonstrated that vindoline has the potential to be used as a therapeutic agent to ameliorate obesity-induced T2DM-associated insulin resistance profile in adipocytes and skeletal muscles.

Iron deficiency is related to lower muscle mass in community-dwelling individuals and impairs myoblast proliferation.

BACKGROUND: Loss of muscle mass is linked with impaired quality of life and an increased risk of morbidity and premature mortality. Iron is essential for cellular processes such as energy metabolism, nucleotide synthesis and numerous enzymatic reactions. As the effects of iron deficiency (ID) on muscle mass and function are largely unknown, we aimed to assess the relation between ID and muscle mass in a large population-based cohort, and subsequently studied effects of ID on cultured skeletal myoblasts and differentiated myocytes. METHODS: In a population-based cohort of 8592 adults, iron status was assessed by plasma ferritin and transferrin saturation, and muscle mass was estimated using 24-h urinary creatinine excretion rate (CER). The relationships of ferritin and transferrin saturation with CER were assessed by multivariable logistic regression. Furthermore, mouse C2C12 skeletal myoblasts and differentiated myocytes were subjected to deferoxamine with or without ferric citrate. Myoblast proliferation was measured with a colorimetric 5-bromo-2'-deoxy-uridine ELISA assay. Myocyte differentiation was assessed using Myh7-stainings. Myocyte energy metabolism, oxygen consumption rate and extracellular acidification rate were assessed using Seahorse mitochondrial flux analysis, and apoptosis rate with fluorescence-activated cell sorting. RNA sequencing (RNAseq) was used to identify ID-related gene and pathway enrichment in myoblasts and myocytes. RESULTS: Participants in the lowest age- and sex-specific quintile of plasma ferritin (OR vs middle quintile 1.62, 95% CI 1.25-2.10, P < 0.001) or transferrin saturation (OR 1.34, 95% CI 1.03-1.75, P = 0.03) had a significantly higher risk of being in the lowest age- and sex-specific quintile of CER, independent of body mass index, estimated GFR, haemoglobin, hs-CRP, urinary urea excretion, alcohol consumption and smoking status. In C2C12 myoblasts, deferoxamine-induced ID reduced myoblast proliferation rate (P-trend <0.001) but did not affect differentiation. In myocytes, deferoxamine reduced myoglobin protein expression (-52%, P < 0.001) and tended to reduce mitochondrial oxygen consumption capacity (-28%, P = 0.10). Deferoxamine induced gene expression of cellular atrophy markers Trim63 (+20%, P = 0.002) and Fbxo32 (+27%, P = 0.048), which was reversed by ferric citrate (-31%, P = 0.04 and -26%, P = 0.004, respectively). RNAseq indicated that both in myoblasts and myocytes, ID predominantly affected genes involved in glycolytic energy metabolism, cell cycle regulation and apoptosis; co-treatment with ferric citrate reversed these effects. CONCLUSIONS: In population-dwelling individuals, ID is related to lower muscle mass, independent of haemoglobin levels and potential confounders. ID impaired myoblast proliferation and aerobic glycolytic capacity, and induced markers of myocyte atrophy and apoptosis. These findings suggest that ID contributes to loss of muscle mass.

Efficacy of Autologous Skeletal Myoblast Cell Sheet Transplantation for Liver Regeneration in Liver Failure.

BACKGROUND: No effective therapies have yet been established for liver regeneration in liver failure. Autologous skeletal myoblast cell sheet transplantation has been proven to improve cardiac function in patients with heart failure, and one of the mechanisms has been reported to be a paracrine effect by various growth factors associated with liver regeneration. Therefore, the present study focused on the effect of myoblast cells on liver regeneration in vitro and in vivo. METHODS: We assessed the effect of myoblast cells on the cells comprising the liver in vitro in association with liver regeneration. In addition, we examined in vivo effect of skeletal myoblast cell sheet transplantation in C57/BL/6 mouse models of liver failure, such as liver fibrosis induced by thioacetamide and hepatectomy. RESULTS: In vitro, the myoblast cells exhibited a capacity to promote the proliferation of hepatic epithelial cells and the angiogenesis of liver sinusoidal endothelial cells, and suppress the activation of hepatic stellate cells. In vivo, sheet transplantation significantly suppressed liver fibrosis in the induced liver fibrosis model and accelerated liver regeneration in the hepatectomy model. CONCLUSIONS: Autologous skeletal myoblast cell sheet transplantation significantly improved the liver failure in the in vitro and in vivo models. Sheet transplantation is expected to have the potential to be a clinically therapeutic option for liver regeneration in liver failure.

