Increased Fusion Index Research Articles

Hesperetin but not ellagic acid increases myosin heavy chain expression and cell fusion in C2C12 myoblasts in the presence of oxidative stress.

Skeletal muscle regeneration is impaired in elderly. An oxidative stress-induced decrease in differentiation capacity of muscle satellite cells is a key factor in this process. The aim of this study is to investigate whether orange polyphenol hesperetin and pomegranate polyphenol ellagic acid enhance myoblast differentiation in the presence and absence of oxidative stress, and to explore underlying mechanisms. C2C12 myoblasts were proliferated for 24 h and differentiated for 120 h while exposed to hesperetin (5, 20, 50 μM), ellagic acid (0.05, 0.1 μM) or a combination (20 μM hesperetin, 0.05 μM ellagic acid) with and without oxidative stress-inducing compound menadione (9 μM) during 24 h of proliferation and during the first 5 h of differentiation. The number of proliferating cells was assessed using fluorescent labeling of incorporated 5-ethynyl-2'-deoxyuridine. Myosin heavy chain expression was assessed by fluorescence microscopy and cell fusion index was calculated. Furthermore, protein expression of phosphorylated p38 and myomixer were assessed using Western blot. None of the compounds induced effects on cell proliferation. Without menadione, 50 μM hesperetin increased fusion index by 12.6% compared to control (p < 0.01), while ellagic acid did not affect measured parameters of differentiation. Menadione treatment did not change myosin heavy chain expression and fusion index. In combination with menadione, 20 μM hesperetin increased myosin heavy chain expression by 35% (p < 0.01) and fusion index by 7% (p = 0.04) compared to menadione. Furthermore, the combination of menadione with hesperetin and ellagic acid increased myosin heavy chain expression by 35% compared to menadione (p = 0.02). Hesperetin and ellagic acid did not change p38 phosphorylation and myomixer expression compared to control, while treatment with menadione increased p38 phosphorylation (p < 0.01) after 5 h and decreased myomixer expression (p = 0.04) after 72 h of differentiation. Hesperetin increased myosin heavy chain expression in the presence of oxidative stress induced by menadione, and increased cell fusion both in the presence and absence of menadione. Ellagic acid did not affect the measured parameters of myoblast differentiation. Therefore, hesperetin should be considered as nutritional prevention or treatment strategy to maintain muscle function in age-related diseases such as sarcopenia. Future research should focus on underlying mechanisms and translation of these results to clinical practice.

Extracellular vesicle miR-206 improves chronic binge alcohol mediated decreased myoblast differentiation in SIV-infected female macaques.

Alcohol misuse in people with HIV (PWH) and chronic binge alcohol (CBA) administration in simian immunodeficiency virus (SIV)-infected macaques are associated with increased physical frailty and impaired functional skeletal muscle mass, respectively. Previous studies by our group demonstrate that muscle-enriched microRNAs (myomiRs) are differentially expressed in skeletal muscle (SKM) from CBA-administered SIV-infected male macaques and their altered expression contributes to impaired differentiation of SKM stem cells, or myoblasts. MicroRNAs can be transported in extracellular vesicles (EVs) to mediate numerous cellular responses through intercellular communication. The current study tested the hypothesis that EV-mediated delivery of miR-206 can ameliorate CBA-mediated decreased myoblast differentiation. Myoblasts were isolated from SKM of female SIV-infected, antiretroviral therapy-treated macaques that received either CBA (2.5g/kg/day, CBA/SIV) or water (VEH/SIV) for 14.5 months. Myotube and myotube derived EV myomiR expression, including miR-206, was lower in the CBA/SIV group. Overexpression of miR-206 decreased histone deacetylase 4 (HDAC4) and paired box 7 (PAX7) expression in myotubes and increased fusion index, a differentiation index, in CBA/SIV-derived myotubes. Similarly, EV-mediated delivery of miR-206 increased both fusion index and myotube density of CBA/SIV-derived myoblasts. These results support the potential therapeutic utility of EVs in delivering myomiRs to improve SKM stem cell differentiation.

