RESUMO
SUMMARY: We are living in an aging society. In 2019, 1 billion individuals were already aged over 60. The number of people in this demographic is predicted to reach 1.4 billion by 2030 and 2.1 billion by 2050 (WHO). In the USA, individuals over 65 represent the fastest growing segment of the population (US census bureau). Similar trends are seen in the UK, with 16.2 million people already aged over 60, equivalent to 24% of the total population (Age UK; https://www.ageuk.org.uk/globalassets/age-uk/documents/reports-and-publications/later_life_uk_factsheet.pdf). Indeed, in the UK, people over the age of 60 outnumbered those under the age of 18, for the first time in 2008. This statistic still prevails today. Because of medical and biopharmaceutical progress, lifespan is increasing rapidly, but healthspan is failing to keep up. If we are to increase healthy living, then we need to begin to understand the mechanisms of how we age across the life course, so that relevant interventions may be developed to facilitate "life in our years," not simply "years in our life." It is reported that only 25% of aging is genetically predetermined. This fits with observations of some families aging very quickly and poorly and others aging slowly and well. If this is indeed the case and the rate of aging is not fixed, then this knowledge provides a significant opportunity to manipulate the impact of environmental influencers of age. With that in mind, it begs the question of what are the mechanisms of aging and is there potential to manipulate this process on an individual-by-individual basis? The focus of this article will be on the process of muscle wasting with aging (sarcopenia) and the potential of exercise and its underlying mechanisms to reverse or delay sarcopenia. There will be a focus on epigenetics in muscle wasting and the capability of exercise to change our skeletal muscle epigenetic profile for the good. The article ends with considerations relating to facial aging, Botox treatment, and gene editing as a tool for plastic surgeons in the future.
Assuntos
Produtos Biológicos , Toxinas Botulínicas Tipo A , Sarcopenia , Idoso , Envelhecimento/genética , Epigênese Genética , Humanos , Pessoa de Meia-Idade , Músculo Esquelético/fisiologia , Atrofia Muscular , Sarcopenia/genética , Sarcopenia/terapiaRESUMO
BACKGROUND: There are few examples of interventions designed to promote physical activity (PA) in adults with Cystic fibrosis (CF). Increasing levels of habitual PA may be more feasible and result in greater compliance than conventional exercise training inventions which give little or no attention to long-term PA behaviour. Despite this there is limited research exploring perceptions of PA among adults with CF. The study aimed to understand the ecological correlates of PA in adults with CF and to involve individuals with CF, their families (where applicable) and clinicians in a formative process to inform the development of an ecological approach to PA promotion in this population. METHODS: An iterative approach was utilised, whereby findings from earlier phases of the research informed subsequent phases. Semi-structured interviews were conducted to explore patients' perceptions of PA, devised using the PRECEDE component of the PRECEDE-PROCEED model. Followed by, focus groups to discuss the perceived barriers, facilitators and opportunities for PA participation and how this information could inform the development and delivery of a PA intervention. Separate focus groups were conducted with individuals with CF (n = 11) and their families and CF MDT members. Thematic analysis was used to construct themes. RESULTS: Physical and mental wellbeing manifested as both barriers and facilitators of PA. CF is characterised by a progressive decline in physical function, which presents as a number of challenging symptoms and set-backs for an individual with CF. PA represents an opportunity for participants to slow the rate of this decline and manage the symptoms associated with the condition. Enjoyment was an important facilitator of PA. Exercise professionals and family reinforce PA behaviour, particularly during adolescence. CONCLUSIONS: PA promotion should form part of routine CF care with additional exercise professional support during adolescence.
