Myogenin mRNA Levels Research Articles

A new insight on alleviating the inhibitory effect of aflatoxin B1 on muscle development in grass carp (Ctenopharyngodon idella): the effect of 4-methylesculetin in vivo and in vitro

Aflatoxin B1 (AFB1), an important fungal toxin, exists mainly in plant feed ingredients and animals consuming feed contaminated with AFB1 will have reduced growth and impaired health condition mainly due to oxidative stress and reduced immunity. Our previous study found that AFB1 caused oxidative damage and inhibited muscle development of zebrafish. 4-methylesculetin (4-ME), a coumarin derivative, is now used in biochemistry and medicine widely because of its antioxidant function. Whether 4-ME could alleviate the inhibition of muscle development in grass carp induced by AFB1 has not been reported. In this experiment, 720 healthy grass carp (11.40 ± 0.01 g) were randomly divided into 4 groups with 3 replicates of 60 fish each, including control group, AFB1 group (60 μg/kg diet AFB1), 4-ME group (10 mg/kg diet 4-ME), and AFB1+4-ME group (60 μg/kg diet AFB1 + 10 mg/kg 4-ME diet), for a 60 d growth experiment. In vitro, we also set up 4 treatment groups in grass carp primary myoblast, including control group, AFB1 group (15 μmol/L AFB1), 4-ME group (0.5 μmol/L 4-ME) and AFB1+4-ME group (15 μmol/L AFB1+0.5 μmol/L 4-ME). The results showed that dietary AFB1 decreased growth performance of grass carp, damaged the ultrastructure and induced oxidative damage in grass carp muscle, and significantly decreased the mRNA and protein expression levels of myogenin (MyoG), myogenic differentiation (MyoD), myosin heavy chain (MYHC), as well as the protein expression levels of laminin β1, fibronectin and collagen Ⅰ (P < 0.05), significantly activated the protein expression levels of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and phosphorylate-38mitogen-activated protein kinase (p38 MAPK) both in grass carp muscle and grass carp primary myoblast (P < 0.05). Supplementation of AFB1 with 4-ME significantly improved the growth performance inhibition and alleviated the muscle fiber development inhibition and extracellular matrix (ECM) degradation in grass carp induced by AFB1. The present results revealed that supplementation of AFB1 contaminated feed with 4-ME reduced the inhibition of growth and muscle development by alleviating AFB1-induced ECM degradation in grass carp, which might be related to the p38 MAPK/uPA/MMP/ECM pathway. The results implied that 4-ME could be used as a valuable mycotoxin scavenger in animal feed.

Curcumin-activated Wnt5a pathway mediates Ca2+ channel opening to affect myoblast differentiation and skeletal muscle regeneration.

Skeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca2+ channel. Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20μL). In addition, we constructed a Wnt5a knockdown C2C12 cell model and a Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80mg/L) and fed to the mice (50mg/kg). Fluorescence microscopy was used to determine the concentration of Ca2+. Western blot and RT-qPCR were used to detect the protein and mRNA levels of Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining. During myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P<0.001) and the Wnt5a signalling pathway was activated. Wnt5a overexpression promoted the expression of MyoD, Myf5, Myogenin, MHC, and Desmin (P<0.05), and conversely, knockdown of Wnt5a inhibited their expression (P<0.001). The Wnt5a pathway mediated the opening of Ca2+ channels, regulated the expression levels of CaN, NFAT2, MyoD, Myf5, Myogenin, MHC, and Desmin (P<0.01) and promoted the differentiation of C2C12 myoblasts and the repair and regeneration of injured skeletal muscle. The expression of Wnt5a, CaN, NFAT2, MyoD, Myogenin, Myf5, and MHC in C2C12 myoblast was significantly increased after curcumin intervention (P<0.05); however, their expression decreased significantly after knocking down Wnt5a on the basis of curcumin intervention (P<0.05). Similarly, in Wnt5a knockout mice, the promotion of muscle regeneration by curcumin was significantly attenuated. Curcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle.