Liposome Loaded Prostaglandin E2 (PGE2) for Muscle Regeneration

Prostaglandin E2 (PGE2) is an FDA approved lipid signaling molecule for range of disorders. Also, it has a significant therapeutic potential for tissue repair and regeneration. However, to date there are no applications of PGE2 specifically designed for the muscle repair and regeneration. Liposome systems offer several advantages for PGE2 delivery, improved drug stability, reduced side effects, and tissue-specific targeting. Our hypothesis is that encapsulating PGE2 in liposome system will significantly enhance its bioavailability and maintain an effective therapeutic level. The research was designed to optimize PGE2 concentration in muscle cells, study its effect on cells differentiation, use the optimized PGE2 concentration for liposome-loading and testing the delivery system in-vitro and in-vivo using muscle injury models. We further investigated the molecular mechanism and signaling pathways triggered by the intracellular elevation of PGE2 in muscle cells. Optimization results revealed that nM concentration of PGE2 was beneficial while μM concentrations were detrimental. All our studies reported here were conducted with 50nM PGE2. 50nM PGE2 significantly enhances proliferation and myogenic differentiation as indicated by increased fusion index ( p=0.02) compared to a control. Scanning Electron Microscopy (SEM) images confirmed the lipid bi-layer formation with core-shell structure of 3μm liposomes containing PGE2. In-vitro studies on the C2C12 muscles cells showed that PGE2-liposome system significantly increased the intracellular concentration of PGE2 compared to a PGE2-vehicle (DMSO) control ( p=0.019), confirming the effcacy of this delivery system. Additionally, 50nM PGE2 significantly regulated myogenic signaling pathways and genes associated with calcium homeostasis, mitochondrial biogenesis, cellular hypertrophy, and oxidative stress. These findings support the notion that PGE2 induces the acceleration of myogenic differentiation which is supported by our initial signaling pathway analysis. Our in-vitro muscle damage model indicated that 50nM PGE2 can significantly attenuate the muscle damage and regenerate myotubes within 48 hours compared to 4 days in normal control. In conclusion, the findings suggest that liposome-PGE2 delivery system can positively influence myogenic differentiation, muscle properties, and cellular signaling pathways, offering promise for improving the delivery of PGE2 to injured skeletal muscles to assist in their regeneration process. This work was directly supported by NIH-National Institutes of Aging P01AG039355 (MB) and the George W. and Hazel M. Jay and Evanston Research Endowments (MB). Authors were supported by NIH Grants: National Institutes of Aging (NINDS) 2-R01NS105621; NIA-R01AG056504, NIA-2R01AG060341, National Institutes of Diabetes, Digestive, and Kidney Diseases Kidney (NIDDK)-1R01DK119066 to MB, and National Institutes of Neurological Disorders and Stroke (NINDS) 2-R01NS105621 to MB. Also, NIH/NIDC 1R01DE031872-01 (VV). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acetyl genistin modulates myotube differentiation and attenuates dexamethasone-induced muscle atrophy through the FoxO1/3 signaling pathway in C2C12 myotubes

Muscle atrophy, a debilitating condition characterized by loss of muscle mass and strength, is a major concern in various clinical settings. Acetyl genistin (AG), a bioactive compound, was evaluated for its role in muscle cell differentiation and its potential protective effects against dexamethasone (dexa)-induced muscle atrophy. Our study demonstrated that AG significantly promoted C2C12 myotube differentiation, as evidenced by enhanced myotube width and increased fusion index. Notably, AG treatment upregulated the expression of myogenic markers, including MHC, MyoD, and MyoG. Moreover, AG displayed protective properties by attenuating dexa-induced muscle atrophy, mainly by suppressing the expression of the atrophy-related genes MAFbx and MuRF1. AG's protective effects are mechanistically attributed to its regulation of the AMPK/FoxO-dependent signaling pathway. Our results highlighted the dual benefits of AG in fostering muscle differentiation and safeguarding against muscle atrophy, positioning it as a promising agent for muscle health and therapeutic applications.

BIO101 stimulates myoblast differentiation and improves muscle function in adult and old mice.

