Cross-talk between motor neurons and myotubes via endogenously secreted neural and muscular growth factors.

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular patho-physiology and development of novel therapies for diseases associated with NM dysfunction. In vivo, the micro-environment surrounding the NMJ has a significant impact on NMJ formation and maintenance via neurotrophic and differentiation factors that are secreted as a result of cross-talk between muscle fibers and motor neurons. Recently we showed the formation of functional NMJs in vitro in a co-culture of immortalized human myoblasts and motor neurons from rat-embryo spinal-cord explants, using a culture medium free from serum and neurotrophic or growth factors. The aim of this study was to assess how functional NMJs were established in this co-culture devoid of exogenous neural growth factors. To investigate this, an ELISA-based microarray was used to compare the composition of soluble endogenously secreted growth factors in this co-culture with an a-neural muscle culture. The levels of seven neurotrophic factors brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-1 (IGF-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and vascular endothelial growth factor (VEGF) were higher (p < 0.05) in the supernatant of NMJ culture compared to those in the supernatant of the a-neural muscle culture. This indicates that the cross-talk between muscle and motor neurons promotes the secretion of soluble growth factors contributing to the local microenvironment thereby providing a favourable regenerative niche for NMJs formation and maturation.

Sex Comparison of Knee Extensor Size, Strength, and Fatigue Adaptation to Sprint Interval Training.

ABSTRACT: Bagley, L, Al-Shanti, N, Bradburn, S, Baig, O, Slevin, M, and McPhee, JS. Sex comparison of knee extensor size, strength, and fatigue adaptation to sprint interval training. J Strength Cond Res 35(1): 64-71, 2021-Regular sprint interval training (SIT) improves whole-body aerobic capacity and muscle oxidative potential, but very little is known about knee extensor anabolic or fatigue resistance adaptations, or whether effects are similar for men and women. The purpose of this study was to compare sex-related differences in knee extensor size, torque-velocity relationship, and fatigability adaptations to 12-week SIT. Sixteen men and 15 women (mean [SEM] age: 41 [±2.5] years) completed measurements of total body composition assessed by dual energy X-ray absorptiometry, quadriceps muscle cross-sectional area (CSAQ) assessed by magnetic resonance imaging, the knee extensor torque-velocity relationship (covering 0-240°·s-1) and fatigue resistance, which was measured as the decline in torque from the first to the last of 60 repeated concentric knee extensions performed at 180°·s-1. Sprint interval training consisted of 4 × 20-second sprints on a cycle ergometer set at an initial power output of 175% of power at V̇o2max, 3 times per week for 12 weeks. Quadriceps muscle cross-sectional area increased by 5% (p = 0.023) and fatigue resistance improved 4.8% (p = 0.048), with no sex differences in these adaptations (sex comparisons: p = 0.140 and p = 0.282, respectively). Knee extensor isometric and concentric torque was unaffected by SIT in both men and women (p > 0.05 for all velocities). Twelve-week SIT, totaling 4 minutes of very intense cycling per week, significantly increased fatigue resistance and CSAQ similarly in men and women, but did not significantly increase torque in men or women. These results suggest that SIT is a time-effective training modality for men and women to increase leg muscle size and fatigue resistance.

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction.

BACKGROUND: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. OBJECTIVE: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. METHODS: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. RESULTS: blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. CONCLUSION: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions.

Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells.

Maladaptive endoplasmic reticulum (ER) stress is associated with modified reactive oxygen species (ROS) generation and mitochondrial abnormalities; and is postulated as a potential mechanism involved in muscle weakness in myositis, an acquired autoimmune neuromuscular disease. This study investigates the impact of ROS generation in an in vitro model of ER stress in skeletal muscle, using the ER stress inducer tunicamycin (24 h) in the presence or absence of a superoxide dismutase/catalase mimetic Eukarion (EUK)-134. Tunicamycin induced maladaptive ER stress, which was mitigated by EUK-134 at the transcriptional level. ER stress promoted mitochondrial dysfunction, described by substantial loss of mitochondrial membrane potential, as well as a reduction in respiratory control ratio, reserve capacity, phosphorylating respiration, and coupling efficiency, which was ameliorated by EUK-134. Tunicamycin induced ROS-mediated biogenesis and fusion of mitochondria, which, however, had high propensity of fragmentation, accompanied by upregulated mRNA levels of fission-related markers. Increased cellular ROS generation was observed under ER stress that was prevented by EUK-134, even though no changes in mitochondrial superoxide were noticeable. These findings suggest that targeting ROS generation using EUK-134 can amend aspects of ER stress-induced changes in mitochondrial dynamics and function, and therefore, in instances of chronic ER stress, such as in myositis, quenching ROS generation may be a promising therapy for muscle weakness and dysfunction.

Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells.

BACKGROUND: Neuromuscular junctions (NMJs) consist of the presynaptic cholinergic motoneuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function. METHODS: This report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development. RESULTS: This unique serum- and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions. CONCLUSION: This coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs.

Soluble Factors Released From Activated T Lymphocytes Regulate C2C12 Myoblast Proliferation and Cellular Signaling, but Effects Are Blunted in the Elderly.

The key objective of this work was to investigate the impact of young and old human lymphocyte secretomes on C2C12 myoblasts regeneration. Conditioned media were harvested from isolated young and older lymphocytes treated with (activated [AC]) or without (nonactivated [NA]), anti-CD3/CD28 activators for 4 days. AC conditioned media from older lymphocytes had decreased levels of amphiregulin (367 ± 208 pg/mL vs 904 ± 323 pg/mL; p = .018) and IGF-I (845 ± 88 ng/mL vs 1100 ± 48 ng/mL; p = .032) compared with younger AC lymphocytes. AC older versus younger lymphocytes had reduced expression of CD25 (24.6 ± 5.5%; p = .0003) and increased expression of FoxP3 (35 ± 15.7%; p = .032). Treatment of C2C12 myoblasts with young AC lymphocytes resulted in decreased expression of MyoD (0.46 ± 0.12; p =.004) and Myogenin (0.34 ± 0.05; p = .010) mRNA, increased activation of MEk1 (724 ± 140 mean fluorescent intensity [MFI]; p =.001) and ERK1/2 (3768 ± 314 MFI; p =.001), and a decreased activation of Akt (74.5 ± 4 MFI; p = .009) and mTOR (61.8 ± 7 MFI; p = .001) compared with old AC lymphocytes. By contrast, C2C12 myoblasts treated with older AC lymphocytes displayed increased expression of MyoD (0.7 ± 0.08; p =.004) and Myogenin (0.68 ± 0.05; p =.010) mRNA, decreased phosphorylation of MEk1 and ERK1/2 (528 ± 80 MFI; p = .008, and 1141 ± 668 MFI; p = .001, respectively), and increased Akt/mTOR activation (171 ± 35 MFI; p = .009, and 184 ± 33 MFI; p = .001, respectively). These data provide new evidence that differences between older and younger lymphocyte secretomes contribute to differential responses of C2C12 myoblasts in culture.

A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.

BACKGROUND: Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. OBJECTIVE: We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. METHODS: Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. RESULTS: Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and ßIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. CONCLUSION: We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy.

Dysregulation of C-X-C motif ligand 10 during aging and association with cognitive performance.

Chronic low-grade inflammation during aging (inflammaging) is associated with cognitive decline and neurodegeneration; however, the mechanisms underlying inflammaging are unclear. We studied a population (n = 361) of healthy young and old adults from the MyoAge cohort. Peripheral levels of C-X-C motif chemokine ligand 10 (CXCL10) was found to be higher in older adults, compared with young, and negatively associated with working memory performance. This coincided with an age-related reduction in blood DNA methylation at specific CpGs within the CXCL10 gene promoter. In vitro analysis supported the role of DNA methylation in regulating CXCL10 transcription. A polymorphism (rs56061981) that altered methylation at one of these CpG sites further associated with working memory performance in 2 independent aging cohorts. Studying prefrontal cortex samples, we found higher CXCL10 protein levels in those with Alzheimer's disease, compared with aged controls. These findings support the association of peripheral inflammation, as demonstrated by CXCL10, in aging and cognitive decline. We reveal age-related epigenetic and genetic factors which contribute to the dysregulation of CXCL10.

Biological impact of advanced glycation endproducts on estrogen receptor-positive MCF-7 breast cancer cells.

