Promotes Muscle Differentiation Research Articles

Differential Effects of Three Medium-Chain Fatty Acids on Mitochondrial Quality Control and Skeletal Muscle Maturation

Nutritional interventions are one focus of sarcopenia treatment. As medium-chain fatty acids (MCFAs) are oxidized in the mitochondria and produce energy through oxidative phosphorylation (OXPHOS), they are key parts of nutritional interventions. We investigated the in vitro effects of three types of MCFA, caprylic acid (C8), capric acid (C10), and lauric acid (C12), in skeletal muscle cells. Compared with C10 and C12, C8 promoted mitophagy through the phosphatase and tensin homolog (PTEN)-induced kinase 1-Parkin pathway and increased the expression of peroxisome proliferator-activated receptor gamma coactivator 1-α and dynamin-related protein 1 to reduce mitochondrial oxidative stress and promote OXPHOS. Furthermore, the expression of myogenic differentiation 1 and myosin heavy chain increased in myotubes, thus promoting muscle differentiation and maturation. These results suggest that C8 improves mitochondrial quality and promotes skeletal muscle maturation; in contrast, C10 and C12 poorly promoted mitochondrial quality control and oxidative stress and suppressed energy production. Future animal experiments are required to establish the usefulness of C8 for nutritional interventions for sarcopenia.

Differential Effects of Three Medium-Chain Fatty Acids on Mitochondrial Quality Control and Skeletal Muscle Maturation

Nutritional interventions are one focus of sarcopenia treatment. As medium-chain fatty acids (MCFAs) are oxidized in the mitochondria and produce energy through oxidative phosphorylation (OXPHOS), they are key parts of nutritional interventions. We investigated the in vitro effects of three types of MCFA, caprylic acid (C8), capric acid (C10), and lauric acid (C12), in skeletal muscle cells. Compared with C10 and C12, C8 promoted mitophagy through the phosphatase and tensin homolog (PTEN)-induced kinase 1-Parkin pathway and increased the expression of peroxisome proliferator-activated receptor gamma coactivator 1-α and dynamin-related protein 1 to reduce mitochondrial oxidative stress and promote OXPHOS. Furthermore, the expression of myogenic differentiation 1 and myosin heavy chain increased in myotubes, thus promoting muscle differentiation and maturation. These results suggest that C8 improves mitochondrial quality and promotes skeletal muscle maturation; in contrast, C10 and C12 poorly promoted mitochondrial quality control and oxidative stress and suppressed energy production. Future animal experiments are required to establish the usefulness of C8 for nutritional interventions for sarcopenia.

PBRM-1/PBAF-regulated genes in a multipotent progenitor in Caenorhabditis elegans.

The Caenorhabditis elegans somatic gonadal precursors (SGPs) are multipotent progenitors that generate all somatic cells of the adult reproductive system. The 2 SGPs originate in the mesodermal layer and are born through a division that produces one SGP and one head mesodermal cell (hmc). One hmc terminally differentiates, and the other dies by programmed cell death. The polybromo-associated BAF (PBAF) chromatin remodeling complex promotes the multipotent SGP fate. The complete loss of PBAF causes lethality, so we used a combination of Cre/lox recombination and GFP nanobody-directed protein degradation to eliminate PBRM-1, the signature subunit of the PBAF complex, from 83 mesodermal cells, including SGPs, body muscles, and the hmc. We used RNA sequencing to identify genes acting downstream of PBAF in these cells and identified 1,955 transcripts that were significantly differentially expressed between pbrm-1(-) and pbrm-1(+) in the mesoderm of L1 larvae. We found that genes involved in muscle cell function were overrepresented; most of these genes had lower expression in the absence of PBRM-1, suggesting that PBAF promotes muscle differentiation. Among the differentially expressed genes were 125 that are normally expressed at higher levels in SGP vs hmc and positively regulated by pbrm-1 and 53 that are normally expressed at higher levels in hmc vs SGP and are negatively regulated by pbrm-1; these are candidate regulators of the SGP/hmc fate decision. We validated one candidate gene using a fluorescent reporter; the hsp-12.3 reporter was derepressed in SGPs in pbrm-1 mutants, suggesting that hsp-12.3 expression is normally repressed by pbrm-1 in SGPs.

Ginsenoside Rc, an Active Component of Panax ginseng, Alleviates Oxidative Stress-Induced Muscle Atrophy via Improvement of Mitochondrial Biogenesis.