Muscle aging is associated with a consistent decrease in the ability of muscle tissue to regenerate following intrinsic muscle degradation, injury or overuse. Age-related imbalance of protein synthesis and degradation, mainly regulated by AKT/mTOR pathway, leads to progressive loss of muscle mass. Maintenance of anabolic and regenerative capacities of skeletal muscles may be regarded as a therapeutic option for sarcopenia and other muscle wasting diseases. Our previous studies have demonstrated that BIO101, a pharmaceutical grade 20-hydroxyecdysone, increases protein synthesis through the activation of MAS receptor involved in the protective arm of renin-angiotensin-aldosterone system. The purpose of the present study was to assess the anabolic and pro-differentiating properties of BIO101 on C2C12 muscle cells in vitro and to investigate its effects on adult and old mice models in vivo. The effects of BIO101 on C2C12 differentiation were assessed using myogenic transcription factors and protein expression of major kinases of AKT/mTOR pathway by Western blot. The in vivo effects of BIO101 have been investigated in BIO101 orally-treated (50mg/kg/day) adult mice (3months) for 28days. To demonstrate potential beneficial effect of BIO101 treatment in a sarcopenic mouse model, we use orally treated 22-month-old C57Bl6/J mice, for 14weeks with vehicle or BIO101. Mice body and muscle weight were recorded. Physical performances were assessed using running capacity and muscle contractility tests. Anabolic properties of BIO101 were confirmed by the rapid activation of AKT/mTOR, leading to an increase of C2C12 myotubes diameters (+26%, P<0.001). Pro-differentiating effects of BIO101 on C2C12 myoblasts were revealed by increased expression of muscle-specific differentiation transcription factors (MyoD, myogenin), resulting in increased fusion index and number of nuclei per myotube (+39% and +53%, respectively, at day 6). These effects of BIO101 were like those of angiotensin (1-7) and were abolished with the use of A779, a MAS receptor specific antagonist. Chronic BIO101 oral treatment induced AKT/mTOR activation and anabolic effects accompanied with improved physical performances in adult and old animals (maximal running distance and maximal running velocity). Our data suggest beneficial anabolic and pro-differentiating effects of BIO101 rendering BIO101 a potent drug candidate for treating sarcopenia and possibly other muscle wasting disorders.

Shear stress in the intervillous space promotes syncytial formation of iPS cells-derived trophoblasts†.

The intervillous space of human placenta is filled with maternal blood, and villous trophoblasts are constantly exposed to the shear stress generated by maternal blood pressure and flow throughout the entire gestation period. However, the effects of shear stress on villous trophoblasts and their biological significance remain unknown. Here, using our recently established naïve human pluripotent stem cells-derived cytotrophoblast stem cells (nCTs) and a device that can apply arbitrary shear stress to cells, we investigated the impact of shear stress on early-stage trophoblasts. After 72h of exposure to 10dyn/cm2 shear stress, nCTs became fused and multinuclear, and mRNA expression of the syncytiotrophoblast (ST) markers, such as glial cell missing 1, endogenous retrovirus group W member 1 envelope, chorionic gonadotropin subunit beta 3, syndecan 1, pregnancy specific beta-1-glycoprotein 3, placental growth factor, and solute carrier family 2 member 1 were significantly upregulated compared to static conditions. Immunohistochemistry showed that shear stress increased fusion index, human chorionic gonadotropin secretion, and human placental lactogen secretion. Increased microvilli formation on the surface of nCTs under flow conditions was detected using scanning electron microscopy. Intracellular cyclic adenosine monophosphate significantly increased under flow conditions. Moreover, transcriptome analysis of nCTs subjected to shear stress revealed that shear stress upregulated ST-specific genes and downregulated CT-specific genes. Collectively, these findings indicate that shear stress promotes the differentiation of nCTs into ST.

IGF-1 Signaling Regulates Mitochondrial Remodeling during Myogenic Differentiation.

Skeletal muscle is essential for locomotion, metabolism, and protein homeostasis in the body. Mitochondria have been considered as a key target to regulate metabolic switch during myo-genesis. The insulin-like growth factor 1 (IGF-1) signaling through the AKT/mammalian target of rapamycin (mTOR) pathway has a well-documented role in promoting muscle growth and regeneration, but whether it is involved in mitochondrial behavior and function remains un-examined. In this study, we investigated the effect of IGF-1 signaling on mitochondrial remodeling during myogenic differentiation. The results demonstrated that IGF-1 signaling stimulated mitochondrial biogenesis by increasing mitochondrial DNA copy number and expression of genes such as Cox7a1, Tfb1m, and Ppargc1a. Moreover, the level of mitophagy in differentiating myoblasts elevated significantly with IGF-1 treatment, which contributed to mitochondrial turnover. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) were identified as two key mediators of IGF-1-induced mitochondrial biogenesis and mitophagy, respectively. In addition, IGF-1 supplementation could alleviate impaired myoblast differentiation caused by mitophagy deficiency, as evidenced by increased fusion index and myosin heavy chain expression. These findings provide new insights into the role of IGF-1 signaling and suggest that IGF-1 signaling can serve as a target for the research and development of drugs and nutrients that support muscle growth and regeneration.