A method for growing and differentiating the C2C12 muscle cell line in the laboratory

Skeletal muscle-related studies have recently been applied in the combat of obesity and metabolic disorders. Culture skeletal muscle cells in in vitro plays an important role in being a promising model for those researches. In the present study, the C2C12 skeletal muscle cells were grown and differentiated in in vitro. The C2C12 myoblasts were grown in Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) for 4 – 5 days. The cells then reached confluent about 70% to 100% and the medium was shifted to DMEM plus 2% horse serum which leads the grown C2C12 cells becoming to differentiated myotubes. Myogenin mRNA levels were found to be significantly higher in the differentiated myotubes than those in the growing cells confirming the successful formation of the differentiated C2C12 cells. These results indicate that the C2C12 cell line is suitable for culture in in vitro to mimic a skeletal muscle microenvironment for further investigations.

Ursolic acid alleviates cancer cachexia by inhibiting STAT3 signaling pathways in C2C12 myotube and CT26 tumor-bearing mouse model

Cancer cachexia, a multi-organ disorder resulting from tumor and immune system interactions, prominently features muscle wasting and affects the survival of patients with cancer. Ursolic acid (UA) is known for its antioxidant, anti-inflammatory, and anticancer properties. However, its impact on cancer cachexia remains unexplored. This study aimed to assess the efficacy of UA in addressing muscle atrophy and organ dysfunction in cancer cachexia and reveal the mechanisms involved. UA dose-dependently ameliorated C2C12 myotube atrophy. Mechanistically, it inhibited the expression of muscle-specific RING finger containing protein 1 (MURF1) and the phosphorylation of signal transducer and activator of transcription 3 (STAT3), and upregulated the mRNA or protein levels of myogenic differentiation antigen and myogenin in cultured C2C12 myotubes treated with conditioned medium. In vivo, UA protected CT26 tumor-bearing mice against loss of body weight, as well as increased skeletal muscle and epididymal fat without affecting tumor growth. Additionally, UA increased food intake in CT26 tumor-bearing mice. The mRNA expression of tumor necrosis-α and interleukin 6 was significantly downregulated in the intestine, gastrocnemius, and heart tissues following 38 d UA administration. UA treatment reversed the levels of myocardial function indicators, including creatine kinase, creatine kinase-MB, lactate dehydrogenase, car-dial troponin T, and glutathione. Finally, UA treatment significantly inhibited the expression of MURF1, the phosphorylation of nuclear factor kappa-B p65, and STAT3 in the gastrocnemius muscle and heart tissues of cachexic mice. Our findings suggest that UA is a promising natural compound for developing dietary supplements for cancer cachexia therapy owing to its anti-catabolic effects.

All-Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion.

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/β-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin-proteasome system involving ZNRF3.

Glutamine supplementation accelerates functional recovery of EDL muscles after injury by modulating the expression of S100 calcium-binding proteins.

The aim of the current study was to investigate the effect of glutamine supplementation on the expression of HSP70 and the calcium-binding proteins from the S100 superfamily in the recovering extensor digitorum longus (EDL) muscle after injury. Two-month-old Wistar rats were subjected to cryolesion of the EDL muscle and then randomly divided into two groups (with or without glutamine supplementation). Starting immediately after the injury, the supplemented group received daily doses of glutamine (1g/kg/day, via gavage) for 3 and 10days orally. Then, muscles were subjected to histological, molecular, and functional analysis. Glutamine supplementation induced an increase in myofiber size of regenerating EDL muscles and prevented the decline in maximum tetanic strength of these muscles evaluated 10days after injury. An accelerated upregulation of myogenin mRNA levels was detected in glutamine-supplemented injured muscles on day 3 post-cryolesion. The HSP70 expression increased only in the injured group supplemented with glutamine for 3days. The increase in mRNA levels of NF-κB, the pro-inflammatory cytokines IL-1β and TNF-α, and the calcium-binding proteins S100A8 and S100A9 on day 3 post-cryolesion in EDL muscles was attenuated by glutamine supplementation. In contrast, the decrease in S100A1 mRNA levels in the 3-day-injured EDL muscles was minimized by glutamine supplementation. Overall, our results suggest that glutamine supplementation accelerates the recovery of myofiber size and contractile function after injury by modulating the expression of myogenin, HSP70, NF-κB, pro-inflammatory cytokines, and S100 calcium-binding proteins.