Loss of skeletal muscle mass and function has detrimental effects on quality of life, morbidity, and mortality, and is particularly relevant in aging societies. The enhancement of mitochondrial function has shown promise in promoting muscle differentiation and function. Ginsenoside Rc (gRc), a major component of ginseng, has various pharmacological activities; however, its effect on muscle loss remains poorly explored. In this study, we examined the effects of gRc on the hydrogen peroxide (H2O2)-induced reduction of cell viability in C2C12 myoblasts and myotubes and H2O2-induced myotube degradation. In addition, we investigated the effects of gRc on the production of intracellular reactive oxygen species (ROS) and mitochondrial superoxide, ATP generation, and peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α) activity in myoblasts and myotubes under H2O2 treatment. Furthermore, to elucidate the mechanism of action of gRc, we conducted a transcriptome analysis of myotubes treated with or without gRc under H2O2 treatment. gRc effectively suppressed H2O2-induced cytotoxicity, intracellular ROS, and mitochondrial superoxide production, restored PGC-1α promoter activity, and increased ATP synthesis. Moreover, gRc significantly affected the expression levels of genes involved in maintaining mitochondrial mass and biogenesis, while downregulating genes associated with muscle degradation in C2C12 myotubes under oxidative stress. We provide compelling evidence supporting the potential of gRc as a promising treatment for muscle loss and weakness. Further investigations of the pharmacological effects of gRc under various pathological conditions of muscle loss will contribute to the clinical development of gRc as a therapeutic intervention.

NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration

BackgroundSatellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin–proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined.ResultsUsing conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter.ConclusionsThese results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.

Modulation of myoblast differentiation by electroactive scaffold morphology and biochemical stimuli

The physico-chemical properties of the scaffold materials used for tissue regeneration strategies have a direct impact on cell shape, adhesion, proliferation, phenotypic and differentiation. Herewith, biophysical and biochemical cues have been widely used to design and develop biomaterial systems for specific tissue engineering strategies. In this context, the patterning of piezoelectric polymers that can provide electroactive stimuli represents a suitable strategy for skeletal muscle tissue engineering applications once it has been demonstrated that mechanoelectrical stimuli promote C2C12 myoblast differentiation. In this sense, this works reports on how C2C12 myoblast cells detect and react to physical and biochemical stimuli based on micropatterned poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) electroactive scaffolds produced by soft lithography in the form of arrays of lines and hexagons (anisotropic and isotropic morphology, respectively) combined with differentiation medium. The scaffolds were evaluated for the proliferation and differentiation of C2C12 myoblast cell line and it is demonstrated that anisotropic microstructures promote muscle differentiation which is further reinforced with the introduction of biochemical stimulus. However, when the physical stimulus is not adequate to the tissue, e.g. isotropic microstructure, the biochemical stimulus has the opposite effect, hindering the differentiation process. Therefore, the proper morphological design of the scaffold combined with biochemical stimulus allows to enhance skeletal muscle differentiation and allows the development of advanced strategies for effective muscle tissue engineering.

Heat Shock Proteins: Important Helpers for the Development, Maintenance and Regeneration of Skeletal Muscles

The skeletal muscle is a highly plastic tissue that shows a remarkable adaptive capacity in response to acute and resistance exercise, and modifies its composition to adapt to use and disuse, a process referred to as muscle plasticity. Heat shock proteins (HSPs), a class of evolutionarily conserved molecular chaperones, have been implicated in the regulation of skeletal muscle plasticity. Here, we summarize key findings supporting the notion that HSPs are important components required to maintain skeletal muscle integrity and functionality. HSPs participate in the transcriptional program required for myogenesis and are activated following muscle exercise and injury. Their dysfunction, either as a consequence of improper expression or genetic mutations, contributes to muscle atrophy and leads to the development of myopathies and peripheral motor neuropathies. Denervation/reinnervation and repeated rounds of nerve degeneration/regeneration have been observed in motor neuropathies, suggesting that an imbalance in HSP expression and function may impair the repair of the neuromuscular junctions. Boosting HSP activity may help preventing muscle atrophy by promoting muscle differentiation and helping the repair of NMJs. Boosting HSP function may also help to combat the development of rhabdomyosarcoma (RMS), a highly aggressive type of pediatric soft tissue sarcoma whose cells have skeletal muscle features but are unable to fully differentiate into skeletal muscle cells.