2,6-Dimethoxy-1,4-benzoquinone increases skeletal muscle mass and performance by regulating AKT/mTOR signaling and mitochondrial function

Background2,6-Dimethoxy-1,4-benzoquinone (DMBQ), a natural phytochemical present in fermented wheat germ, has been reported to exert anti-cancer, anti-inflammatory, and anti-adipogenic effects. However, the effect of DMBQ on muscle hypertrophy and myoblast differentiation has not been elucidated. PurposeWe investigated the effect of DMBQ on skeletal muscle mass and muscle function and then determined the possible mechanism of DMBQ. MethodsTo examine myogenic differentiation and hypertrophy, confluent C2C12 cells were incubated in differentiation medium with or without various concentrations of DMBQ for 4 days. In animal experiments, C57BL/6 mice were fed DMBQ-containing AIN-93 diet for 7 weeks. Grip strength, treadmill, microscopic evaluation of muscle tissue, western blotting, and quantitative real-time PCR were performed. ResultsDMBQ significantly increased fusion index, myotube size, and the protein expression of myosin heavy chain (MHC). DMBQ increased the phosphorylation of protein kinase B (AKT) and p70 ribosomal protein S6 kinase (S6K), whereas the phosphorylation of these proteins was abolished by the phosphoinositide 3-kinase inhibitor LY294002 in C2C12 cells. In addition, DMBQ treatment increased peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), which programs mitochondrial biogenesis, protein levels compared with control C2C12 cells. DMBQ significantly increased maximal respiration and spare respiratory capacity in C2C12 cells. In animal experiments, DMBQ increased skeletal muscle weights and skeletal muscle fiber size compared with the control group values. In addition, the DMBQ group showed increased grip strength and running distance on an accelerating treadmill. The protein expression of total MHC, MHC1, MHC2A, and MHC2B in skeletal muscle was upregulated by DMBQ supplementation. We found that DMBQ increased the phosphorylation of AKT and mammalian target of rapamycin (mTOR), as well as downstream S6K and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in skeletal muscle. DMBQ also stimulated mRNA expression of PGC1α, accompanied by an increase in mitochondrial DNA content, oxidative phosphorylation (OXPHOS) proteins, and oxidative enzyme activity. ConclusionCollectively, DMBQ was shown to increase skeletal muscle mass and performance by regulating the AKT/mTOR signaling pathway and enhancing mitochondrial function, which might be useful for the treatment and prevention of skeletal muscle atrophy.

Modulation of Ki67 and myogenic regulatory factor expression by tocotrienol-rich fraction ameliorates myogenic program of senescent human myoblasts.

IntroductionReplicative senescence results in dysregulation of cell proliferation and differentiation, which plays a role in the regenerative defects observed during age-related muscle atrophy. Vitamin E is a well-known antioxidant, which potentially ameliorates a wide range of age-related manifestations. The aim of this study was to determine the effects of tocotrienol-rich fraction (TRF) in modulating the expression of proliferation- and differentiation-associated proteins in senescent human myoblasts during the differentiation phase.Material and methodsHuman skeletal muscle myoblasts were cultured until senescence. Young and senescent cells were treated with TRF for 24 h before and after differentiation induction, followed by evaluation of cellular morphology and efficiency of differentiation. Expression of cell proliferation marker Ki67 protein and myogenic regulatory factors MyoD and myogenin were determined.ResultsOur findings showed that treatment with TRF significantly improved the morphology of senescent myoblasts. Promotion of differentiation was observed in young and senescent myoblasts with TRF treatment as shown by the increased fusion index and larger size of myotubes. Increased Ki67 and myogenin expression with TRF treatment was also observed in senescent myoblasts, suggesting amelioration of the myogenic program by TRF during replicative senescence.ConclusionsTRF modulates the expression of regulatory factors related to proliferation and differentiation in senescent human myoblasts and could be beneficial for ameliorating the regenerative defects during aging.