5′-CMP and 5′-UMP promote myogenic differentiation and mitochondrial biogenesis by activating myogenin and PGC-1α in a mouse myoblast C2C12 cell line

Ribonucleotides are basic monomeric building blocks for RNA considered as conditionally essential nutrients. They are normally produced in sufficient quantity, but can become insufficient upon stressful challenges. The administration of pyrimidine nucleotides, such as cytidine-5′-monophosphate (5′-CMP) and uridine-5′-monophosphate (5′-UMP), enables rats to endure prolonged exercise. However, the underlying mechanisms have remained elusive. To investigate these mechanisms, we studied the effect of 5′-CMP and 5′-UMP on muscular differentiation and mitochondrial biogenesis in myoblast C2C12 cells. 5′-CMP and 5′-UMP were found to increase the mRNA levels of myogenin, which is a myogenic regulatory protein expressed during the final differentiation step and fusion of myoblasts into myotubes. 5′-CMP and 5′-UMP also promoted myoblast differentiation into myotube cells. 5′-CMP and 5′-UMP further increased the mRNA levels of PGC-1α which regulates mitochondrial biogenesis and skeletal muscle fiber type. In addition, 5′-CMP and 5′-UMP increased mitochondrial DNA copy number and enhanced mRNA levels of slow-muscle myosin heavy chains. Moreover, cytidine and uridine, nucleosides corresponding to 5′-CMP and 5′-UMP, markedly promoted myotube formation in C2C12 cells. Considering the metabolism and absorption of nucleotides, the active bodies underlying the effects observed with 5′-CMP and 5′-UMP could be cytidine and uridine. In conclusion, our results indicate that 5′-CMP and 5′-UMP can promote myogenic differentiation and mitochondrial biogenesis, as well as increase slow-twitch fiber via the activation of myogenin and PGC-1α. In addition, 5′-CMP and 5′-UMP may be considered as safe and effective agents to enhance muscle growth and improve the endurance in skeletal muscles.

Fibrous protein composite scaffolds (3D) for tissue regeneration: An in vitro study on skeletal muscle regeneration

The present study explores the differentiation of myoblasts in bioengineered 3D composite scaffolds containing keratin and gelatin. Based on the composition and rheological properties three different scaffolds namely HM1, HM2 and HM3 were prepared, characterized and employed for the present study. The scaffolds were then subjected to C2C12 myoblasts differentiation under in vitro conditions as per the standard protocols. Results reveal a wide variation in the elastic modulus, water uptake and degradation rate of the scaffolds significantly impact the myogenesis processes. Composite scaffolds HM2 and HM3 ease the myogenesis compared to HM1, wherein, results in nil myogenesis. Among HM2 and HM3, accelerated myogenesis and the significant expression of myogenin mRNA levels along with extensive myotube development were observed in the HM3 scaffold. In conclusion, scaffolds modulus play a vital role in myogenesis and the observations of the present study provide a possible strategy for better skeletal muscle regeneration using composite scaffolds.

Spexin Promotes the Proliferation and Differentiation of C2C12 Cells In Vitro-The Effect of Exercise on SPX and SPX Receptor Expression in Skeletal Muscle In Vivo.

