RESUMO
Elucidating the epigenetic mechanisms underlying muscle mass determination and skeletal muscle wasting holds the potential of identifying molecular pathways that constitute possible drug targets. Here, we report that the methyltransferase SMYD3 modulates myostatin and c-Met transcription in primary skeletal muscle cells and C2C12 myogenic cells. SMYD3 targets the myostatin and c-Met genes and participates in the recruitment of the bromodomain protein BRD4 to their regulatory regions through protein-protein interaction. By recruiting BRD4, SMYD3 favors chromatin engagement of the pause-release factor p-TEFb (positive transcription elongation factor) and elongation of Ser2-phosphorylated RNA polymerase II (PolIISer2P). Reducing SMYD3 decreases myostatin and c-Met transcription, thus protecting from glucocorticoid-induced myotube atrophy. Supporting functional relevance of the SMYD3/BRD4 interaction, BRD4 pharmacological blockade by the small molecule JQ1 prevents dexamethasone-induced myostatin and atrogene up-regulation and spares myotube atrophy. Importantly, in a mouse model of dexamethasone-induced skeletal muscle atrophy, SMYD3 depletion prevents muscle loss and fiber size decrease. These findings reveal a mechanistic link between SMYD3/BRD4-dependent transcriptional regulation, muscle mass determination, and skeletal muscle atrophy and further encourage testing of small molecules targeting specific epigenetic regulators in animal models of muscle wasting.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/genética , Miostatina/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Animais , Linhagem Celular , Quinase 9 Dependente de Ciclina/metabolismo , Dexametasona/farmacologia , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/genética , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/induzido quimicamente , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including ß-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both ß-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.
Assuntos
Fatores Biológicos/metabolismo , Pontos de Checagem do Ciclo Celular , Miócitos Cardíacos/metabolismo , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismo , Regeneração/fisiologia , Adulto , Animais , Diferenciação Celular , Dano ao DNA , Humanos , Masculino , Miócitos Cardíacos/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Ratos Sprague-DawleyRESUMO
Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨm ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨm , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨm by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨm , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria.
Assuntos
Metabolismo Energético/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Mitofagia/fisiologia , Neurônios/metabolismo , Animais , Células Cultivadas , Camundongos , Proteínas Quinases/metabolismoRESUMO
Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. Here, we report that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7+/Myf5â» stem cells and Pax7+/Myf5+ committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7+ cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Gênica/genética , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Imunoprecipitação da Cromatina , Proteína Potenciadora do Homólogo 2 de Zeste , Citometria de Fluxo , Imunofluorescência , Histona-Lisina N-Metiltransferase/genética , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Camundongos , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Complexo Repressor Polycomb 2RESUMO
PURPOSE OF REVIEW: To discuss how innate muscle stem-cell metabolism and nutrient availability can provide temporal regulation of chromatin accessibility and transcription. RECENT FINDINGS: Fluorescence-activated cell sorting coupled with whole transcriptome sequencing revealed for the first time that quiescent and proliferating skeletal muscle stem cells exhibit a process of metabolic reprogramming, from fatty-acid oxidation during quiescence to glycolysis during proliferation. Using a combination of immunofluorescence and chromatin immunoprecipitation sequencing, this shift in metabolism has been linked to altered availability of key metabolites essential for histone (de)acetylation and (de)methylation, including acetyl-CoA, s-adenosylmethionine and α-ketoglutarate. Importantly, these changes in metabolite availability have been linked to muscle stem-cell function. SUMMARY: Together, these results provide greater insight into how muscle stem cells interact with their local environment, with important implications for metabolic diseases, skeletal muscle regeneration and cell-transplantation therapies.
Assuntos
Cromatina/genética , Dieta , Epigênese Genética , Histonas/metabolismo , Músculo Esquelético/citologia , Estado Nutricional , Células-Tronco/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Cromatina/metabolismo , Humanos , Ácidos Cetoglutáricos/metabolismo , Metilação , Músculo Esquelético/metabolismo , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina/metabolismo , TranscriptomaRESUMO
Silent information regulators (SIRTs) have been shown to deacetylate a range of metabolic enzymes, including those in glycolysis and the Krebs cycle, and thus alter their activity. SIRTs require NAD(+) for their activity, linking cellular energy status to enzyme activity. To examine the impact of SIRT1 modulation on oxidative metabolism, this study tests the effect of ligands that are either SIRT-activating compounds (resveratrol and SRT1720) or SIRT inhibitors (EX527) on the metabolism of (13)C-enriched substrates by guinea pig brain cortical tissue slices with (13)C and (1)H nuclear magnetic resonance spectroscopy. Resveratrol increased lactate labeling but decreased incorporation of (13)C into Krebs cycle intermediates, consistent with effects on AMPK and inhibition of the F0/F1-ATPase. By testing with resveratrol that was directly applied to astrocytes with a Seahorse analyzer, increased glycolytic shift and increased mitochondrial proton leak resulting from interactions of resveratrol with the mitochondrial electron transport chain were revealed. SRT1720, by contrast, stimulated incorporation of (13)C into Krebs cycle intermediates and reduced incorporation into lactate, although the inhibitor EX527 paradoxically also increased Krebs cycle (13)C incorporation. In summary, the various SIRT1 modulators show distinct acute effects on oxidative metabolism. The strong effects of resveratrol on the mitochondrial respiratory chain and on glycolysis suggest that caution should be used in attempts to increase bioavailability of this compound in the CNS.