Assuntos
Fibrose Cística , Adolescente , Adulto , Fibrose Cística/terapia , Exercício Físico , Grupos Focais , Humanos , Atividade Motora , Cooperação do PacienteRESUMO
BACKGROUND: Cell assays are important for investigating the mechanisms of ageing, including losses in protein homeostasis and 'proteostasis collapse'. We used novel isotopic labelling and proteomic methods to investigate protein turnover in replicatively aged (>140 population doublings) murine C2C12 myoblasts that exhibit impaired differentiation and serve as a model for age-related declines in muscle homeostasis. METHODS: The Absolute Dynamic Profiling Technique for Proteomics (Proteo-ADPT) was used to investigate proteostasis in young (passage 6-10) and replicatively aged (passage 48-50) C2C12 myoblast cultures supplemented with deuterium oxide (D2 O) during early (0-24 h) or late (72-96 h) periods of differentiation. Peptide mass spectrometry was used to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. RESULTS: Young cells exhibited a consistent ~25% rise in protein accretion over the 96-h experimental period. In aged cells, protein accretion increased by 32% (P < 0.05) during early differentiation, but then fell back to baseline levels by 96-h. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P < 0.05, FDR < 5% interaction between age × differentiation stage) different changes in abundance between young and aged cells at early and later periods of differentiation, including proteins associated with translation, glycolysis, cell-cell adhesion, ribosomal biogenesis, and the regulation of cell shape. During early differentiation, heat shock and ribosomal protein abundances increased in aged cells due to suppressed degradation rather than heightened synthesis. For instance, HS90A increased at a rate of 10.62 ± 1.60 ng/well/h in aged which was significantly greater than the rate of accretion (1.86 ± 0.49 ng/well/h) in young cells. HS90A synthesis was similar in young (21.23 ± 3.40 ng/well/h) and aged (23.69 ± 1.13 ng/well/h), but HS90A degradation was significantly (P = 0.05) greater in young (19.37 ± 2.93 ng/well/h) versus aged (13.06 ± 0.76 ng/well/h) cells. During later differentiation the HS90A degradation (8.94 ± 0.38 ng/well/h) and synthesis (7.89 ± 1.28 ng/well/h) declined and were significantly less than the positive net balance between synthesis and degradation (synthesis = 28.14 ± 3.70 ng/well/h vs. degradation = 21.49 ± 3.13 ng/well/h) in young cells. CONCLUSIONS: Our results suggest a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts. The quality of key chaperone proteins, including HS90A, HS90B and HSP7C was reduced in aged cells and may account for the disruption to cell signalling required for the later stages of differentiation and fusion.
Assuntos
Proteoma , Proteômica , Animais , Óxido de Deutério/metabolismo , Camundongos , Mioblastos/metabolismo , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
PURPOSE: Sedentary behaviour is negatively associated with mood and cognition, yet how acute sitting contributes to these overall associations is unknown. Since sitting heightens inflammation and impairs cerebrovascular function, this study investigated the hypothesis that these sitting-induced changes are related to impaired mood and cognition. METHODS: Twenty-five healthy desk workers (18 male, 28.3 ± 7.5 years, BMI: 24.2 ± 3.3 kgâm-2) were recruited. During laboratory visit one, participants were familiarised with cognitive performance tests measuring executive function, attention and working memory. During laboratory visit two, participants completed 6 h of continuous, uninterrupted sitting. At baseline and after 6 h, serum markers of inflammation, middle cerebral artery blood flow velocity (MCAv), cerebrovascular carbon dioxide reactivity (CVR), dynamic cerebral autoregulation (CA), cognitive performance and mood (positive and negative affect, alert, contented and calm) were assessed. Data were analysed using paired-samples t tests and correlation analyses. RESULTS: Following sitting, C-reactive protein (∆-1.0 µg/ml) and tissue plasminogen activator (∆-360.4 pg/ml) decreased (p < 0.05), MCAv reduced (∆-2.9 cmâs-1, p = 0.012) and normalised gain increased in the very low frequency range, indicating impaired CA (∆ + 0.22%·mmHg-1, p = 0.016). Positive affect (∆-4.6, p < 0.001), and alert (∆-10.6 p = 0.002) and contented (∆-7.4, p = 0.006) mood states also decreased following sitting. No significant changes in interleukin-6, tumour necrosis factor-alpha, von Willebrand factor, CVR or cognitive performance were observed (p > 0.05). The observed changes in inflammation and cerebrovascular function were not related to changes in mood (p > 0.05). CONCLUSION: Alterations in inflammation or cerebrovascular function following six hours of prolonged, uninterrupted sitting are not related to the observed reductions in mood, indicating other mechanisms underlie the relationship between acute sitting and mood disturbances.
RESUMO
The estimated cost of acute injuries in college-level sport in the USA is â¼1.5 billion dollars per year, without taking into account the cost of follow up rehabilitation. In addition to this huge financial burden, without appropriate diagnosis and relevant interventions, sport injuries may be career-ending for some athletes. With a growing number of females participating in contact based and pivoting sports, middle aged individuals returning to sport and natural injuries of ageing all increasing, such costs and negative implications for quality of life will expand. For those injuries, which cannot be predicted and prevented, there is a real need, to optimise repair, recovery and function, post-injury in the sporting and clinical worlds. The 21st century has seen a rapid growth in the arena of regenerative medicine for sporting injuries, in a bid to progress recovery and to facilitate return to sport. Such interventions harness knowledge relating to stem cells as a potential for injury repair. While the field is rapidly growing, consideration beyond the stem cells, to the factors they secrete, should be considered in the development of effective, affordable treatments.