HuR Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating Myomaker mRNA Stability.

Human antigen R (HuR) is an RNA-binding protein that contributes to a wide variety of biological processes and diseases. HuR has been demonstrated to regulate muscle growth and development, but its regulatory mechanisms are not well understood, especially in goats. In this study, we found that HuR was highly expressed in the skeletal muscle of goats, and its expression levels changed during longissimus dorsi muscle development in goats. The effects of HuR on goat skeletal muscle development were explored using skeletal muscle satellite cells (MuSCs) as a model. The overexpression of HuR accelerated the expression of myogenic differentiation 1 (MyoD), Myogenin (MyoG), myosin heavy chain (MyHC), and the formation of myotubes, while the knockdown of HuR showed opposite effects in MuSCs. In addition, the inhibition of HuR expression significantly reduced the mRNA stability of MyoD and MyoG. To determine the downstream genes affected by HuR at the differentiation stage, we conducted RNA-Seq using MuSCs treated with small interfering RNA, targeting HuR. The RNA-Seq screened 31 upregulated and 113 downregulated differentially expressed genes (DEGs) in which 11 DEGs related to muscle differentiation were screened for quantitative real-time PCR (qRT-PCR) detection. Compared to the control group, the expression of three DEGs (Myomaker, CHRNA1, and CAPN6) was significantly reduced in the siRNA-HuR group (p < 0.01). In this mechanism, HuR bound to Myomaker and increased the mRNA stability of Myomaker. It then positively regulated the expression of Myomaker. Moreover, the rescue experiments indicated that the overexpression of HuR may reverse the inhibitory impact of Myomaker on myoblast differentiation. Together, our findings reveal a novel role for HuR in promoting muscle differentiation in goats by increasing the stability of Myomaker mRNA.

The MyoD1 Promoted Muscle Differentiation and Generation by Activating CCND2 in Guanling Cattle.

Simple SummaryMuscle proliferation and differentiation is a complex process, which is mainly regulated by the myogenic regulatory factors (MRFs) gene family. As one of its family members, myogenic differentiation 1 (MyoD1) is an important regulator of myoblast differentiation and fibrogenesis, and its main function is to regulate the proliferation of myoblasts and the directed development of myogenic cell lines. In this study, CRISPR/CAS9 technology was used to construct the MyoD1 gene knockout MDBK cell lines, and then the transcriptome of MDBK cells was analyzed based on high-throughput sequencing technology. The differentially expressed genes in the transcriptome and the functional enrichment of differentially expressed genes were analyzed. The differentially expressed genes in the transcriptome and the functional enrichment were analyzed to provide experimental data for further studies on the mechanism of MyoD1 regulating CCND2 in myocyte differentiation.The purpose of this study was to analyze the transcriptome of MyoD1 gene knockout MDBK cells (bovine kidney cells) using high-throughput sequencing. For the first time, CRISPR/CAS9 technology was used to construct a MyoD1 knockout in MDBK cells and transcriptome sequence analysis was used to examine MyoD1-related target gene expression. Transcriptome sequencing indicated the presence of 723 differentially expressed genes (DEGs) by comparing wild type and MyoD1 knockout MDBK cells and included 178 upregulated and 72 downregulated genes. The DEGs are mainly enriched in Pl-3-kinase and AKT, p53 signaling pathways. Quantitative RT-PCR confirmed that PDE1B, ADAMTS1, DPT, and CCND2 were highly expressed in the leg muscle, longissimus dorsi, and shoulder of Guanling cattle, and CCND2 was inhibited after MyoD1 knockout, suggesting it may be a key downstream gene of MyoD1 and associated with muscle formation and differentiation in Guanling cattle. This provides experimental data for subsequent studies on the regulatory mechanisms of muscle differentiation in Guanling cattle.

