RESUMEN
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 VÌo2max, 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.
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Entrenamiento de Intervalos de Alta Intensidad , Adulto , Femenino , Humanos , Rodilla , Articulación de la Rodilla/diagnóstico por imagen , Masculino , Fatiga Muscular , Fuerza Muscular , Músculo Esquelético , TorqueRESUMEN
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.
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Neoplasias de la Mama/metabolismo , Productos Finales de Glicación Avanzada/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama/patología , Femenino , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Células MCF-7 , Proteínas de Neoplasias/genéticaRESUMEN
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.
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Apoptosis/efectos de los fármacos , Ácido Eicosapentaenoico/administración & dosificación , Mioblastos/metabolismo , Mioblastos/patología , Regeneración/efectos de los fármacos , Animales , Línea Celular , Ratones , Mioblastos/efectos de los fármacos , Miositis , Palmitatos/administración & dosificación , Factor de Necrosis Tumoral alfa/administración & dosificaciónRESUMEN
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.
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Diferenciación Celular , Activación de Linfocitos , Linfocitos/metabolismo , Mioblastos/citología , Adulto , Animales , Ciclo Celular , Línea Celular , Proliferación Celular , Forma de la Célula , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Ratones , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/metabolismo , Miogenina/genética , Miogenina/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Adulto JovenRESUMEN
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.
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Regulación de la Expresión Génica , Sarcopenia/patología , Anciano , Antropometría , Biopsia , Composición Corporal , Estudios de Cohortes , Estudios Transversales , Inglaterra , Perfilación de la Expresión Génica , Humanos , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Fuerza Muscular , Músculo Esquelético/metabolismo , Miostatina/metabolismo , Reacción en Cadena de la Polimerasa , Receptores Tipo I de Interleucina-1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
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Movimiento Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mioblastos/enzimología , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular , Inhibidores Enzimáticos/química , Ratones , Mioblastos/citología , Fosfohidrolasa PTEN/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidoresRESUMEN
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.
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Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Interleucina-6/farmacología , Mioblastos Esqueléticos/metabolismo , Animales , Muerte Celular , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/efectos de los fármacos , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de CitocinasRESUMEN
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 â¼21 ± 0.7% apoptosis, which was reduced (â¼50%; P < 0.05) when administered with IGF-I. Likewise, Sirt1 expression was elevated following 10 ng ml(-1) TNF-α administration, but was reduced (â¼30%; 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.
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Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Sirtuina 1/metabolismo , Estilbenos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Diferenciación Celular/fisiología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Silenciador del Gen , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Modelos Animales , Mioblastos Esqueléticos/citología , ARN Interferente Pequeño/farmacología , Resveratrol , Sirtuina 1/efectos de los fármacos , Sirtuina 1/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
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.
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Envejecimiento , Diferenciación Celular , Mioblastos/citología , Animales , Secuencia de Bases , Ciclo Celular , Línea Celular , Cartilla de ADN , Citometría de Flujo , Ratones , Microscopía Fluorescente , Músculo Esquelético/citología , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Mioblastos/enzimología , Mioblastos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Potential roles for undifferentiated skeletal muscle stem cells or satellite cells in muscle hypertrophy and repair have been reported, however, the capacity, the mode and the mechanisms underpinning migration have not been investigated. We hypothesised that damaged skeletal myoblasts would elicit a mesenchymal-like migratory response, which could be precisely tracked and subsequently manipulated. METHODS: We therefore established a model of mechanical damage and developed a MATLAB(TM) tool to measure the migratory capacity of myoblasts in a non-subjective manner. RESULTS: Basal migration following damage was highly directional, with total migration distances of 948µm ± 239µm being recorded (average 0-24 hour distances: 491µm ± 113µm and 24-48 hour distances: 460µm ± 218µm). Pharmacological inhibition of MEK or PI3-K using PD98059 (20µM) or LY294002 (5µm), resulted in significant reduction of overall cell migration distances of 38% (p<0.001) and 39.5% (p<0.0004), respectively. Using the semi-automated cell tracking using MATLAB(TM) program we validated that not only was migration distance reduced as a consequence of reduced cell velocity, but critically also as a result of altered directionality of migration. CONCLUSION: These studies demonstrate that murine myoblasts in culture migrate and provide a good model for studying responsiveness to damage in vitro. They illustrate for the first time the powerful tool that MATLAB(TM) provides in determining that both velocity and directional capacity influence the migratory potential of cellular movement with obvious implications for homing and for metastases.
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Automatización , Movimiento Celular , Mioblastos/citología , Animales , Línea Celular , Medios de Cultivo , Ratones , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacosRESUMEN
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.