SPX (spexin) and its receptors GalR2 and GalR3 (galanin receptor subtype 2 and galanin receptor subtype 3) play an important role in the regulation of lipid and carbohydrate metabolism in human and animal fat tissue. However, little is still known about the role of this peptide in the metabolism of muscle. The aim of this study was to determine the impact of SPX on the metabolism, proliferation and differentiation of the skeletal muscle cell line C2C12. Moreover, we determined the effect of exercise on the SPX transduction pathway in mice skeletal muscle. We found that increased SPX, acting via GalR2 and GalR3 receptors, and ERK1/2 phosphorylation stimulated the proliferation of C2C12 cells (p < 0.01). We also noted that SPX stimulated the differentiation of C2C12 by increasing mRNA and protein levels of differentiation markers Myh, myogenin and MyoD (p < 0.01). SPX consequently promoted myoblast fusion into the myotubule (p < 0.01). Moreover, we found that, in the first stage (after 2 days) of myocyte differentiation, GalR2 and GalR3 were involved, whereas in the last stage (day six), the effect of SPX was mediated by the GalR3 isoform. We also noted that exercise stimulated SPX and GalR2 expression in mice skeletal muscle as well as an increase in SPX concentration in blood serum. These new insights may contribute to a better understanding of the role of SPX in the metabolism of skeletal muscle.

Knockdown of optineurin controls C2C12 myoblast differentiation via regulating myogenin and MyoD expressions

Mutations in optineurin (OPTN) have been identified in a small proportion of sporadic and familial amyotrophic lateral sclerosis (ALS) cases. Recent evidences suggest that OPTN would be involved in not only the pathophysiological mechanisms of motor neuron death of ALS but also myofiber degeneration of sporadic inclusion body myositis. However, the detailed role of OPTN in muscle remains unclear. Initially, we showed that OPTN expression levels were significantly increased in the denervated muscles of mice, suggesting that OPTN may be involved in muscle homeostasis. To reveal the molecular role of OPTN in muscle atrophy, we used cultured C2C12 myotubes treated with tumor necrosis factor-like inducer of apoptosis (TWEAK) as an in vitro model of muscle atrophy. Our data showed that OPTN had no effect on the process of muscle atrophy in this model. On the other hand, we found that myogenic differentiation was affected by OPTN. Immunoblotting analysis showed that OPTN protein levels gradually decreased during C2C12 differentiation. Furthermore, OPTN knockdown inhibited C2C12 differentiation, accompanied by reduction of mRNA and protein expression levels of myogenin and MyoD. These findings suggested that OPTN may have a novel function in muscle homeostasis and play a role in the pathogenesis of neuromuscular diseases.

Selenium alleviates the negative effect of heat stress on myogenic differentiation of C2C12 cells with the response of selenogenome

With the globe warming, heat stress (HS) has frequently affected animal production. Selenium (Se) is an essential trace element for animals and exerts most of its biological functions through selenoproteins. We previously demonstrated that the damage to C2C12 cells by HS accompanied with the response of selenoprotein encoding genes and proteins. The objective of this study was to investigate whether selenium supplementation (sodium selenite, SS and selenomethionine, SeMet) could alleviate the negative effect of heat stress on the differentiation of C2C12 cells, and interpret the potential corresponding selenoproteins response. The differentiated cells were cultured for 4 and 8 days under different condition: at 37 °C, 41.5 °C and 41.5 °C with 0.5 μmol Se/L SS or SeMet, and the HSP70, cell apoptosis, selenoproteins and cell differentiation-related gene or protein were detected. The result showed that HS up-regulated (P < 0.05) mRNA and protein levels of HSP70 and gene expression of AMPKα1 and AMPKα2, and down-regulated (P < 0.05) mRNA or protein levels of MYOGENIN and MYOD. Meanwhile, up to 15 and 17 selenoprotein genes expression were significantly changed response to 4-and 8-days HS challenge, respectively. Relative to the HS group, SS and SeMet supplementation down-regulated the mRNA and protein abundance of HSP70 to different degrees, and partly recovered (P < 0.05) the mRNA or protein abundance of MYOGENIN and MYOD at 4th and 8th day. Especially, 16 and 10 selenoprotein genes expression in cells affected by HS were altered by SS and SeMet supplementation, respectively. Both SS and SeMet supplementation modestly increased (P < 0.05) protein levels of GPX1 and SELENON in cells under HS. In summary, Se supplementation partly alleviated the negative impact of HS on myogenic differentiation of C2C12 cells and the process may associate with the alternation of selenoprotein expression pattern, and SeMet exhibits better effect than SS.