Assuntos
Traumatismos em Atletas , Qualidade de Vida , Medicina Regenerativa , Volta ao Esporte , Esportes , Transplante de Células-Tronco , Traumatismos em Atletas/economia , Traumatismos em Atletas/terapia , Feminino , Humanos , Pessoa de Meia-Idade , Células-Tronco , Estados UnidosRESUMO
UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3ß/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3ß activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.
Assuntos
Metabolismo Energético , Músculo Esquelético/enzimologia , Atrofia Muscular/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Rugby League (RL) match-play causes muscle damage, inflammation and symptoms of fatigue. To facilitate recovery, nutritional interventions are often employed, including Montmorency cherry juice (MC). We assessed the effects of MC on recovery following RL match-play in eleven male professional RL players who played in two matches (7-days apart) with MC or placebo (PLB) supplemented for 5-days pre-match, matchday and 2-days post-match. Blood was collected 48h pre-match, half-time, within 30-mins of full-time and 48h post-match to assess Interleukin concentrations (IL-6, -8 -10). Self-reported sleep, fatigue, mood, stress, and muscle-soreness were assessed 24h pre and 24 and 48h post-matches with muscle function assessed 48h pre and 48h post-match. No differences in distance covered (6334 ± 1944 Vs 6596 ± 1776m) and total collisions (28 ± 11 Vs 29 ± 13) were observed between both matches. There was a small albeit significant increase in IL-6, -8 and -10 concentrations pre to post-match in both PLB (IL-6: 0.83 ± 0.92 Vs 2.91 ± 1.40, IL-8: 2.16 ± 1.22 Vs 3.91 ± 1.61 and IL-10: 2.51 ± 2.14 Vs 0.61 ± 0.50 pg.mL-1) and MC groups (IL-6: 0.53 ± 0.53 Vs 2.24 ± 1.73, IL-8: 1.85 ± 0.96 Vs 3.46 ± 1.12 and IL-10: 0.48 ± 0.50 Vs 2.54 ± 2.10 pg.mL-1), although there were no significant differences between groups (P<0.05). Likewise, there was a small but significant increase in muscle soreness (P=0.01) and reduction in CMJ (P=0.003) with no significant differences between groups. No significant changes in sleep, fatigue or mood (P>0.05) were observed pre to post-match or between groups. These data suggest MC does not affect the modest changes observed in cytokine responses and markers of recovery from RL match-play.Keywords: Team Sport, Nutrition, Performance, Recovery.
Assuntos
Futebol Americano/lesões , Sucos de Frutas e Vegetais , Músculo Esquelético/fisiopatologia , Mialgia/prevenção & controle , Miosite/prevenção & controle , Prunus avium , Adolescente , Afeto , Biomarcadores/sangue , Fadiga/fisiopatologia , Humanos , Interleucina-10/sangue , Interleucina-6/sangue , Interleucina-8/sangue , Masculino , Músculo Esquelético/patologia , Angústia Psicológica , Sono/fisiologiaRESUMO
BACKGROUND: Physical activity (PA) is important in the management of Cystic Fibrosis (CF) and is associated with a number of beneficial effects. PA assessment is not commonplace or consistent in clinical practice, therefore understanding of PA in adults with CF remains limited. The purpose of this review was to evaluate PA levels in this population and compare PA to global recommendations and non-CF peers. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines were utilised to inform the review process. Original research was identified and screened against inclusion/exclusion criteria. Quality was assessed, data extracted and a narrative synthesis undertaken to describe the findings. RESULTS: Adults with CF did not achieve recommended PA guidelines and step count targets in 5/8 studies where assessment was possible. No significant differences in PA were found between CF and non-CF peers in 3/5 studies. Associations between PA and improved lung function were inconsistent with 4/9 studies finding a positive association. Evidence for an association between PA and higher exercise capacity was stronger with all 4 studies reviewed reporting a positive association. Quality ratings were low across all studies. CONCLUSIONS: PA in adults with CF is largely comparable to their non-CF peers, despite being insufficiently active to achieve PA recommendations. Assessment tools used and outcomes reported are variable, many of which do not provide sufficient information to assess relevant components of PA. There is a requirement for high quality studies designed specifically to explore PA in adults with CF, ideally employing standardised PA assessment methods.