Abstract IA020: Reprogramming of myogenic transcription factors in rhabdomyosarcoma

Abstract Rhabdomyosarcoma, a pediatric malignancy with partial resemblance to undifferentiated skeletal muscle, is characterized by high expression of myogenic-lineage transcription factors such as MYOD1 and MYOG. Despite high expression of these transcription factors, which in normal muscle result in differentiation, RMS cells fail to differentiate, suggesting the presence of factors that inhibit their normal differentiation-promoting functions. In this talk, I will present data that the key muscle transcriptional regulator, SIX1, which in development activates the myogenic regulatory factors (MRFs) and promotes muscle differentiation, in fact inhibits differentiation in fusion-negative (FN) RMS. SIX1 holds FN-RMS cells in a progenitor-like state by altering the chromatin landscape and causing MYOD1, a key MRF, to preferentially bind to regulatory regions of genes permissive to growth rather than differentiation. Loss of SIX1 results in re-localization of MYOD1 to promoters/enhancers of genes associated with differentiation, and further results in increased binding of MYOG at such loci. Altered binding of MYOD1 and MYOG in response to SIX1 loss results in marked inhibition of RMS growth in vivo, via induction of differentiation. These data suggest that SIX1 acts as a master regulatory factor controlling the fate of RMS cells. Data will be presented that suggest dynamic actions of SIX1 and its co-factors throughout normal muscle differentiation, whereby high levels of SIX1 expressed in early muscle differentiation may mimic a transcriptional state seen in RMS. We hypothesize that the specific levels of SIX1, combined with a unique combination of transcriptional co-factors, reprogram genome-wide binding of MRFs to different promoter/enhancer sites at specific developmental time points, and that RMS is trapped in an early developmental state where SIX1 represses differentiation via genome-wide alterations in MRF binding that favor growth. Understanding the co-factors that work with SIX1 to alter chromatin state and MRF binding may enable the discovery of novel targets whose inhibition could serve as a relatively non-toxic treatment to restore normal developmental processes and inhibit RMS progression. Citation Format: Heide L. Ford, Jessica Y. Hsu, Etienne P. Danis, Stephanie Nance, Jenean H. O’Brien, Annika L. Gustafson, Veronica M. Wessells, Andrew E. Goodspeed, Jared C. Talbot, Sharon L. Amacher, Paul Jedlicka, Joshua C. Black, James C. Costello, Adam D. Durbin, Kristin B. Artinger. Reprogramming of myogenic transcription factors in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA020.

MOTS-c promotes muscle differentiation in vitro

MOTS-c (mitochondrial open reading frame of the 12 S rRNA-c) is a newly discovered peptide that has been shown to have a protective role in whole-body metabolic homeostasis. This could be a consequence of the effect of MOTS-c on muscle tissue. Here, we investigated the role of MOTS-c in the differentiation of human (LHCN-M2) and murine (C2C12) muscle progenitor cells. Cells were treated with peptides at the onset of differentiation or after myotubes had been formed. We identified in silico a putative Src Homology 2 (SH2) binding motif in the YIFY region of the MOTS-c sequence, and created a Y8F mutant MOTS-c peptide to explore the role of this region. In both cellular models, treatment with wild-type MOTS-c peptide increased myotube formation whereas treatment with the Y8F peptide did not. MOTS-c wild-type, but not Y8F peptide, also protected against interleukin-6 (IL-6)-induced reduction of nuclear myogenin staining in myocytes. Thus, we investigated whether MOTS-c interacts with the IL-6/Janus kinase/ Signal transducer and activator of transcription 3 (STAT3) pathway, and found that MOTS-c, but not the Y8F peptide, blocked the transcriptional activity of STAT3 induced by IL-6. Altogether, our findings suggest that, in muscle cells, MOTS-c interacts with STAT3 via the putative SH2 binding motif in the YIFY region to reduce STAT3 transcriptional activity, which enhances myotube formation. This newly discovered mechanism of action highlights MOTS-c as a potential therapeutic target against muscle-wasting in several diseases.

Identification of protein markers for skeletal muscle‐derived extracellular vesicles (SkM‐EVs) by quantitative proteomics reveals how SkM‐EVs function in vivo