PUFA Treatment Affects C2C12 Myocyte Differentiation, Myogenesis Related Genes and Energy Metabolism.

Polyunsaturated fatty acids (PUFAs) are the main components of cell membrane affecting its fluidity, signaling processes and play a vital role in muscle cell development. The effects of docosahexaenoic acid (DHA) on myogenesis are well known, while the effects of arachidonic acid (AA) are largely unclear. The purpose of this study is to evaluate the effect of two PUFAs (DHA and AA) on cell fate during myogenic processes, Wnt signaling and energy metabolism by using the C2C12 cells. The cells were treated with different concentrations of AA or DHA for 48 h during the differentiation period. PUFA treatment increased mRNA level of myogenic factor 5 (Myf5), which is involved in early stage of myoblast proliferation. Additionally, PUFA treatment prevented myoblast differentiation, indicated by decreased myotube fusion index and differentiation index in parallel with reduced mRNA levels of myogenin (MyoG). After PUFA withdrawal, some changes in cell morphology and myosin heavy chain mRNA levels were still observed. Expression of genes associated with Wnt signaling pathway, and energy metabolism changed in PUFA treatment in a dose and time dependent manner. Our data suggests that PUFAs affect the transition of C2C12 cells from proliferation to differentiation phase by prolonging proliferation and preventing differentiation.

Role of plasminogen activator inhibitor-1 in muscle wasting induced by a diabetic state in female mice.

Muscle wasting is a complication in patients with diabetes and leads to a reduced quality of life. However, the detailed mechanisms of diabetes-induced muscle wasting remain unknown. Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that suppresses plasminogen activator activity, is involved in the pathophysiology of various diseases, including diabetes. In the present study, we examined the role of endogenous PAI-1 in the decrease in muscle mass and the impaired grip strength induced by the diabetic state by employing streptozotocin (STZ)-treated PAI-1-deficient female mice. The analyses of skeletal muscles and grip strength were performed in PAI-1-deficient and wild-type mice 4 weeks after the induction of a diabetic state by STZ administration. PAI-1 deficiency did not affect muscle mass in the lower limbs measured by quantitative computed tomography or tissue weights of the tibialis anterior, gastrocnemius and soleus muscles of female mice with or without STZ treatment. On the other hand, PAI-1 deficiency significantly aggravated grip strength decreased by STZ in female mice. PAI-1 deficiency did not affect the mRNA levels of Pax7, MyoD, myogenin or myosin heavy chain in either the tibialis anterior or soleus muscles of female mice with or without STZ treatment. In conclusion, we revealed for the first time that PAI-1 deficiency aggravates grip strength impaired by the diabetic state in female mice, although it did not affect diabetes-decreased muscle mass.

Acute environmental hypoxia potentiates satellite cell-dependent myogenesis in response to resistance exercise through the inflammation pathway in human.