Assuntos
Fibrose Cística , Tolerância ao Exercício , Exercício Físico , Autoeficácia , Adulto , Fibrose Cística/fisiopatologia , Fibrose Cística/psicologia , Fibrose Cística/terapia , Exercício Físico/fisiologia , Exercício Físico/psicologia , Indicadores Básicos de Saúde , Humanos , Avaliação de Resultados em Cuidados de Saúde , Resultado do TratamentoRESUMO
The bioengineering of skeletal muscle tissue in-vitro has enabled researchers to more closely mimic the in-vivo skeletal muscle niche. The three-dimensional (3-D) structure of the tissue engineered systems employed to date enable the generation of highly aligned and differentiated myofibers within a representative biological matrix. The use of electrical stimulation to model concentric contraction, via innervation of the myofibers, and the use of mechanical loading to model passive lengthening or stretch has begun to provide a manipulable environment to investigate the cellular and molecular responses following exercise mimicking stimuli in-vitro. Currently available bioreactor systems allow either electrical stimulation or mechanical loading to be utilized at any given time. In the present manuscript, we describe in detail the methodological procedures to create 3-D bioengineered skeletal muscle using both cell lines and/or primary human muscle derived cells from a tissue biopsy, through to modeling exercising stimuli using a bioreactor that can provide both electrical stimulation and mechanical loading simultaneously within the same in-vitro system.
Assuntos
Técnicas de Cultura de Células , Exercício Físico , Músculo Esquelético/fisiologia , Engenharia Tecidual , Animais , Engenharia Biomédica , Biópsia , Reatores Biológicos , Linhagem Celular , Criopreservação , Estimulação Elétrica , Humanos , Esferoides CelularesRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Eicosapentaenoico/administração & dosagem , Mioblastos/metabolismo , Mioblastos/patologia , Regeneração/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos , Mioblastos/efeitos dos fármacos , Miosite , Palmitatos/administração & dosagem , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
Skeletal muscle mass, quality and adaptability are fundamental in promoting muscle performance, maintaining metabolic function and supporting longevity and healthspan. Skeletal muscle is programmable and can 'remember' early-life metabolic stimuli affecting its function in adult life. In this review, the authors pose the question as to whether skeletal muscle has an 'epi'-memory? Following an initial encounter with an environmental stimulus, we discuss the underlying molecular and epigenetic mechanisms enabling skeletal muscle to adapt, should it re-encounter the stimulus in later life. We also define skeletal muscle memory and outline the scientific literature contributing to this field. Furthermore, we review the evidence for early-life nutrient stress and low birth weight in animals and human cohort studies, respectively, and discuss the underlying molecular mechanisms culminating in skeletal muscle dysfunction, metabolic disease and loss of skeletal muscle mass across the lifespan. We also summarize and discuss studies that isolate muscle stem cells from different environmental niches in vivo (physically active, diabetic, cachectic, aged) and how they reportedly remember this environment once isolated in vitro. Finally, we will outline the molecular and epigenetic mechanisms underlying skeletal muscle memory and review the epigenetic regulation of exercise-induced skeletal muscle adaptation, highlighting exercise interventions as suitable models to investigate skeletal muscle memory in humans. We believe that understanding the 'epi'-memory of skeletal muscle will enable the next generation of targeted therapies to promote muscle growth and reduce muscle loss to enable healthy aging.