BackgroundSkeletal muscle serves as a secretory organ; it releases various soluble factors called myokine. They act as mediators for cell‐cell communications in autocrine, paracrine, and endocrine fashions. Extracellular vesicles (EVs) have been proposed to be a partial paracrine and/or endocrine effectors by facilitating the exchange of biological cargoes among cells and tissue. Some in vitro study showed that skeletal muscle released EVs and skeletal muscle‐derived EVs (SkM‐EVs) could induce biological effects in recipient cells. However, whether the skeletal muscle actively releases EVs in vivo, how much proportion of plasma EVs are derived from this tissue, and where SkM‐EVs are delivered and exert their roles remain largely unknown. Here, we aimed to identify SkM‐EV marker proteins by quantitative proteomics on human and mouse myocyte‐derived EVs to evaluate SkM‐EVs abundance in vivo. In addition, we explored whether SkM‐EVs isolated from mice could exert biological functions.MethodsProteomic analysis of EVs released from mouse C2C12 myotubes and human iPS‐derived myocytes was performed to identify potential SkM‐EVs markers. The levels of SkM‐EVs in plasma and muscle interstitium were evaluated by the abundance of identified SkM‐EVs protein markers. Based on previous methods, we developed a modified protocol to isolated SkM‐EVs from muscle interstitium. We confirmed that muscle interstitium‐EVs were incorporated into C2C12 myoblast and investigated the expression levels of myogenesis‐related genes.Results&amp; DiscussionWe first determined proteomic profiles of EVs released from C2C12 myoblasts, C2C12 myotubes, and hiPSC‐derived myocytes and identified several proteins that serve as potential markers for SkM‐EVs. Among them, we identified ATP2A1, β‐enolase, and desmin as reliable SkM‐EV marker proteins. Using these protein markers, we confirmed that SkM‐EVs were present in muscle interstitium but little in the blood, which were supported by our electron microscopy. Unexpectedly, the abundance of SkM‐EVs present in plasma and muscle interstitium was not changed in response to acute exercise. Furthermore, we showed that SkM‐EVs accumulated in muscle interstitium space were incorporated into C2C12 myoblast and promoted muscle differentiation by regulating gene expression of Pax7 and myosin heavy chain at least partially through myomiRs (miRs‐1, ‐206, ‐431, and ‐486). We expect that molecular characterization and in vivo distribution of SkM‐EVs could provide a conceptually new basis how skeletal muscle EVs exert their functions.

Yeast hydrolysate ameliorates dexamethasone-induced muscle atrophy by suppressing MuRF-1 expression in C2C12 cells and C57BL/6 mice

• Effect of YH on C2C12 cell and DEX-induced muscle atrophy mouse were conducted. • YH can promote muscle differentiation in C2C12 cell. • YH prevented muscle atrophy via downregulating MuRF-1, FoxO3a, Atrogin-1 in vitro . • YH prevented skeletal muscle loss and downregulates MuRF-1 in DEX-induced muscle atrophy mouse model. In this study, we evaluated the effects of yeast ( Saccharomyces cerevisiae ) hydrolysate (YH) on muscle differentiation and muscle atrophy inhibitory activity in C2C12 cells and atrophy inhibitory activity in a dexamethasone (DEX)-induced muscle atrophy mouse model. Jenner-Giemsa staining revealed that the length and area of ​​myotubes increased in the YH groups. Moreover, the gene expression of myogenic differentiation 1 and myogenin was significantly increased in the YH groups. In the DEX-induced muscle atrophy C2C12 model, the expression of muscle ring-finger protein-1 (MuRF-1), forkhead box class O3a, and Atrogin-1 were significantly reduced in the YH groups. Furthermore, in the muscle-atrophy mice model, grip strength and lean mass were increased by 1.28-fold and 3.5%, respectively compared to the control in the YH group (300 mg/kg). These results were confirmed by the downregulation of MuRF-1 in muscle. In conclusion, YH promoted muscle cell differentiation and exhibited an inhibitory effect on DEX-induced muscle atrophy.

Donepezil, an anti-Alzheimer's disease drug, promotes differentiation and regeneration in injured skeletal muscle through the elevation of the expression of myogenic regulatory factors

We previously demonstrated that donepezil, an anti-Alzheimer's disease drug, improved skeletal muscle atrophy by enhancing the angiogenesis of endothelial cells and activating the proliferation of satellite cells in a mouse model of peripheral arterial disease. However, the effect of donepezil on muscle differentiation during regeneration remains unclear. Therefore, we measured the expressions of myogenic regulatory factors and late muscle differentiation markers in donepezil-treated C2C12 myoblast cells before and after the induction of cell differentiation. The results indicate that the expressions of myogenin, troponin T (TnT) and myosin heavy chain (MyHC) were significantly increased and myotube formation was accelerated in donepezil-treated cells under the differentiation condition. However, the promotive effect of donepezil on muscle differentiation could not be reproduced by the addition of acetylcholine (ACh) and was not disrupted after treatment with ACh receptor blockers. Moreover, other kinds of acetylcholinesterase inhibitors failed to promote muscle differentiation in C2C12 cells. These results indicate that the specific characteristics of donepezil in the promotion of muscle differentiation are independent of its acetylcholinesterase-inhibitory action. We further found that donepezil induced an incremental shift of the cross-sectional area of myofibers and elevated the expressions of myogenin, TnT and MyHC in a mouse model of cardiotoxin injury. These results suggest that donepezil promotes the differentiation of muscle regeneration upon injury via the elevation of the expressions of myogenic regulatory factors and late muscle differentiation markers. Our findings suggest that donepezil can be a useful therapeutic agent for injured skeletal muscle treatment.