P.130MyoscreenTM, a drug discovery platform for disease modeling and the screening of compounds restoring muscle health

Effective treatments are missing for degenerative muscle disorders such as DMD because the current in vitro muscle pathological models lack physiology relevance and pharmacology predictivity. We recently reported MyoscreenTM human patient-derived skeletal myotubes that demonstrate sarcomeric organization, membrane-localized dystrophin and acetylcholine receptor (AChR) expression, response to chemical and electrical stimulation and pharmacologically relevant drug responses. The objective of the present study was to employ our extensive panel of patient derived myoblasts for disease modeling and assays pertinent to muscle biology such as myotube differentiation efficiency, AChR clustering, calcium homeostasis and contraction. To do so, a high-content screen for compounds that stimulate healthy myotube differentiation was performed. Hits were confirmed in several donors and potency determined in dose-response studies. Functional assays exploited calcium imaging techniques and the FDSS/µCELL from Hamamatsu for electrical stimulation. Out of 2560 pharmacologically active or FDA-approved drugs, 29 (1.1%) increased fusion index and/or myotube size by >40%. Molecules with previously established links to regeneration processes were identified (eg. thyroid hormone analogues), as well as CYT9733 that efficiently enhanced healthy and dystrophic myotube formation with an EC50 of 0.002-2µM. Other effects of CYT9733 include increased AChR clusters and intracellular calcium flux during excitation-contraction coupling upon ACh or electrical stimulation, intensifying myotube contraction. CYT9733 is an especially good candidate molecule for regulating changes in muscle regeneration phenotype and of further interest for preclinical testing. The results illustrate predictivity of this disease modeling and drug discovery platform, baptized MyoScreen, its capacity to select active compounds and provide early investigation of mechanisms of action involved.

All-trans retinoic acid regulates sheep primary myoblast proliferation and differentiation in vitro

Vitamin A and its metabolite, retinoic acid (RA), play key roles in cell differentiation and organ morphogenesis. The objective of this study was to investigate the effect of RA on sheep primary myoblast proliferation and differentiation. Sheep primary myoblasts were isolated and treated with all-trans retinoic acid (ATRA, 10 nM and 100 nM) and vehicle. The results showed that 10 nM ATRA sufficiently inhibited cell proliferation, which might be through downregulation of cyclin D1 (P < 0.05) and cyclin-dependent kinase 4 (P < 0.05) and proliferating cell nuclear antigen protein (P < 0.05) abundance. Moreover, compared with control cells, both 10 nM and 100 nM ATRA promoted myotube formation and increased fusion index (P < 0.05), which was associated with elevated myogenin mRNA content (P < 0.05). As expected, both myogenin (P < 0.01) and myosin heavy chain (P < 0.05) protein levels were increased by ATRA. Interestingly, ATRA treatment increased H3K4me3 and decreased H3K27me3 enrichment in the myogenin promoter region (P < 0.05). Meanwhile, 100 nM ATRA stimulated 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-deoxyglucose uptake (P < 0.05) and upregulated glucose transporter 4 expression at both mRNA and protein levels (P < 0.05). Although ATRA did not alter p38 content, phospho-p38 content was increased (P < 0.01). In addition, ATRA treatment activated the mTOR signaling pathway (P < 0.05). Taken together, these results demonstrated that ATRA plays an important role in regulating sheep myoblast proliferation and myogenic differentiation and suggested vitamin A as a potential target for manipulating muscle growth efficiency in sheep industry.

Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion.

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

Radicicol enhances the regeneration of skeletal muscle injured by crotoxin via decrease of NF-kB activation

This study evaluated cellular and molecular effects of radicicol, a heat shock protein (HSP) inducer, on the regeneration of skeletal muscle injured by crotoxin, the main toxin isolated from Crotalus durissus terrificus venom. Regenerating muscles treated with radicicol had decreased NF-kB activation. Differentiating myoblasts treated with radicicol showed reduced number of NF-kB positive nuclei and increased fusion index. The results suggest that radicicol enhances regeneration of muscle by attenuating NF-kB activation and increasing myogenic differentiation.

Chlorella vulgaris Improves the Regenerative Capacity of Young and Senescent Myoblasts and Promotes Muscle Regeneration.