Assuntos
Envelhecimento/genética , Epigênese Genética , Exercício Físico/fisiologia , Doenças Metabólicas/genética , Músculo Esquelético/fisiologia , Animais , Humanos , Nicho de Células-Tronco/genéticaRESUMO
Tumour Necrosis Factor-Alpha (TNF-α) is chronically elevated in conditions where skeletal muscle loss occurs. As l-glutamine can dampen the effects of inflamed environments, we investigated the role of l-glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF-α (20 ng · ml(-1) ) ± l-glutamine (20 mM). TNF-α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf-I, and Igfbp5. Furthermore, when administered to mature myotubes, TNF-α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf-I, Igfbp2, and glutamine synthetase and parallel increases in Fox03, Cfos, p53, and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non-significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF-α. Importantly, 20 mM l-glutamine reduced p38 MAPK activity in TNF-α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. l-glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf-Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and restored atrophic gene expression of Fox03 back to baseline in TNF-α conditions. In conclusion, l-glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. l-glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. J. Cell. Physiol. 9999: 231: 2720-2732, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Glutamina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Mioblastos Esqueléticos/patologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Atrofia , Fusão Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
Sufficient quantity and quality of skeletal muscle is required to maintain lifespan and healthspan into older age. The concept of skeletal muscle programming/memory has been suggested to contribute to accelerated muscle decline in the elderly in association with early life stress such as fetal malnutrition. Further, muscle cells in vitro appear to remember the in vivo environments from which they are derived (e.g. cancer, obesity, type II diabetes, physical inactivity and nutrient restriction). Tumour-necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is chronically elevated in sarcopenia and cancer cachexia. Higher TNF-α levels are strongly correlated with muscle loss, reduced strength and therefore morbidity and earlier mortality. We have extensively shown that TNF-α impairs regenerative capacity in mouse and human muscle derived stem cells [Meadows et al. (J Cell Physiol 183(3):330-337, 2000); Foulstone et al. (J Cell Physiol 189(2):207-215, 2001); Foulstone et al. (Exp Cell Res 294(1):223-235, 2004); Stewart et al. (J Cell Physiol 198(2):237-247, 2004); Al-Shanti et al. (Growth factors (Chur, Switzerland) 26(2):61-73, 2008); Saini et al. (Growth factors (Chur, Switzerland) 26(5):239-253, 2008); Sharples et al. (J Cell Physiol 225(1):240-250, 2010)]. We have also recently established an epigenetically mediated mechanism (SIRT1-histone deacetylase) regulating survival of myoblasts in the presence of TNF-α [Saini et al. (Exp Physiol 97(3):400-418, 2012)]. We therefore wished to extend this work in relation to muscle memory of catabolic stimuli and the potential underlying epigenetic modulation of muscle loss. To enable this aim; C2C12 myoblasts were cultured in the absence or presence of early TNF-α (early proliferative lifespan) followed by 30 population doublings in the absence of TNF-α, prior to the induction of differentiation in low serum media (LSM) in the absence or presence of late TNF-α (late proliferative lifespan). The cells that received an early plus late lifespan dose of TNF-α exhibited reduced morphological (myotube number) and biochemical (creatine kinase activity) differentiation vs. control cells that underwent the same number of proliferative divisions but only a later life encounter with TNF-α. This suggested that muscle cells had a morphological memory of the acute early lifespan TNF-α encounter. Importantly, methylation of myoD CpG islands were increased in the early TNF-α cells, 30 population doublings later, suggesting that even after an acute encounter with TNF-α, the cells have the capability of retaining elevated methylation for at least 30 cellular divisions. Despite these fascinating findings, there were no further increases in myoD methylation or changes in its gene expression when these cells were exposed to a later TNF-α dose suggesting that this was not directly responsible for the decline in differentiation observed. In conclusion, data suggest that elevated myoD methylation is retained throughout muscle cells proliferative lifespan as result of early life TNF-α treatment and has implications for the epigenetic control of muscle loss.
Assuntos
Adaptação Fisiológica/genética , Adaptação Fisiológica/imunologia , Epigênese Genética/genética , Epigênese Genética/imunologia , Fibras Musculares Esqueléticas/imunologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Animais , Humanos , Fenômenos Imunogenéticos/genética , Modelos Genéticos , Modelos ImunológicosRESUMO
Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating socio-economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin-like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS-1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging.