Selective Inhibition of Genomic and Non-Genomic Effects of Thyroid Hormone Regulates Muscle Cell Differentiation and Metabolic Behavior.

Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the β3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.

Effect of Deer Antler Extract on Muscle Differentiation and 5-Aminoimidazole-4-Carboxamide Ribonucleoside (AICAR)-Induced Muscle Atrophy in C2C12 Cells.

The effect of deer antler extract on muscle differentiation and muscle atrophy were evaluated to minimize muscle loss following aging. Various deer antler extracts (HWE, hot water extract of deer antler; FE, HWE of fermented deer antler; ET, enzyme-assisted extract of deer antler; UE, extract prepared by ultrasonication of deer antler) were evaluated for their effect on muscle differentiation and inhibition of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced muscle atrophy in C2C12 cells. Morphological changes according to the effect of antler extracts on muscle differentiation were confirmed by Jenner-Giemsa staining. In addition, the expression levels of genes related to muscle differentiation and atrophy were confirmed through qRT-PCR. In the presence of antler extracts, the length and thickness of myotubes and myogenin differentiation 1 (MyoD1) and myogenic factor 5 (Myf5) gene expression were increased compared to those in the control group (CON). Gene expression of AMP-activated protein kinase (AMPK), MyoD1, and myogenin, along with the muscle atrophy factors muscle RING finger-1 (MuRF-1) and forkhead box O3a (FoxO3a) upon addition of deer antler extracts to muscle-atrophied C2C12 cells was determined by qRT-PCR after treatment with AICAR. The expression of MuRF-1 and FoxO3a decreased in the groups treated with antler extracts compared to that in the group treated with AICAR alone. In addition, gene expression of MyoD1 and myogenin in the muscle atrophy cell model was significantly increased compared that into the CON. Therefore, our findings indicate that antler extract can increase the expression of MyoD1, Myf5 and myogenin, inhibit muscle atrophy, and promote muscle differentiation.

MiR-194-Loaded Gelatin Nanospheres Target MEF2C to Suppress Muscle Atrophy in a Mechanical Unloading Model.

Muscle atrophy usually occurs under mechanical unloading, which increases the risk of injury to reduce the functionality of the moving system, while there is still no effective therapy until now. It was found that miR-194 was significantly downregulated in a muscle atrophy model, and its target protein was the myocyte enhancer factor 2C (MEF2C). miR-194 could promote muscle differentiation and also inhibit ubiquitin ligases, thus miR-194 could be used as a nucleic acid drug to treat muscle atrophy, whereas miRNA was unstable in vivo, limiting its application as a therapeutic drug. A gelatin nanosphere (GN) delivery system was applied for the first time to load exogenous miRNA here. Exogenous miR-194 was loaded in GNs and injected into the muscle atrophy model. It demonstrated that the muscle fiber cross-sectional area, in situ muscle contractile properties, and myogenic markers were increased significantly after treatment. It proposed miR-194 loaded in GNs as an effective treatment for muscle atrophy by promoting muscle differentiation and inhibiting ubiquitin ligase activity. Moreover, the developed miRNA delivery system, taking advantage of its tunable composition, degradation rate, and capacity to load various drug molecules with high dosage, is considered a promising platform to achieve precise treatment of muscle atrophy-related diseases.