Acute environmental hypoxia may potentiate muscle hypertrophy in response to resistance training but the mechanisms are still unknown. To this end, twenty subjects performed a 1-leg knee extension session (8 sets of 8 repetitions at 80% 1 repetition maximum, 2-min rest between sets) in normoxic or normobaric hypoxic conditions (FiO2 14%). Muscle biopsies were taken 15 min and 4 hours after exercise in the vastus lateralis of the exercised and the non-exercised legs. Blood samples were taken immediately, 2h and 4h after exercise. In vivo, hypoxic exercise fostered acute inflammation mediated by the TNFα/NF-κB/IL-6/STAT3 (+333%, +194%, + 163% and +50% respectively) pathway, which has been shown to contribute to satellite cells myogenesis. Inflammation activation was followed by skeletal muscle invasion by CD68 (+63%) and CD197 (+152%) positive immune cells, both known to regulate muscle regeneration. The role of hypoxia-induced activation of inflammation in myogenesis was confirmed in vitro. Acute hypoxia promoted myogenesis through increased Myf5 (+300%), MyoD (+88%), myogenin (+1816%) and MRF4 (+489%) mRNA levels in primary myotubes and this response was blunted by siRNA targeting STAT3. In conclusion, our results suggest that hypoxia could improve muscle hypertrophic response following resistance exercise through IL-6/STAT3-dependent myogenesis and immune cells-dependent muscle regeneration.

Activation of protein synthesis, regeneration, and MAPK signaling pathways following repeated bouts of eccentric cycling

The aim of this study was to examine the activation of skeletal muscle signaling pathways related to protein synthesis and the gene expression of regeneration/degradation markers following repeated bouts of eccentric cycling. Nine untrained men (25.4 ± 1.9 yr) performed two 30-min eccentric cycling bouts (ECC1, ECC2) at 85% of maximal concentric workload, separated by 2 wk. Muscle biopsies were taken from the vastus lateralis before and 2 h after each bout. Indirect markers of muscle damage were assessed before and 24-48 h after exercise. Changes in the Akt/mammalian target of rapamycin (mTOR)/rbosomal protein S6 kinase 1 (S6K1)/ribosomal protein S6 (rpS6) and MAPK signaling pathways were measured by Western blot and changes in mRNA expression of IL-6 and IL-1β, and myogenic regulatory factors (MRFs) were measured by real-time PCR. ECC1 induced greater increases in indirect markers of muscle damage compared with ECC2. Phosphorylation of S6K1 and rpS6 increased after both exercise bouts (P < 0.05), whereas phosphorylation of mTOR increased after ECC2 only (P = 0.03). Atrogin-1 mRNA expression decreased after ECC1 and ECC2 (P < 0.05) without changes in muscle RING-finger protein-1 mRNA. Basal mRNA levels of myoblast determination protein-1 (MyoD), MRF4, and myogenin were higher 2 wk after ECC1 (P < 0.05). MRF4 mRNA increased after ECC1 and ECC2 (P < 0.05), whereas MyoD mRNA expression increased only after ECC1 (P = 0.03). Phosphorylation of JNK and p38 MAPK increased after both exercise bouts (P < 0.05), similar to IL-6 and IL-1β mRNA expression. All together, these results suggest that differential regulation of the mTOR pathway and MRF expression could mediate the repeated bout effect observed between an initial and secondary bout of eccentric exercise.

The influence of direct and indirect fibroblast cell contact on human myogenic cell behavior and gene expression in vitro.

Underpinning skeletal muscle plasticity is the interplay between many cell types, of which fibroblasts are emerging as potent players, both negatively in the development of fibrosis but also positively in stimulating muscle repair through enhancing myogenesis. The mechanisms behind this interaction however remain unknown. To investigate this, waste hamstring muscle tissue was obtained from eight healthy young men undergoing reconstructive anterior cruciate ligament surgery and primary myoblasts and fibroblasts were isolated. Myoblasts were cultured alone or with fibroblasts, either in direct or indirect contact (separated by an insert with a permeable membrane). The myogenesis parameters proliferation, differentiation, and fusion were determined from immunostained cells, while, in replicate samples, gene expression levels of GAPDH, Ki67, Pax7, MyoD, myogenin, myomaker, MHC-Iβ, TCF7L2, COL1A1, and p16 were determined by RT-PCR. We found only trends for an influence of skeletal muscle fibroblasts on myogenic cell proliferation and differentiation. While greater mRNA levels of GAPDH, Pax7, MyoD, myogenin, and MHC-Iβ were observed in myogenic cells in indirect contact with fibroblasts (insert) when compared with cells cultured alone, a similar effect of an empty insert was also observed. In conclusion we find very little influence of skeletal muscle fibroblasts on myoblasts derived from the same tissue, although it cannot be excluded that a different outcome would be seen under less optimal myogenic growth conditions.NEW & NOTEWORTHY Using passage one primary myoblasts and fibroblasts isolated from human skeletal muscle, we found only a trend for an effect of skeletal muscle fibroblasts on myogenic cell proliferation and differentiation. This is contrary to previous reports and raises the possibility that fibroblasts of different tissue origins exert distinct roles.