Diabetes mellitus potentiates the risk of breast cancer. We have previously described the pro-tumorigenic effects of advanced glycation endproducts (AGEs) on estrogen receptor (ER)-negative MDA-MB-231 breast cancer cell line mediated through the receptor for AGEs (RAGE). However, a predominant association between women with ER-positive breast cancer and type 2 diabetes mellitus has been reported. Therefore, we have investigated the biological impact of AGEs on ER-positive human breast cancer cell line MCF-7 using in vitro cell-based assays including cell count, migration, and invasion assays. Western blot, FACS analyses and quantitative real time-PCR were also performed. We found that AGEs at 50-100µg/mL increased MCF-7 cell proliferation and cell migration associated with an enhancement of pro-matrix metalloproteinase (MMP)-9 activity, without affecting their poor invasiveness. However, 200µg/mL AGEs inhibited MCF-7 cell proliferation through induction of apoptosis indicated by caspase-3 cleavage detected using Western blotting. A phospho-protein array analysis revealed that AGEs mainly induce the phosphorylation of extracellular-signal regulated kinase (ERK)1/2 and cAMP response element binding protein-1 (CREB1), both signaling molecules considered as key regulators of AGEs pro-tumorigenic effects. We also showed that AGEs up-regulate RAGE and ER expression at the protein and transcript levels in MCF-7 cells, in a RAGE-dependent manner after blockade of AGEs/RAGE interaction using neutralizing anti-RAGE antibody. Throughout the study, BSA had no effect on cellular processes. These findings pave the way for future studies investigating whether the exposure of AGEs-treated ER-positive breast cancer cells to estrogen could lead to a potentiation of breast cancer development and progression.

Omega-3 fatty acid EPA improves regenerative capacity of mouse skeletal muscle cells exposed to saturated fat and inflammation.

Sarcopenic obesity is characterised by high fat mass, low muscle mass and an elevated inflammatory environmental milieu. We therefore investigated the effects of elevated inflammatory cytokine TNF-α (aging/obesity) and saturated fatty acid, palmitate (obesity) on skeletal muscle cells in the presence/absence of EPA, a-3 polyunsaturated fatty acid with proposed anti-inflammatory, anti-obesity activities. In the present study we show that palmitate was lipotoxic, inducing high levels of cell death and blocking myotube formation. Cell death under these conditions was associated with increased caspase activity, suppression of differentiation, reductions in both creatine kinase activity and gene expression of myogenic factors; IGF-II, IGFBP-5, MyoD and myogenin. However, inhibition of caspase activity via administration of Z-VDVAD-FMK (caspase-2), Z-DEVD-FMK (caspase-3) and ZIETD-KMK (caspase 8) was without effect on cell death. By contrast, lipotoxicity associated with elevated palmitate was reduced with the MEK inhibitor PD98059, indicating palmitate induced cell death was MAPK mediated. These lipotoxic conditions were further exacerbated in the presence of inflammation via TNF-α co-administration. Addition of EPA under cytotoxic stress (TNF-α) was shown to partially rescue differentiation with enhanced myotube formation being associated with increased MyoD, myogenin, IGF-II and IGFBP-5 expression. EPA had little impact on the cell death phenotype observed in lipotoxic conditions but did show benefit in restoring differentiation under lipotoxic plus cytotoxic conditions. Under these conditions Id3 (inhibitor of differentiation) gene expression was inversely linked with survival rates, potentially indicating a novel role of EPA and Id3 in the regulation of apoptosis in lipotoxic/cytotoxic conditions. Additionally, signalling studies indicated the combination of lipo- and cyto-toxic effects on the muscle cells acted through ceramide, JNK and MAPK pathways and blocking these pathways using PD98059 (MEK inhibitor) and Fumonisin B1 (ceramide inhibitor) significantly reduced levels of cell death. These findings highlight novel pathways associated with in vitro models of lipotoxicity (palmitate-mediated) and cytotoxicity (inflammatory cytokine mediated) in the potential targeting of molecular modulators of sarcopenic obesity.

Regenerative function of immune system: Modulation of muscle stem cells.