Early-senescent bone marrow mesenchymal stem cells promote C2C12 cell myogenic differentiation by preventing the nuclear translocation of FOXO3

AimsMouse bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells with self-renewal and differentiation abilities. Since the effects of senescent BMSCs on C2C12 cells are not fully clear, the present study aimed to elucidate these effects. Main methodsSenescence-associated β-galactosidase staining and western blotting were performed to confirm the senescence of BMSCs. Immunofluorescence and western blotting were used to assess myoblast differentiation in each group. The role of the AKT/P70 signaling pathway and forkhead box O3 (FOXO3) nuclear translocation was explored by western blotting. BMSC-derived exosomes were injected into the tibialis anterior of mice, and RT-qPCR was used to assess the role of exosomes in promoting muscle differentiation. Key findingsConditioned medium (CM) from early-senescent BMSCs promoted myogenic differentiation in vitro, which was detected as enhanced expression of myosin heavy chain (MHC), myogenin (MYOG), and myogenic differentiation 1 (MyoD). The AKT signaling pathway was found to be regulated by CM, which inhibited FOXO3 nuclear translocation. RT-qPCR analysis results showed that MHC, MyoD, and MYOG mRNA expression increased in the tibialis anterior of mice after exosome injection. SignificanceThe present study demonstrated that early-senescent BMSCs accelerated C2C12 cell myogenic differentiation, and the transcription factor, FOXO3, was the target of senescent cells. Collectively, our results suggest that the AKT/P70 signaling pathway mediates the effect of BMSCs on neighboring cells.

The functional analysis of transiently upregulated miR-101 suggests a "braking" regulatory mechanism during myogenesis.

Skeletal muscle differentiation is a highly coordinated process that involves many cellular signaling pathways and microRNAs (miRNAs). A group of muscle-specific miRNAs has been reported to promote myogenesis by suppressing key signaling pathways for cell growth. However, the functional role and regulatory mechanism of most non-muscle-specific miRNAs with stage-specific changes during differentiation are largely unclear. Here, we describe the functional characterization of miR-101a/b, a pair of non-muscle-specific miRNAs that show the largest change among a group of transiently upregulated miRNAs during myogenesis in C2C12 cells. The overexpression of miR-101a/b inhibits myoblast differentiation by suppressing the p38/MAPK, Interferon Gamma, and Wnt pathways and enhancing the C/EBP pathway. Mef2a, a key protein in the p38/MAPK pathway, was identified as a direct target of miR-101a/b. Interestingly, we found that the long non-coding RNA (lncRNA) Malat1, which promotes muscle differentiation, interacts with miR-101a/b, and this interaction competes with Mef2a mRNA to relieve the inhibition of the p38/MAPK pathway during myogenesis. These results uncovered a "braking" role in differentiation of transiently upregulated miRNAs and provided new insights into the competing endogenous RNA (ceRNA) regulatory mechanism in myoblast differentiation and myogenesis.

LncMGPF is a novel positive regulator of muscle growth and regeneration.

BackgroundLong non‐coding RNAs (lncRNAs) play critical regulatory roles in diverse biological processes and diseases. While a large number of lncRNAs have been identified in skeletal muscles until now, their function and underlying mechanisms in skeletal myogenesis remain largely unclear.MethodsWe characterized a novel functional lncRNA designated lncMGPF (lncRNA muscle growth promoting factor) using RACE, Northern blot, fluorescence in situ hybridization and quantitative real‐time PCR. Its function was determined by gene overexpression, interference, and knockout experiments in C2C12 myoblasts, myogenic progenitor cells, and an animal model. The molecular mechanism by which lncMGPF regulates muscle differentiation was mainly examined by cotransfection experiments, luciferase reporter assay, RNA immunoprecipitation, RNA pull‐down, and RNA stability analyses.ResultsWe report that lncMGPF, which is highly expressed in muscles and positively regulated by myoblast determination factor (MyoD), promotes myogenic differentiation of muscle cells in vivo and in vitro. lncMGPF knockout in mice substantially decreases growth rate, reduces muscle mass, and impairs muscle regeneration. Overexpression of lncMGPF in muscles can rescue the muscle phenotype of knockout mice and promote muscle growth of wild‐type mice. Mechanistically, lncMGPF promotes muscle differentiation by acting as a molecular sponge of miR‐135a‐5p and thus increasing the expression of myocyte enhancer factor 2C (MEF2C), as well as by enhancing human antigen R‐mediated messenger RNA stabilization of myogenic regulatory genes such as MyoD and myogenin (MyoG). We confirm that pig lncRNA AK394747 and human lncRNA MT510647 are homologous to mouse lncMGPF, with conserved function and mechanism during myogenesis.ConclusionsOur data reveal that lncMGPF is a novel positive regulator of myogenic differentiation, muscle growth and regeneration in mice, pigs, and humans.