Angiotensin-(1\u20137) exerts a protective action in a rat model of ventilator-induced diaphragmatic dysfunction

BackgroundVentilator-induced diaphragmatic dysfunction (VIDD) is a common event during mechanical ventilation (MV) leading to rapid muscular atrophy and contractile dysfunction. Recent data show that renin-angiotensin system is involved in diaphragmatic skeletal muscle atrophy after MV. In particular, angiotensin-II can induce marked diaphragm muscle wasting, whereas angiotensin-(1–7) (Ang-(1–7)) could counteract this activity. This study was designed to evaluate the effects of the treatment with Ang-(1–7) in a rat model of VIDD with neuromuscular blocking agent infusion. Moreover, we studied whether the administration of A-779, an antagonist of Ang-(1–7) receptor (Mas), alone or in combination with PD123319, an antagonist of AT2 receptor, could antagonize the effects of Ang-(1–7).MethodsSprague-Dawley rats underwent prolonged MV (8 h), while receiving an iv infusion of sterile saline 0.9% (vehicle) or Ang-(1–7) or Ang-(1–7) + A-779 or Ang-(1–7) + A-779 + PD123319. Diaphragms were collected for ex vivo contractility measurement (with electric stimulation), histological analysis, quantitative real-time PCR, and Western blot analysis.ResultsMV resulted in a significant reduction of diaphragmatic contractility in all groups of treatment. Ang-(1–7)-treated rats showed higher muscular fibers cross-sectional area and lower atrogin-1 and myogenin mRNA levels, compared to vehicle treatment. Treatment with the antagonists of Mas and Ang-II receptor 2 (AT2R) caused a significant reduction of muscular contractility and an increase of atrogin-1 and MuRF-1 mRNA levels, not affecting the cross-sectional fiber area and myogenin mRNA levels.ConclusionsSystemic Ang-(1–7) administration during MV exerts a protective role on the muscular fibers of the diaphragm preserving muscular fibers anatomy, and reducing atrophy. The involvement of Mas and AT2R in the mechanism of action of Ang-(1–7) still remains controversial.

Leucine promotes differentiation of porcine myoblasts through the protein kinase B (Akt)/Forkhead box O1 signalling pathway.

Leucine, one of the branched-chain amino acids, is the only amino acid to regulate protein turnover in skeletal muscle. Leucine not only increases muscle protein synthesis, but also decreases muscle protein degradation. It is well documented that leucine plays a positive role in differentiation of murine muscle cells. However, the role of leucine on porcine myoblast differentiation and its mechanism remains unclear. In this study, porcine myoblasts were induced to differentiate with differentiation medium containing different concentrations of leucine, and wortmannin was used to interdict the activity of protein kinase B (Akt). We found that leucine increased the number of myosin heavy chain-positive cells and creatine kinase activity. Moreover, leucine increased the mRNA and protein levels of myogenin and myogenic determining factor (MyoD). In addition, leucine increased the levels of phosphorylated Akt/Akt and phosphorylated Forkhead box O1 (P-FoxO1)/FoxO1, as well as decreased the protein level of FoxO1. However, wortmannin, a specific repressor of PI3K/Akt signalling pathway, attenuated the positive role of leucine on porcine myoblast differentiation. Our results suggest that leucine promotes porcine myoblast differentiation through the Akt/FoxO1 signalling pathway.