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Factor I del Crecimiento Similar a la Insulina/metabolismo , Neuronas Motoras/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular , Células Cultivadas , Humanos , Unión Neuromuscular/metabolismo , RatasRESUMEN
Reduced muscle mass and increased susceptibility to TNF-induced degradation accompany inflamed ageing and chronic diseases. Furthermore, C(2) myoblasts display diminished differentiation and increased susceptibility to TNF-alpha-induced cell death versus subcloned C(2)C(12) cells, providing relevant models to assess: differentiation (creatine kinase), growth (protein), death (trypan-blue) and anabolic/catabolic parameters (RT-PCR) over 72 h +/- TNF-alpha (20 ng ml(-1)). At 48 and 72 h, respectively, larger myotubes and significantly higher CK activity (320.26 +/- 6.82 vs. 30.71 +/- 2.5, P < 0.05; 544.94 +/- 27.7 vs. 39.4 +/- 3.37 mU mg ml(-1), P < 0.05), fold increases in myoD (21.45 +/- 3.12 vs. 3.97 +/- 1.76, P < 0.05; 31.07 +/- 3.1 vs. 6.82 +/- 1.93, P < 0.05) and myogenin mRNA (241.8 +/- 40 vs. 36.80 +/- 19.3, P < 0.05; 440 +/- 100.5 vs. 201.1 +/- 86, P < 0.05) were detected in C(2)C(12) versus C(2). C(2)C(12) showed significant increases in IGF-I mRNA (243.05 +/- 3.87 vs. 105.75 +/- 21.95, P < 0.05), reduced proliferation and significantly lower protein expression (1.21 +/- 0.28 vs. 1.79 +/- 0.29 mg ml(-1), P < 0.05) at 72 h versus C(2) cells. Significant temporal reductions in C(2)C(12) IGFBP2 mRNA (28.02 +/- 15.44, 13.82 +/- 8.07, 6.92 +/- 4.37, P < 0.05) contrasted increases in C(2)s (4.31 +/- 3.31, 13.02 +/- 9.92, 82.9 +/- 58.9, P < 0.05) at 0, 48 and 72 h, respectively. TNF-alpha increased cell death in C(2)s (2.67 +/- 1.54%, 34.42 +/- 5.39%, 29.71 +/- 5.79% (0, 48, 72 h), P < 0.05), yet was without effect in C(2)C(12)s at 48 h but caused a small significant increase at 72 h (9.88 +/- 4.02% (TNF-alpha) vs. 6.17 +/- 0.749% (DM), 72 h). TNF-alpha and TNFRI mRNA were unchanged; however, larger reductions in IGF-I (8.2- and 7.5-fold vs. 4.5- and 4.1-fold (48, 72 h)), IGF-IR (2-fold vs. no-significant reduction (72 h)) and IGFBP5 (3.24 vs. 1.38 (48 h) and 2.21 vs. 1.71 (72 h), P < 0.05) mRNA were observed in C(2) versus C(2)C(12) with TNF-alpha. This investigation provides insight into regulators of altered basal hypertrophy and TNF-induced atrophy, providing a model for future investigation into therapeutic initiatives for ageing/wasting disorders.
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Envejecimiento/fisiología , Atrofia/metabolismo , Hipertrofia/metabolismo , Músculo Esquelético/citología , Mioblastos/fisiología , Animales , Atrofia/patología , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Creatina Quinasa/metabolismo , Hipertrofia/patología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Miogenina/genética , Miogenina/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
IGF-I positively impacts on muscle anabolism/regeneration. Using C2 skeletal myoblasts, we previously reported high dose TNF-alpha -induced (10 ng.ml(-1)) cell death is rescued by IGF-I. However, non-myotoxic low dose TNF-alpha (1.25 ng.ml(-1)) elicits a MAPK-mediated apoptotic response when co-incubated with IGF-I (1.5 ng.ml(-1)). Our aim was to investigate these conflicting roles of IGF-I in our model. Insulin array and qRT-PCR identified Adra1d as a potential regulatory gene that was up-regulated in survival and down-regulated under apoptotic conditions. TNF-alpha administration (1.25 or 10 ng.ml(-1)) induced significant decreases ( approximately 50% both incubations) in Adra1d expression relative to DM. IGF-I addition to high dose TNF-alpha (10 ng.ml(-1)) induced myoblast survival and matched a significant (P < 0.05) increase in Adra1d expression. By contrast, IGF-I addition to low dose TNF-alpha (1.25 ng.ml(-1)) induced elevated death resulting in a significant (P < 0.05) decline ( approximately 55%) in Adra1d expression. Pre-administration of PD98059 (20 uM), which rescues death induced by co-incubation of low dose TNF-alpha with IGF-I, Adra1d levels were again comparable to DM control. Since Adra1d was elevated following incubations that induced myoblast survival, we investigated effects of Adra1d siRNA gene silencing under these conditions. Adra1d knockdown resulted in significantly higher levels of cell death under all incubations suggesting Adra1d expression is essential for skeletal muscle cell survival.