Assuntos
Envelhecimento/efeitos dos fármacos , Restrição Calórica , Proteínas Alimentares/administração & dosagem , Longevidade/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Envelhecimento/genética , Animais , Dieta , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Insulina/genética , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Longevidade/genética , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Transdução de Sinais , Sirtuínas/genética , Sirtuínas/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Virus replication efficiency is influenced by two conflicting factors, kinetics of the cellular interferon (IFN) response and induction of an antiviral state versus speed of virus replication and virus-induced inhibition of the IFN response. Disablement of a virus's capacity to circumvent the IFN response enables both basic research and various practical applications. However, such IFN-sensitive viruses can be difficult to grow to high-titer in cells that produce and respond to IFN. The current default option for growing IFN-sensitive viruses is restricted to a limited selection of cell-lines (e.g. Vero cells) that have lost their ability to produce IFN. This study demonstrates that supplementing tissue-culture medium with an IFN inhibitor provides a simple, effective and flexible approach to increase the growth of IFN-sensitive viruses in a cell-line of choice. We report that IFN inhibitors targeting components of the IFN response (TBK1, IKK2, JAK1) significantly increased virus replication. More specifically, the JAK1/2 inhibitor Ruxolitinib enhances the growth of viruses that are sensitive to IFN due to (i) loss of function of the viral IFN antagonist (due to mutation or species-specific constraints) or (ii) mutations/host cell constraints that slow virus spread such that it can be controlled by the IFN response. This was demonstrated for a variety of viruses, including, viruses with disabled IFN antagonists that represent live-attenuated vaccine candidates (Respiratory Syncytial Virus (RSV), Influenza Virus), traditionally attenuated vaccine strains (Measles, Mumps) and a slow-growing wild-type virus (RSV). In conclusion, supplementing tissue culture-medium with an IFN inhibitor to increase the growth of IFN-sensitive viruses in a cell-line of choice represents an approach, which is broadly applicable to research investigating the importance of the IFN response in controlling virus infections and has utility in a number of practical applications including vaccine and oncolytic virus production, virus diagnostics and techniques to isolate newly emerging viruses.
Assuntos
Técnicas de Cultura de Células/métodos , Interferons/antagonistas & inibidores , Pirazóis/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Meios de Cultura , Cães , Proteínas de Fluorescência Verde , Humanos , Interferons/metabolismo , Células Madin Darby de Rim Canino , Vírus do Sarampo/fisiologia , Camundongos , Vírus da Caxumba/fisiologia , Nitrilas , Orthomyxoviridae/fisiologia , Pirimidinas , Coelhos , Vírus Sinciciais Respiratórios/fisiologia , Suínos , Células VeroRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Sarcopenia/patologia , Idoso , Antropometria , Biópsia , Composição Corporal , Estudos de Coortes , Estudos Transversais , Inglaterra , Perfilação da Expressão Gênica , Humanos , Interferon gama/metabolismo , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Força Muscular , Músculo Esquelético/metabolismo , Miostatina/metabolismo , Reação em Cadeia da Polimerase , Receptores Tipo I de Interleucina-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular , Ativação Linfocitária , Linfócitos/metabolismo , Mioblastos/citologia , Adulto , Animais , Ciclo Celular , Linhagem Celular , Proliferação de Células , Forma Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/metabolismo , Miogenina/genética , Miogenina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Adulto JovemRESUMO
We investigated the ability of testosterone (T) to restore differentiation in multiple population doubled (PD) murine myoblasts, previously shown to have a reduced differentiation in monolayer and bioengineered skeletal muscle cultures vs. their parental controls (CON) (Sharples et al., 2011, 2012 [7,26]). Cells were exposed to low serum conditions in the presence or absence of T (100nM)±PI3K inhibitor (LY294002) for 72h and 7 days (early and late muscle differentiation respectively). Morphological analyses were performed to determine myotube number, diameter (µm) and myonuclear accretion as indices of differentiation and myotube hypertrophy. Changes in gene expression for myogenin, mTOR and myostatin were also performed. Myotube diameter in CON and PD cells increased from 17.32±2.56µm to 21.02±1.89µm and 14.58±2.66µm to 18.29±3.08µm (P≤0.05) respectively after 72h of T exposure. The increase was comparable in both PD (+25%) and CON cells (+21%) suggesting a similar intrinsic ability to respond to exogenous T administration. T treatment also significantly increased myonuclear accretion (% of myotubes expressing 5+ nuclei) in both cell types after 7 days exposure (P≤0.05). Addition of PI3K inhibitor (LY294002) in the presence of T attenuated these effects in myotube morphology (in both cell types) suggesting a role for the PI3K pathway in T stimulated hypertrophy. Finally, PD myoblasts showed reduced responsiveness to T stimulated mRNA expression of mTOR vs. CON cells and T also reduced myostatin expression in PD myoblasts only. The present study demonstrates testosterone administration improves hypertrophy in myoblasts that basally display impaired differentiation and hypertrophic capacity vs. their parental controls, the action of testosterone in this model was mediated by PI3K/Akt pathway.
Assuntos
Hipertrofia/induzido quimicamente , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Testosterona/farmacologia , Animais , Células Cultivadas , Cromonas/farmacologia , Camundongos , Morfolinas/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.