Early molecular response and microanatomical changes in the masseter muscle and mandibular head after botulinum toxin intervention in adult mice

BackgroundMasseter muscle paralysis induced by botulinum toxin type A (BoNTA) evokes subchondral bone loss in mandibular heads of adult rats and growing mice after 4 weeks. However, the primary cellular and molecular events leading to altered bone remodeling remain unexplored. Thus, the aim of the current work has been to assess the molecular response that precedes the early microanatomical changes in the masseter muscle and subchondral bone of the mandibular head in adult mice after BoNTA intervention. MethodsA pre-clinical in vivo study was performed by a single intramuscular injection of 0.2 U BoNTA in the right masseter (experimental) of adult BALB/c mice. The contralateral masseter was injected with vehicle (control). Changes in mRNA levels of molecular markers of bone loss or muscle atrophy/regeneration were addressed by qPCR at day 2 or 7, respectively. mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL) was assessed in mandibular heads, whilst mRNA levels of Atrogin-1/MAFbx, MuRF-1 and Myogenin were addressed in masseter muscles. In order to identify the early microanatomical changes at day 14, fiber diameters in transversal sections of masseter muscles were quantified, and histomorphometric analysis was used to determine the bone per tissue area and the trabecular thickness of subchondral bone of the mandibular heads. ResultsAn increase of up to 4-fold in RANKL mRNA levels were detected in mandibular heads of the BoNTA-injected sides as early as 2 days after intervention. Moreover, a 4–6 fold increase in Atrogin-1/MAFbx and MuRF-1 and an up to 25 fold increase in Myogenin mRNA level were detected in masseter muscles 7 days after BoNTA injections. Masseter muscle mass, as well as individual muscle fiber diameter, were significantly reduced in BoNTA-injected side after 14 days post-intervention. At the same time, in the mandibular heads from the treated side, the subchondral bone loss was evinced by a significant reduction in bone per tissue area (−40%) and trabecular thickness (−55%). ConclusionsOur results show that masseter muscle paralysis induced by BoNTA leads to significant microanatomical changes by day 14, preceded by molecular changes as early as 2 days in bone, and 7 days in muscle. Therefore, masseter muscle atrophy and subchondral bone loss detected at 14 days are preceded by molecular responses that occur during the first week after BoNTA intervention.

Extracellular Vesicles Released by Oxidatively Injured or Intact C2C12 Myotubes Promote Distinct Responses Converging toward Myogenesis.

Myogenic differentiation is triggered, among other situations, in response to muscle damage for regenerative purposes. It has been shown that during myogenic differentiation, myotubes release extracellular vesicles (EVs) which participate in the signalling pattern of the microenvironment. Here we investigated whether EVs released by myotubes exposed or not to mild oxidative stress modulate the behaviour of targeted differentiating myoblasts and macrophages to promote myogenesis. We found that EVs released by oxidatively challenged myotubes (H2O2-EVs) are characterized by an increased loading of nucleic acids, mainly DNA. In addition, incubation of myoblasts with H2O2-EVs resulted in a significant decrease of myotube diameter, myogenin mRNA levels and myosin heavy chain expression along with an upregulation of proliferating cell nuclear antigen: these effects collectively lead to an increase of recipient myoblast proliferation. Notably, the EVs from untreated myotubes induced an opposite trend in myoblasts, that is, a slight pro-differentiation effect. Finally, H2O2-EVs were capable of eliciting an increased interleukin 6 mRNA expression in RAW264.7 macrophages. Notably, this is the first demonstration that myotubes communicate with surrounding macrophages via EV release. Collectively, the data reported herein suggest that myotubes, depending on their conditions, release EVs carrying differential signals which could contribute to finely and coherently orchestrate the muscle regeneration process.