CircRNA Profiling of Skeletal Muscle in Two Pig Breeds Reveals CircIGF1R Regulates Myoblast Differentiation via miR-16.

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation.

mt-Ty 5'tiRNA regulates skeletal muscle cell proliferation and differentiation.

In this study, we sought to determine the role of tRNA-derived fragments in the regulation of gene expression during skeletal muscle cell proliferation and differentiation. We employed cell culture to examine the function of mt-Ty 5' tiRNAs. Northern blotting, RT-PCR as well as RNA-Seq, were performed to determine the effects of mt-Ty 5' tiRNA loss and gain on gene expression. Standard and transmission electron microscopy (TEM) were used to characterize cell and sub-cellular structures. mt-Ty 5'tiRNAs were found to be enriched in mouse skeletal muscle, showing increased levels in later developmental stages. Gapmer-mediated inhibition of tiRNAs in skeletal muscle C2C12 myoblasts resulted in decreased cell proliferation and myogenic differentiation; consistent with this observation, RNA-Seq, transcriptome analyses, and RT-PCR revealed that skeletal muscle cell differentiation and cell proliferation pathways were also downregulated. Conversely, overexpression of mt-Ty 5'tiRNAs in C2C12 cells led to a reversal of these transcriptional trends. These data reveal that mt-Ty 5'tiRNAs are enriched in skeletal muscle and play an important role in myoblast proliferation and differentiation. Our study also highlights the potential for the development of tiRNAs as novel therapeutic targets for muscle-related diseases.

HDAC8 regulates protein kinase D phosphorylation in skeletal myoblasts in response to stress signaling.

Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination of myogenic transcription factors. Myogenic transcription is tightly regulated by various intracellular signaling pathways, which include members of the protein kinase D (PKD) family. PKD is a family of serine-threonine kinases that regulate gene expression, protein secretion, cell proliferation, differentiation and inflammation. PKD is a unique PKC family member that shares distant sequence homology to calcium-regulated kinases and plays an important role in muscle physiology. In this report, we show that class I histone deacetylase (HDAC) inhibition, and in particular HDAC8 inhibition, attenuated PKD phosphorylation in skeletal C2C12 myoblasts in response to phorbol ester, angiotensin II and dexamethasone signaling independent of changes in total PKD protein expression. As class I HDACs and PKD signaling are requisite for myocyte differentiation, these data suggest that HDAC8 functions as a potential feedback regulator of PKD phosphorylation to control myogenic gene expression.

The dose-response effects of arachidonic acid on primary human skeletal myoblasts and myotubes.

Background: Cellular inflammatory response, mediated by arachidonic acid (AA) and cyclooxygenase, is a highly regulated process that leads to the repair of damaged tissue. Recent studies on murine C2C12 cells have demonstrated that AA supplementation leads to myotube hypertrophy. However, AA has not been tested on primary human muscle cells. Therefore, the purpose of this study was to determine whether AA supplementation has similar effects on human muscle cells. Methods: Proliferating and differentiating human myoblasts were exposed to AA in a dose-dependent manner (50-0.80 µM) for 48 (myoblasts) or 72 (myotubes) hours. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and cell counting; myotube area was determined by immunocytochemistry and confocal microscopy; and anabolic signaling pathways were evaluated by western blot and RT-PCR. Results: Our data show that the treatment of primary human myoblasts treated with 50 µM and 25 µM of AA led to the release of PGE2 and PGF2α at levels higher than those of control-treated cells (p < 0.001 for all concentrations). Additionally, 50 µM and 25 µM of AA suppressed myoblast proliferation, myotube area, and myotube fusion. Anabolic signaling indicated reductions in total and phosphorylated TSC2, AKT, S6, and 4EBP1 in myoblasts at 50 µM of AA (p < 0.01 for all), but not in myotubes. These changes were not affected by COX-2 inhibition with celecoxib. Conclusion: Together, our data demonstrate that high concentrations of AA inhibit myoblast proliferation, myotube fusion, and myotube hypertrophy, thus revealing potential deleterious effects of AA on human skeletal muscle cell health and viability.

Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy.