Asunto(s)
Mioblastos Esqueléticos/citología , Receptores Adrenérgicos alfa 1/metabolismo , Animales , Apoptosis , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Flavonoides/farmacología , Técnicas de Silenciamiento del Gen , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mioblastos Esqueléticos/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Adrenérgicos alfa 1/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
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.
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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.
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Muscle damage with a lack of regeneration, manifests itself in several life-threatening diseases, including cancer cachexia, congestive heart failure, AIDS and sepsis. Often misdiagnosed as a condition simply of weight loss, cachexia is actually a highly complex metabolic disorder involving features of anorexia, anaemia, lipolysis and insulin resistance. A significant loss of lean body mass arises from such conditions, resulting in wasting of skeletal muscle. Unlike starvation, the weight loss seen in chronic illnesses arises equally from loss of muscle and of fat. The cachectic state is particularly problematic in cancer, typifying poor prognosis and often lowering responses to chemotherapy and radiation treatment. More than half of cancer patients suffer from cachexia, and strikingly, nearly one-third of cancer deaths are related to cachexia rather than the tumour burden. In considering this disorder, we are faced with a conundrum; how is it possible for uncontrolled growth to prevail in the tumour, in the face of unrestrained tissue loss in our muscles? Consistently, the catabolic state has been associated with a shift in the homeostatic balance between muscle synthesis and degradation mediated by the actions of growth factors and cytokines. Indeed, tumour necrosis factor-alpha (TNF-alpha) levels are raised in several animal models of cachectic muscle wasting, whereas the insulin-like growth factor (IGF) system acts potently to regulate muscle development, hypertrophy and maintenance. This concept of skeletal muscle homeostasis, often viewed as the net balance between two separate processes of protein synthesis and degradation has however changed. More recently, the view is that these two biochemical processes are not occurring independently of each other but in fact are finely co-ordinated by a web of intricate signalling networks. This review, therefore, aims to discuss data currently available regarding the mechanisms of degeneration and regeneration with specific emphasis on the potential and controversial cross-talk which may exist between anabolic growth factors (e.g. IGF-I) and catabolic cytokines (e.g. TNF-alpha). Also importantly, the potential impact at a cellular level of exercise, diet and age will be addressed. Finally, the ability to 'hi-jack' signalling pathways traditionally believed to be for growth and survival or death will be reviewed. It is anticipated that such a review will highlight significant gaps in our knowledge of the cachectic state as well as provide caution with regards to therapeutics suggesting total block on inflammatory processes such as that associated with TNF-alpha action.
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Caquexia/etiología , Citocinas/fisiología , Sustancias de Crecimiento/fisiología , Caquexia/fisiopatología , Muerte Celular/fisiología , Humanos , Modelos Biológicos , Músculo Esquelético/fisiopatología , Fosfatidilinositol 3-Quinasas/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal , Somatomedinas/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Ubiquitina/fisiologíaRESUMEN
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.
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Proliferación Celular/fisiología , Mioblastos/citología , Transducción de Señal/fisiología , Linfocitos T/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Factores Biológicos/metabolismo , Células Cultivadas , Humanos , Activación de Linfocitos , Masculino , Adulto JovenRESUMEN
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.
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OBJECTIVE: The concept of skeletal muscle homeostasis--often viewed as the net balance between two separate processes, namely protein degradation and protein synthesis--are not occurring independently of each other, but are finely co-ordinated by a web of intricate signalling networks. MATERIALS AND METHODS: Using rodent muscle cell lines we have investigated TNF-alpha/IGF-I interactions, in an attempt to mimic and understand mechanisms underlying the wasting process. RESULTS AND CONCLUSION: When myoblast cells are incubated with TNF-alpha (10 ng ml(- 1)) maximal damage ( approximately 21% +/- 0.7 myoblast death, p < 0.05) was induced. Co-incubation of TNF-alpha (10 ng ml(- 1)) with IGF-I resulted in cell survival ( approximately 50% reduction in myoblast death, p < 0.05), however, myotube formation was not evident. In contrast, a novel role of IGF-I has been identified whereby co-incubation of muscle cells with IGF-I (1.5 ng ml(- 1)) and a non-apoptotic dose of TNF-alpha (1.25 ng ml(- 1); sufficient to block differentiation) unexpectedly were shown not to rescue a block on differentiation but to facilitate significant myoblast death (p < 0.05). Interestingly, pre-administration of PD98059, a MAPK signal-blocking agent followed by co-incubation of 1.25 ng ml(- 1) TNF-alpha and 1.5 ng ml(- 1) IGF-I, reduced death to baseline levels (p < 0.05). We show for the first time that IGF-I can be apoptotic in the absence of TNF-alpha-induced cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mioblastos Esqueléticos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Apoptosis/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Creatina Quinasa/análisis , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Immunoblotting , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/análisis , Mioblastos Esqueléticos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.