Ageing is characterised by progressive deterioration of physiological systems and the loss of skeletal muscle mass is one of the most recognisable, leading to muscle weakness and mobility impairments. This review highlights interactions between the immune system and skeletal muscle stem cells (widely termed satellite cells or myoblasts) to influence satellite cell behaviour during muscle regeneration after injury, and outlines deficits associated with ageing. Resident neutrophils and macrophages in skeletal muscle become activated when muscle fibres are damaged via stimuli (e.g. contusions, strains, avulsions, hyperextensions, ruptures) and release high concentrations of cytokines, chemokines and growth factors into the microenvironment. These localised responses serve to attract additional immune cells which can reach in excess of 1×10(5) immune cell/mm(3) of skeletal muscle in order to orchestrate the repair process. T-cells have a delayed response, reaching peak activation roughly 4 days after the initial damage. The cytokines and growth factors released by activated T-cells play a key role in muscle satellite cell proliferation and migration, although the precise mechanisms of these interactions remain unclear. T-cells in older people display limited ability to activate satellite cell proliferation and migration which is likely to contribute to insufficient muscle repair and, consequently, muscle wasting and weakness. If the factors released by T-cells to activate satellite cells can be identified, it may be possible to develop therapeutic agents to enhance muscle regeneration and reduce the impact of muscle wasting during ageing and disease.

The lymphocyte secretome from young adults enhances skeletal muscle proliferation and migration, but effects are attenuated in the secretome of older adults.

Older people experience skeletal muscle wasting, in part due to impaired proliferative capacity of quiescent skeletal muscle satellite cells which can be reversed by exposure to young blood. To investigate the role of immune cells in muscle regeneration, we isolated lymphocytes from whole blood of young and older healthy volunteers and cultured them with, or without, anti-CD3/CD28 activators to induce release of cytokines, interleukins, and growth factors into the media. The secreted proteins were collected to prepare a conditioned media, which was subsequently used to culture C2C12 myoblasts. The conditioned media from the activated young lymphocytes increased the rate of proliferation of myoblasts by around threefold (P < 0.005) and caused an approximate fourfold (P < 0.005) increase in migration compared with nonactivated lymphocyte control media. These responses were characterized by minimal myotube formation (2%), low fusion index (5%), low myosin heavy chain content, and substantial migration. In contrast, myoblasts treated with conditioned media from activated old lymphocytes exhibited a high degree of differentiation, and multi-nucleated myotube formation that was comparable to control conditions, thus showing no effect on proliferation or migration of myoblasts. These results indicate that secreted proteins from lymphocytes of young people enhance the muscle cell proliferation and migration, whereas secreted proteins from lymphocytes of older people may contribute to the attenuated skeletal muscle satellite cell proliferation and migration.

Lean mass, muscle strength and gene expression in community dwelling older men: findings from the Hertfordshire Sarcopenia Study (HSS).

Sarcopenia is associated with adverse health outcomes. This study investigated whether skeletal muscle gene expression was associated with lean mass and grip strength in community-dwelling older men. Utilising a cross-sectional study design, lean muscle mass and grip strength were measured in 88 men aged 68-76 years. Expression profiles of 44 genes implicated in the cellular regulation of skeletal muscle were determined. Serum was analysed for circulating cytokines TNF (tumour necrosis factor), IL-6 (interleukin 6, IFNG (interferon gamma), IL1R1 (interleukin-1 receptor-1). Relationships between skeletal muscle gene expression, circulating cytokines, lean mass and grip strength were examined. Participant groups with higher and lower values of lean muscle mass (n = 18) and strength (n = 20) were used in the analysis of gene expression fold change. Expression of VDR (vitamin D receptor) [fold change (FC) 0.52, standard error for fold change (SE) ± 0.08, p = 0.01] and IFNG mRNA (FC 0.31; SE ± 0.19, p = 0.01) were lower in those with higher lean mass. Expression of IL-6 (FC 0.43; SE ± 0.13, p = 0.02), TNF (FC 0.52; SE ± 0.10, p = 0.02), IL1R1 (FC 0.63; SE ± 0.09, p = 0.04) and MSTN (myostatin) (FC 0.64; SE ± 0.11, p = 0.04) were lower in those with higher grip strength. No other significant changes were observed. Significant negative correlations between serum IL-6 (R = -0.29, p = 0.005), TNF (R = -0.24, p = 0.017) and grip strength were demonstrated. This novel skeletal muscle gene expression study carried out within a well-characterized epidemiological birth cohort has demonstrated that lower expression of VDR and IFNG is associated with higher lean mass, and lower expression of IL-6, TNF, IL1R1 and myostatin is associated with higher grip strength. These findings are consistent with a role of proinflammatory factors in mediating lower muscle strength in community-dwelling older men.