Cellular senescence leads to the depletion of myogenic progenitors and decreased regenerative capacity. We show that the small molecule 2,6-disubstituted purine, reversine, can improve some well-known hallmarks of cellular aging in senescent myoblast cells. Reversine reactivated autophagy and insulin signaling pathway via upregulation of Adenosine Monophosphate-activated protein kinase (AMPK) and Akt2, restoring insulin sensitivity and glucose uptake in senescent cells. Reversine also restored the loss of connectivity of glycolysis to the TCA cycle, thus restoring dysfunctional mitochondria and the impaired myogenic differentiation potential of senescent myoblasts. Altogether, our data suggest that cellular senescence can be reversed by treatment with a single small molecule without employing genetic reprogramming technologies.

Tangeretin alleviates Tunicamycin-induced endoplasmic reticulum stress and associated complications in skeletal muscle cells.

Endoplasmic reticulum (ER) stress and associated oxidative stress are involved in the genesis and progression of skeletal muscle diseases such as myositis and atrophy or muscle wasting. Targeting the ER stress and associated downstream pathways can aid in the development of better treatment strategies for these diseases with limited therapeutic approaches. There is a growing interest in identifying natural products against ER stress due to the lower toxicity and cost effectiveness. In the present study, we investigated the protective effect of Tangeretin, a citrus methoxyflavone found in citrus peels against Tunicamycin (pharmacological ER stress inducer)-induced ER stress and associated complications in rat skeletal muscle L6 cell lines. Treatment with Tunicamycin for a period of 24 h resulted in the upregulation of ER stress marker proteins, ER resident oxidoreductases and cellular reactive oxygen species (ROS). Co-treatment with Tangeretin was effective in alleviating Tunicamycin-induced ER stress and associated redox-related complications by significantly downregulating the unfolded protein response (UPR), ER resident oxidoreductase proteins, cellular ROS and improving the antioxidant enzyme activity. Tunicamycin also induced upregulation of phosphorylated p38 MAP Kinase and loss of mitochondrial membrane potential. Tangeretin significantly reduced the levels of phosphorylated p38 MAP Kinase and improved the mitochondrial membrane potential. From the results, it is evident that Tangeretin can be explored further as a potential candidate for skeletal muscle diseases involving protein misfolding and ER stress.

Decrease in the expression of muscle-specific miRNAs, miR-133a and miR-1, in myoblasts with replicative senescence.

Muscles that are injured or atrophied by aging undergo myogenic regeneration. Although myoblasts play a pivotal role in myogenic regeneration, their function is impaired with aging. MicroRNAs (miRNAs) are also involved in myogenic regeneration. MiRNA (miR)-1 and miR-133a are muscle-specific miRNAs that control the proliferation and differentiation of myoblasts. In this study, we determined whether miR-1 and miR-133a expression in myoblasts is altered with cellular senescence and involved in senescence-impaired myogenic differentiation. C2C12 murine skeletal myoblasts were converted to a replicative senescent state by culturing to a high passage number. Although miR-1 and miR-133a expression was largely induced during myogenic differentiation, expression was suppressed in cells at high passage numbers (passage 10 and/or passage 20). Although the senescent myoblasts exhibited a deterioration of myogenic differentiation, transfection of miR-1 or miR-133a into myoblasts ameliorated cell fusion. Treatment with the glutaminase 1 inhibitor, BPTES, removed senescent cells from C2C12 myoblasts with a high passage number, whereas myotube formation and miR-133a expression was increased. In addition, primary cultured myoblasts prepared from aged C57BL/6J male mice (20 months old) exhibited a decrease in miR-1 and miR-133a levels compared with younger mice (3 months old). The results suggest that replicative senescence suppresses muscle-specific miRNA expression in myoblasts, which contributes to the senescence-related dysfunction of myogenic regeneration.

Requirement for PINCH in skeletal myoblast differentiation.

PINCH, an adaptor of focal adhesion complex, plays essential roles in multiple cellular processes and organogenesis. Here, we ablated PINCH1 or both of PINCH1 and PINCH2 in skeletal muscle progenitors using MyoD-Cre. Double ablation of PINCH1 and PINCH2 resulted in early postnatal lethality with reduced size of skeletal muscles and detachment of diaphragm muscles from the body wall. PINCH mutant myofibers failed to undergo multinucleation and exhibited disrupted sarcomere structures. The mutant myoblasts in culture were able to adhere to newly formed myotubes but impeded in cell fusion and subsequent sarcomere genesis and cytoskeleton organization. Consistent with this, expression of integrin ß1 and some cytoskeleton proteins and phosphorylation of ERK and AKT were significantly reduced in PINCH mutants. However, N-cadherin was correctly expressed at cell adhesion sites in PINCH mutant cells, suggesting that PINCH may play a direct role in myoblast fusion. Expression of MRF4, the most highly expressed myogenic factor at late stages of myogenesis, was abolished in PINCH mutants that could contribute to observed phenotypes. In addition, mice with PINCH1 being ablated in myogenic progenitors exhibited only mild centronuclear myopathic changes, suggesting a compensatory role of PINCH2 in myogenic differentiation. Our results revealed a critical role of PINCH proteins in myogenic differentiation.