Sarcopenia is characterized by the loss of muscle mass, strength, and function with ageing. With increasing life expectancy, greater attention has been given to counteracting the effects of sarcopenia on the growing elderly population. Chlorella vulgaris, a microscopic, unicellular, green alga with the potential for various pharmaceutical uses, has been widely studied in this context. This study is aimed at determining the effects of C. vulgaris on promoting muscle regeneration by evaluating myoblast regenerative capacity in vitro. Human skeletal myoblast cells were cultured and underwent serial passaging into young and senescent phases and were then treated with C. vulgaris, followed by the induction of differentiation. The ability of C. vulgaris to promote myoblast differentiation was analysed through cellular morphology, real-time monitoring, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation, myogenin expression, and cell cycle profiling. The results obtained showed that senescent myoblasts exhibited an enlarged and flattened morphology, with increased SA-β-gal expression, reduced myogenic differentiation, decreased expression of myogenin, and an increased percentage of cells in the G0/G1 phase. Treatment with C. vulgaris resulted in decreased SA-β-gal expression and promotion of myogenic differentiation, as observed via an increased fusion index, maturation index, myotube size, and surface area and an increased percentage of cells that stained positive for myogenin. In conclusion, C. vulgaris improves the regenerative capacity of young and senescent myoblasts and promotes myoblast differentiation, indicating its potential to promote muscle regeneration.

FHL3 negatively regulates the differentiation of skeletal muscle satellite cells in chicken.

As a member of four and a half LIM domain proteins, FHL3 gene-encoded protein (FHL3) plays an important role in skeletal muscle development and contraction. In this study, we determined the potential role of FHL3 in the proliferation and differentiation of primary satellite cells in chicken. RT-qPCR results showed that FHL3 mRNA was highly expressed in skeletal muscle in 12 chicken tissues. Four cell proliferation assays (CCK8 assay; EDU staining assay; flow cytometric detection of cell cycle assay; and detection of cell proliferation marker genes Ki67 and N-Ras assay) revealed that FHL3 knockdown had no effect on the proliferation rate of chicken satellite cells. FHL3 knockdown promoted the differentiation of satellite cells into myotubes, as evidenced by increased fusion index, number of nuclei per myotube, Myog, Myh7, Myf5, and Mrf4 mRNA expressions, and myog and myosin heavy chain protein expressions of myogenic markers (P < 0.05). These results showed that the FHL3 was a negative regulator of the differentiation and fusion of chicken satellite cells into myotubes. However, FHL3 expression was increased during the differentiation of chicken satellite cells into myotubes. The study suggested that FHL3 might have different functions in chicken myotubes compared with that in chicken satellite cells.

Oxytocin is involved in steroid hormone–stimulated bovine satellite cell proliferation and differentiation in vitro

Sex steroid hormones are used in the meat industry due to their ability to regulate muscle hypertrophy. However, the mechanisms underlying their action are not fully elucidated. Recent reports demonstrate that steroid hormones increase oxytocin (OXT) expression in skeletal muscle, indicating that OXT may play a role in satellite cell activity. This hypothesis was tested using steroid hormones (17β-estradiol [E2]; trenbolone acetate [TBA]), tamoxifen (TAM), OXT, and atosiban (A: OXT receptor inhibitor) applied to bovine satellite cells (BSCs) to investigate BSC regulation by OXT. Oxytocin alone increased fusion index (P < 0.05) but not BSC proliferation. Oxytocin reduced (P < 0.05) apoptotic nuclei and stimulated migration rate (P < 0.05). Similarly, E2 and TBA increased (P < 0.05) BSC proliferation rate, fusion index, and migration and decreased (P < 0.05) apoptotic nuclei. 17β-Estradiol or TBA supplemented with A had lower (P < 0.05) BSC proliferation rate, fusion index, and migration and more (P < 0.05) apoptotic nuclei compared with E2 or TBA alone. Furthermore, OXT expression increased (P < 0.05) in E2 or TBA-treated proliferating BSC. Oxytocin, E2, and TBA increased (P < 0.05) MyoD and MyoG expression in proliferating BSC. During BSC differentiation, OXT expression increased (P < 0.05) with E2 or TBA treatments. MyoG expression increased (P < 0.05) in OXT, E2, and TBA compared with control. However, A, OXT + A, TAM, TAM + OXT, E2 + TAM, E2 + A, and TBA + A decreased (P < 0.05) MyoG expression during BSC differentiation. These results indicate that OXT is involved in steroid hormone–stimulated BSC activity.