Activated lymphocytes secretome inhibits differentiation and induces proliferation of C2C12 myoblasts.

BACKGROUND/AIMS: ageing is associated with a marked decline in immune function which may contribute to the local environment that can influence the regenerative process of skeletal muscle cells. METHODS: Herein, we focused on determining the effect of an activated immune system secretome on myoblast differentiation and proliferation as possible means to attenuate adverse effects of muscle aging. C2C12 myoblasts were used as model to assess the impact of lymphocyte conditioned media (CM) following anti-CD3/IL-2 activation. RESULTS: Myoblasts cultured with activated lymphocytes CM exhibited reduced morphological and biochemical differentiation (98±20, p<0.005) and increased entry to the S Phase of the cell cycle (61%±7, p<0.001), when compared with myoblasts cultured with non-activated lymphocytes CM. Associated with increased proliferation and reduced differentiation, muscle specific transcription factors MyoD and myogenin were significantly reduced in C2C12 treated with activated lymphocytes CM vs control CM, respectively (myoD: 0.5±0.12 fold reduction P<0.005); myogenin: 0.38±0.08 fold reduction; p<0.005). Moreover, key protein of proliferation pERK1/2 increased (46±11U/ml, p<0.05) whereas mediator of differentiation pAkt decreased (21±12U/ml, p<0.05) in C2C12 treated with activated vs. non-activated CM. CONCLUSION: our data demonstrate that, following activation, secretome of the immune system cells elicit marked regulatory effects on skeletal muscle growth and differentiation; enhancing the former with the loss of the latter.

Identification and characterization of novel Kirrel isoform during myogenesis.

Somatic cell fusion is an essential component of skeletal muscle development and growth and repair from injury. Additional cell types such as trophoblasts and osteoclasts also require somatic cell fusion events to perform their physiological functions. Currently we have rudimentary knowledge on molecular mechanisms regulating somatic cell fusion events in mammals. We therefore investigated during in vitro murine myogenesis a mammalian homolog, Kirrel, of the Drosophila Melanogaster genes Roughest (Rst) and Kin of Irre (Kirre) which regulate somatic muscle cell fusion during embryonic development. Our results demonstrate the presence of a novel murine Kirrel isoform containing a truncated cytoplasmic domain which we term Kirrel B. Protein expression levels of Kirrel B are inverse to the occurrence of cell fusion events during in vitro myogenesis which is in stark contrast to the expression profile of Rst and Kirre during myogenesis in Drosophila. Furthermore, chemical inhibition of cell fusion confirmed the inverse expression pattern of Kirrel B protein levels in relation to cell fusion events. The discovery of a novel Kirrel B protein isoform during myogenesis highlights the need for more thorough investigation of the similarities and potential differences between fly and mammals with regards to the muscle cell fusion process.

Phospho-tyrosine phosphatase inhibitor Bpv(Hopic) enhances C2C12 myoblast migration in vitro. Requirement of PI3K/AKT and MAPK/ERK pathways.

Muscle progenitor cell migration is an important step in skeletal muscle myogenesis and regeneration. Migration is required for muscle precursors to reach the site of damage and for the alignment of myoblasts prior to their fusion, which ultimately contributes to muscle regeneration. Limited spreading and migration of donor myoblasts are reported problems of myoblast transfer therapy, a proposed therapeutic strategy for Duchenne Muscular Dystrophy, warranting further investigation into different approaches for improving the motility and homing of these cells. In this article, the effect of protein phospho-tyrosine phosphatase and PTEN inhibitor BpV(Hopic) on C2C12 myoblast migration and differentiation was investigated. Applying a wound healing migration model, it is reported that 1 µM BpV(Hopic) is capable of enhancing the migration of C2C12 myoblasts by approximately 40 % in the presence of myotube conditioned media, without significantly affecting their capacity to differentiate and fuse into multinucleated myotubes. Improved migration of myoblasts treated with 1 µM BpV(Hopic) was associated with activation of PI3K/AKT and MAPK/ERK pathways, while their inhibition with either LY294002 or UO126, respectively, resulted in a reduction of C2C12 migration back to control levels. These results propose that bisperoxovanadium compounds may be considered as potential tools for enhancing the migration of myoblasts, while not reducing their differentiation capacity and underpin the importance of PI3K/AKT and MAPK/ERK signalling for the process of myogenic progenitor migration.