Celecoxib impairs primary human myoblast proliferation and differentiation independent of cyclooxygenase 2 inhibition.

The use of non-steroidal anti-inflammatory drugs (NSAIDs) for treatment of musculoskeletal injuries is commonplace in the general, athletic, and military populations. While NSAIDs have been studied in a variety of tissues, the effects of NSAIDs on skeletal muscle have not been fully defined. To address this, we investigated the degree to which the cyclooxygenase (COX)-2-selective NSAID celecoxib affects muscle cell proliferation, differentiation, anabolic signaling, and mitochondrial function in primary human skeletal myoblasts and myotubes. Primary muscle cells were treated with celecoxib or NS-398 (a pharmacological inhibitor of COX-2) as a control. Celecoxib administration significantly reduced myoblast proliferation, viability, fusion, and myotube area in a dose-dependent manner, whereas NS-398 had no effect on any of these outcomes. Celecoxib treatment was also associated with reduced phosphorylation of ribosomal protein S6 in myoblasts, and reduced phosphorylation of AKT, p70S6K, S6, and ERK in myotubes. In contrast, NS-398 did not alter phosphorylation of these molecules in myoblasts or myotubes. In myoblasts, celecoxib significantly reduced mitochondrial membrane potential and respiration, as evidenced by the decreased citric acid cycle (CAC) intermediates cis-aconitic acid, alpha-keto-glutarate acid, succinate acid, and malic acid. Similar results were observed in myotubes, although celecoxib also reduced pyruvic acid, citric acid, and fumaric acid. NS-398 did not affect CAC intermediates in myoblasts or myotubes. Together, these data reveal that celecoxib inhibits proliferation, differentiation, intracellular signaling, and mitochondrial function in primary human myoblasts and myotubes independent of its function as a COX-2 inhibitor.

Ginsenoside Rd ameliorates muscle wasting by suppressing the signal transducer and activator of transcription 3 pathway.

BACKGROUND: The effects of some drugs, aging, cancers, and other diseases can cause muscle wasting. Currently, there are no effective drugs for treating muscle wasting. In this study, the effects of ginsenoside Rd (GRd) on muscle wasting were studied. METHODS: Tumour necrosis factor-alpha (TNF-α)/interferon-gamma (IFN-γ)-induced myotube atrophy in mouse C2C12 and human skeletal myoblasts (HSkM) was evaluated based on cell thickness. Atrophy-related signalling, reactive oxygen species (ROS) level, mitochondrial membrane potential, and mitochondrial number were assessed. GRd (10 mg/kg body weight) was orally administered to aged mice (23-24 months old) and tumour-bearing (Lewis lung carcinoma [LLC1] or CT26) mice for 5 weeks and 16 days, respectively. Body weight, grip strength, inverted hanging time, and muscle weight were assessed. Histological analysis was also performed to assess the effects of GRd. The evolutionary chemical binding similarity (ECBS) approach, molecular docking, Biacore assay, and signal transducer and activator of transcription (STAT) 3 reporter assay were used to identify targets of GRd. RESULTS: GRd significantly induced hypertrophy in the C2C12 and HSkM myotubes (average diameter 50.8 ± 2.6% and 49.9% ± 3.7% higher at 100 nM, vs. control, P ≤ 0.001). GRd treatment ameliorated aging- and cancer-induced (LLC1 or CT26) muscle atrophy in mice, which was evidenced by significant increases in grip strength, hanging time, muscle mass, and muscle tissue cross-sectional area (1.3-fold to 4.6-fold, vs. vehicle, P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). STAT3 was found to be a possible target of GRd by the ECBS approach and molecular docking assay. Validation of direct interaction between GRd and STAT3 was confirmed through Biacore analysis. GRd also inhibited STAT3 phosphorylation and STAT3 reporter activity, which led to the inhibition of STAT3 nuclear translocation and the suppression of downstream targets of STAT3, such as atrogin-1, muscle-specific RING finger protein (MuRF-1), and myostatin (MSTN) (29.0 ± 11.2% to 84.3 ± 30.5%, vs. vehicle, P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). Additionally, GRd scavenged ROS (91.7 ± 1.4% reduction at 1 nM, vs. vehicle, P ≤ 0.001), inhibited TNF-α-induced dysregulation of ROS level, and improved mitochondrial integrity (P ≤ 0.05; P ≤ 0.01; P ≤ 0.001). CONCLUSIONS: GRd ameliorates aging- and cancer-induced muscle wasting. Our findings suggest that GRd may be a novel therapeutic agent or adjuvant for reversing muscle wasting.