153. Third Generation Antisense Targeted to Double Homeobox Protein 4 (DUX4) Reduced DUX4 Expression and Improved Differentiation of FSHD Myoblasts

Facioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of double homeobox protein 4 (DUX4) gene at chromosome 4q35. To date there is no effective treatment for the disease. In the current study, we have evaluated selected Third Generation Antisense compounds (3GAs) targeted to DUX4 for their effectiveness in knocking-down DUX4 in FSHD myoblasts. In addition, we examined the effects of the treatments on myogenic differentiation of FSHD myoblasts. Multiple 3GA compounds (3GA1-5) showed significant knock-down of DUX4 in the FSHD myoblasts, as examined by quantitative RT-PCR. We evaluated two 3GA candidates (3GA3 and 3GA5) at 4 different concentrations, 2.5 nM, 5nM, 25nM, and 50nM, and observed dose-response effects. One of the 3GA compounds completely knocked-down DUX4 expression at 50nM. The effects of 3GAs at25nM on cell differentiation were determined by fusion index, myotube size and number of atrophic myotubes after 7 days of muscle differentiation in culture. Our results showed that the treatments increased fusion index and reduced the number of atrophic myotubes. The studies showed that 3GAs successfully knocked-down DUX4, which improved the myogenic capacity of FSHD myoblasts. The findings demonstrated that the 3GAs are promising therapeutic molecules to be further developed for FSHD treatment.

Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression.

Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C2C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C2C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids.

Low Intensity Exercise Training Improves Skeletal Muscle Regeneration Potential.

Purpose: The aim of this study was to determine whether 12 days of low-to-moderate exercise training at low altitude (598 m a.s.l.) improves skeletal muscle regeneration in sedentary adult women.Methods: Satellite cells were obtained from the vastus lateralis skeletal muscle of seven women before and after this exercise training at low altitude. They were investigated for differentiation aspects, superoxide anion production, antioxidant enzymes, mitochondrial potential variation after a depolarizing insult, intracellular Ca2+ concentrations, and micro (mi)RNA expression (miR-1, miR-133, miR-206).Results: In these myogenic populations of adult stem cells, those obtained after exercise training, showed increased Fusion Index and intracellular Ca2+ concentrations. This exercise training also generally reduced superoxide anion production in cells (by 12–67%), although not in two women, where there was an increase of ~15% along with a reduced superoxide dismutase activity. miRNA expression showed an exercise-induced epigenetic transcription profile that was specific according to the reduced or increased superoxide anion production of the cells.Conclusions: The present study shows that low-to-moderate exercise training at low altitude improves the regenerative capacity of skeletal muscle in adult women. The differentiation of cells was favored by increased intracellular calcium concentration and increased the fusion index. This low-to-moderate training at low altitude also depicted the epigenetic signature of cells.

Role of H2 Calponin in Myoblast Differentiation, Fusion and Myogenesis

Actin cytoskeleton plays a pivotal role in myoblast motility, adhesion, fusion and differentiation into myotubes. However, how the actin cytoskeleton is regulated during myogenesis is largely unknown. H2-calponin is a regulatory protein that stabilizes the actin cytoskeleton and regulates cell motility and adhesion. In the present study, we investigated the role of h2 calponin in the regulation of myoblast migration, differentiation and fusion. The results showed that h2-calponin is expressed at significant levels in undifferentiated mouse primary satellite cells and C2C12 myoblasts. In migrating satellite cells and C2C12 myoblasts, h2-calponin co-localizes with actin stress fibers at both the leading edge and the tail regions. In newly fused myotubes derived from primary satellite cells, h2-calponin is mainly present at the ends of the myotubes. The level of h2-calponin significantly decreases during satellite cell and myoblast differentiation and fusion, and becomes undetectable in mature myocytes. Primary satellite cells isolated from h2-calponin gene knockout mice had significantly decreased cell spreading area indicating reduced substrate adhesion and increased rate of cell proliferation as compared with wild type controls. Biochemical and histological studies revealed that skeletal muscles of h2-calponin knockout mice retain normal muscle weight and myofilament protein compositions. Myotubes derived from h2-calponin-null satellite cells showed apparently normal sarcomere striations and expression of myofilament proteins. Therefore, deletion of h2-calponin does not abolish the maximum capacity of myoblast function and myogenesis. However, in vitro differentiation studies revealed that h2-calponin-hull myoblasts had higher efficiency of fusion, as indicated by increased fusion index, than wild type controls. The data suggest that the function of h2-calponin provides a novel mechanism in regulating actin cytoskeleton activity during myoblast differentiation, fusion and myogenesis.