The role of insulin-like-growth factor binding protein 2 (IGFBP2) and phosphatase and tensin homologue (PTEN) in the regulation of myoblast differentiation and hypertrophy.

The complex actions of the insulin-like-growth factor binding proteins (IGFBPs) in skeletal muscle are becoming apparent, with IGFBP2 being implicated in skeletal muscle cell proliferation and differentiation (Ernst et al., 1992; Sharples et al., 2010). Furthermore, PTEN signalling has been linked to IGFBP2 action in other cell types by co-ordinating downstream Akt signalling, a known modulator of myoblast differentiation. The present study therefore aimed to determine the interaction between IGFBP2 and PTEN on myoblast differentiation. It has previously been established that C2C12 cells have high IGFBP2 gene expression upon transfer to low serum media, and that expression reduces rapidly as cells differentiate over 72 h [1]. Wishing to establish a potential role for IGFBP2 in this model, a neutralising IGFBP2 antibody was administered to C2C12 myoblasts upon initiation of differentiation. Myoblasts subsequently displayed reduced morphological differentiation (myotube number), biochemical differentiation (creatine kinase) and myotube hypertrophy (myotube area) with an early reduction in Akt phosphorylation. Knock-down of phosphatase and tensin homologue (PTEN) using siRNA in the absence of the neutralising antibody did not improve differentiation or hypertrophy vs. control conditions, however, in the presence of the neutralising IGFBP2 antibody, differentiation was restored and importantly hypertrophy exceeded that of control levels. Overall, these data suggest that; 1) reduced early availability of IGFBP2 can inhibit myoblast differentiation at later time points, 2) knock-down of PTEN levels can restore myoblast differentiation in the presence of neutralising IGFBP2 antibody, and 3) PTEN inhibition acts as a potent inducer of myotube hypertrophy when the availability of IGFBP2 is reduced in C2C12 myoblasts.

Sirtuin 1 regulates skeletal myoblast survival and enhances differentiation in the presence of resveratrol.

Sirtuin 1 also known as NAD-dependent deacetylase sirtuin 1, is a protein that in humans is encoded by the Sirt1 gene. Sirt1 is an enzyme that deacetylates proteins that contribute to cellular regulation and is a key regulator of cell defenses and survival in response to stress. Deletion of Sirt1 abolishes the increase in lifespan induced by calorie restriction or sublethal cytokine stress, indicating that Sirt1 promotes longevity and survival. We have demonstrated that administration of a sublethal dose of tumour necrosis factor-α (TNF-α; 1.25 ng ml(-1)) inhibits myotube formation, and co-incubation with insulin-like growth factor I (IGF-I; 1.5 ng ml(-1)) facilitates C2 myoblast death rather than rescuing differentiation. A higher dose of TNF-α (10 ng ml(-1)) resulted in significant apoptosis, which was rescued by IGF-I (1.5 ng ml(-1); 50% rescue; P < 0.05). We aimed to investigate the role of Sirt1 in the conflicting roles of IGF-I. Quantitative real-time PCR revealed that Sirt1 expression was elevated in myoblasts following incubation of 10 ng ml(-1) TNF-α or 1.25 ng ml(-1) TNF-α plus IGF-I (fivefold and 7.2-fold increases versus control, respectively; P < 0.05). A dose of 10 ng ml(-1) TNF-α induced ∼21 ± 0.7% apoptosis, which was reduced (∼50%; P < 0.05) when administered with IGF-I. Likewise, Sirt1 expression was elevated following 10 ng ml(-1) TNF-α administration, but was reduced (∼30%; P < 0.05) in the presence of IGF-I. C2C12 myoblasts, a subclone of the C2 cell line produced for their differentiation potential and used to examine intrinsic ageing, unlike C2 cells, do not die in the presence of TNF-α and do not upregulate Sirt1. As conditions that induced the greatest myoblast stress/damage resulted in elevated Sirt1 expression, we investigated the effects of Sirt1 gene silencing. Treatment with 10 ng ml(-1) TNF-α or co-incubation with 1.25 ng ml(-1) TNF-α and 1.5 ng ml(-1) IGF-I resulted in apoptosis (20.33 ± 2.08 and 19 ± 2.65%, respectively), which was increased when myoblasts were pretreated with Sirt1 small interfering RNA (31 ± 2.65 and 27.33 ± 2.52%, respectively; P < 0.05) and was reduced (14.33 ± 3.05%, P < 0.05 and 12.78 ± 4.52%, P = 0.054) by resveratrol, which also significantly rescued the block on differentiation. In conclusion, Sirt1 expression increases in conditons of stress, potentially serving to reduce or dampen myoblast death.