A regenerative niche for stem cells.

Production of hyaluronic acid allows regenerative signaling in muscle stem cells after injury.

JMJD3 activated hyaluronan synthesis drives muscle regeneration in an inflammatory environment.

Muscle stem cells (MuSCs) reside in a specialized niche that ensures their regenerative capacity. Although we know that innate immune cells infiltrate the niche in response to injury, it remains unclear how MuSCs adapt to this altered environment for initiating repair. Here, we demonstrate that inflammatory cytokine signaling from the regenerative niche impairs the ability of quiescent MuSCs to reenter the cell cycle. The histone H3 lysine 27 (H3K27) demethylase JMJD3, but not UTX, allowed MuSCs to overcome inhibitory inflammation signaling by removing trimethylated H3K27 (H3K27me3) marks at the Has2 locus to initiate production of hyaluronic acid, which in turn established an extracellular matrix competent for integrating signals that direct MuSCs to exit quiescence. Thus, JMJD3-driven hyaluronic acid synthesis plays a proregenerative role that allows MuSC adaptation to inflammation and the initiation of muscle repair.

CREB1 promotes proliferation and differentiation by mediating the transcription of CCNA2 and MYOG in bovine myoblasts.

The cAMP response element binding protein 1 (CREB1) is an important nuclear transcription factor in eukaryotes. To explore the potential role of CREB1 on Qinchuan bovine skeletal myoblasts, we investigated the function of CREB1 on proliferation and differentiation. In this study, we found that CREB1 promoted cell proliferation by promoting DNA synthesis in S phase and cell division in G2 phase and promoted myogenic differentiation process in bovine myoblasts. Through dual luciferase experiments, we found that CREB1 can bind to the proximal promoter regions of CCNA2 and MyoG, indicating that CREB1 can play a positive regulatory role in the proliferation and differentiation of myoblasts by mediating the transcription of CCNA2 and MyoG. In addition, through downstream target gene analysis and transcriptome sequencing, we found that CREB1 plays a role in cell proliferation, myogenic differentiation, skeletal muscle repair and other related pathways.

Aqueous extracts of Corni Fructus protect C2C12 myoblasts from DNA damage and apoptosis caused by oxidative stress.

BACKGROUND: Although the various pharmacological effects of Corni Fructus are highly correlated with its antioxidant activity, the blocking effect against oxidative stress in muscle cells is not clear. The purpose of this study was to investigate the effect of aqueous extracts of Corni Fructus (CFE) against oxidative stress caused by hydrogen peroxide (H2O2) in murine skeletal C2C12 myoblasts. METHODS AND RESULTS: MTT assay for cell viability, DCF-DA staining for reactive oxygen species (ROS) production, Comet assay for DNA damage, annexin V-FITC and PI double staining for apoptosis, JC-1 staining and caspase assay for monitor mitochondrial integrity, and western blotting for related protein levels were conducted in H2O2 oxidative stressed C2C12 cells. Our results showed that CFE pretreatment significantly ameliorated the loss of cell viability and inhibited apoptosis in H2O2-treated C2C12 cells in a concentration-dependent manner. DNA damage induced by H2O2 was also markedly attenuated in the presence of CFE, which was associated with suppression of ROS generation. In addition, H2O2 reduced mitochondrial membrane potential and caused downregulation of Bcl-2 and upregulation of Bax expression, although these were abrogated by CFE pretreatment. Moreover, CFE blocked H2O2-induced cytosolic release of cytochrome c, activation of caspase-9 and caspase-3, and degradation of poly (ADP-ribose) polymerase. CONCLUSION: Taken together, the present results demonstrate that CFE could protect C2C12 cells from H2O2-induced damage by eliminating ROS generation, thereby blocking mitochondria-mediated apoptosis pathway. These results indicate that CFE has therapeutic potential for the prevention and treatment of oxidative stress-mediated myoblast injury.