Inhibitory effects of IL-6 on IGF-1 activity in skeletal myoblasts could be mediated by the activation of SOCS-3.

In elderly people, low and high levels of insulin-like growth factor 1 (IGF-1) and interleukin-6 (IL-6), respectively, are well documented and may contribute to reduced muscle mass and poor muscle function of ageing and suggesting a biological interactions between IGF-1 and IL-6. However, the dual effect of IGF-1/IL-6 on skeletal muscle differentiation and proliferation has not been fully investigated. We therefore hypothesised that IL-6 impairs the biological activity of IGF-1 in skeletal muscle through inhibiting its signalling pathways, ERK1/2 and Akt. Our aim was to examine the combined effects of these factors on models of muscle wasting, with objectives to examine skeletal muscle differentiation and proliferation using the murine C2 skeletal muscle cell line. Cells were cultured with DM, IGF-1 and IL-6 alone (control treatments), or co-cultured with IGF-1/IL-6. Co-incubation of C2 cells in IGF-1 plus IL-6 resulted in maximal cell death (22 ± 4%; P < 0.005) compared with control treatments (14 ± 2.9%). This was also confirmed by cyclin D1 expression levels in co-incubation treatments (7 ± 3.5%; P < 0.05) compared with control treatments (≈ 23%). The expression levels of myogenic-specific transcriptional factor mRNAs (myoD and myogenin) were also significantly (P < 0.005) reduced by 70% and 90%, respectively, under the co-incubation regimes, compared with control treatments. Signalling investigations showed significant phosphorylation reduction by 20%, (P < 0.05) of ERK1/2 and Akt in co-incubation treatments relative to either treatment alone. Expression studies for SOCS-3 (1.6-fold ± 0.08, P < 0.05) and IRS-1 (0.65-fold ± 0.13 P < 0.005) mRNAs showed significant elevation and reduction for both genes, respectively, in co-treatments relative to control treatments. These data may suggest that IL-6 exerts its inhibitory effects on IGF-1 signalling pathways (ERK1/2 and Akt) through blocking its receptor substrate IRS-1 by SOCS-3.

Reduction of myoblast differentiation following multiple population doublings in mouse C2 C12 cells: a model to investigate ageing?

Ageing skeletal muscle displays declines in size, strength, and functional capacity. Given the acknowledged role that the systemic environment plays in reduced regeneration (Conboy et al. [2005] Nature 433: 760-764), the role of resident satellite cells (termed myoblasts upon activation) is relatively dismissed, where, multiple cellular divisions in-vivo throughout the lifespan could also impact on muscular deterioration. Using a model of multiple population doublings (MPD) in-vitro thus provided a system in which to investigate the direct impact of extensive cell duplications on muscle cell behavior. C(2) C(12) mouse skeletal myoblasts (CON) were used fresh or following 58 population doublings (MPD). As a result of multiple divisions, reduced morphological and biochemical (creatine kinase, CK) differentiation were observed. Furthermore, MPD cells had significantly increased cells in the S and decreased cells in the G1 phases of the cell cycle versus CON, following serum withdrawal. These results suggest continued cycling rather than G1 exit and thus reduced differentiation (myotube atrophy) occurs in MPD muscle cells. These changes were underpinned by significant reductions in transcript expression of: IGF-I and myogenic regulatory factors (myoD and myogenin) together with elevated IGFBP5. Signaling studies showed that decreased differentiation in MPD was associated with decreased phosphorylation of Akt, and with later increased phosphorylation of JNK1/2. Chemical inhibition of JNK1/2 (SP600125) in MPD cells increased IGF-I expression (non-significantly), however, did not enhance differentiation. This study provides a potential model and molecular mechanisms for deterioration in differentiation capacity in skeletal muscle cells as a consequence of multiple population doublings that would potentially contribute to the ageing process.