Assuntos
Antioxidantes/farmacologia , Encéfalo , Ácido Láctico/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estilbenos/farmacologia , Animais , Área Sob a Curva , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Carbazóis/farmacologia , Isótopos de Carbono/metabolismo , Relação Dose-Resposta a Droga , Feminino , Cobaias , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Imageamento por Ressonância Magnética , Masculino , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Resveratrol , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/metabolismoRESUMO
Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.
Assuntos
Adenilato Quinase/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Glucose/metabolismo , Mitocôndrias/metabolismo , Sirtuína 3/fisiologia , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Animais , Células Cultivadas , DNA Mitocondrial/metabolismo , Transporte de Elétrons , Metabolismo Energético , Privação de Alimentos , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Genoma Mitocondrial , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células NIH 3T3 , Sirtuína 3/genética , Sirtuína 3/metabolismoRESUMO
PURPOSE OF REVIEW: To discuss recent findings regarding the role of the histone deacetylase sirtuin SirT1 in anabolic and catabolic signaling pathways in skeletal muscle. RECENT FINDINGS: Since its discovery as a regulator of peroxisome proliferator-activated receptor γ-coactivator 1α transcriptional activity, SirT1 has received much attention for its role in the regulation of tissue-specific and whole body regulation of metabolic processes. Although these early seminal discoveries identified SirT1 as a central player in metabolism, it is only more recently that we have begun to understand the complexities of SirT1 signaling. In addition to peroxisome proliferator-activated receptorgamma-coactivator 1α, SirT1 has been found to regulate the activity and/or transcription of protein kinase B, mammalian target of rapamycin, forkhead box O 1 and 3a and myogenic determination factor, all of which are central players in the regulation of energy status in skeletal muscle, via actions on catabolic and anabolic signaling. SUMMARY: SirT1-mediated regulation of skeletal muscle metabolism (and indeed, whole body metabolism) likely occurs at numerous levels, from cell membrane receptors to transcription factors and histones. The end result of these regulatory actions of SirT1 is to maintain cellular homeostasis.
Assuntos
Homeostase , Músculo Esquelético/metabolismo , Sirtuína 1/fisiologia , Animais , Atrofia , Metabolismo Energético/fisiologia , Regulação da Expressão Gênica , Humanos , Hipertrofia/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/fisiologia , Biogênese de Organelas , PPAR gama/metabolismo , Transdução de Sinais , Fatores de TranscriçãoRESUMO
Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle wasting and weakness, leading to premature death from respiratory and/or cardiac failure. A clinically relevant question is whether myostatin inhibition can improve function of the diaphragm, which exhibits a severe and progressive pathology comparable with that in DMD. We hypothesized that antibody-directed myostatin inhibition would improve the pathophysiology of diaphragm muscle strips from young mdx mice (when the pathology is mild) and adult mdx mice (when the pathology is quite marked). Five weeks treatment with a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg/week) increased muscle mass (P < 0.05) and increased diaphragm median fiber cross-sectional area (CSA, P < 0.05) in young C57BL/10 and mdx mice, compared with saline-treated controls. PF-354 had no effect on specific force (sPo, maximum force normalized to muscle CSA) of diaphragm muscle strips from young C57BL/10 mice, but increased sPo by 84% (P < 0.05) in young mdx mice. In contrast, 8 weeks of PF-354 treatment did not improve muscle mass, median fiber CSA, collagen infiltration, or sPo of diaphragm muscle strips from adult mdx mice. PF-354 antibody-directed myostatin inhibition completely restored the functional capacity of diaphragm strips to control levels when treatment was initiated early, but not in the later stages of disease progression, suggesting that such therapies may only have a limited window of efficacy for DMD and related conditions.
Assuntos
Envelhecimento , Diafragma/patologia , Distrofia Muscular Animal/metabolismo , Miostatina/química , Animais , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/patologia , Miostatina/antagonistas & inibidores , Miostatina/metabolismo , Fatores de TempoRESUMO
OBJECTIVES: Preferential damage to fast, glycolytic myofibers is common in many muscle-wasting diseases, including Duchenne muscular dystrophy (DMD). Promoting an oxidative phenotype could protect muscles from damage and ameliorate the dystrophic pathology with therapeutic relevance, but developing efficacious strategies requires understanding currently unknown biological roles for dystrophin and utrophin in dystrophic muscle adaptation and plasticity. METHODS: Combining whole transcriptome RNA sequencing and mitochondrial proteomics with assessments of metabolic and contractile function, we investigated the roles of dystrophin and utrophin in fast-to-slow muscle remodeling with low-frequency electrical stimulation (LFS, 10 Hz, 12 h/d, 7 d/wk, 28 d) in mdx (dystrophin null) and dko (dystrophin/utrophin null) mice, two established preclinical models of DMD. RESULTS: Novel biological roles in adaptation were demonstrated by impaired transcriptional activation of estrogen-related receptor alpha-responsive genes supporting oxidative phosphorylation in dystrophic muscles. Further, utrophin expression in dystrophic muscles was required for LFS-induced remodeling of mitochondrial respiratory chain complexes, enhanced fiber respiration, and conferred protection from eccentric contraction-mediated damage. CONCLUSIONS: These findings reveal novel roles for dystrophin and utrophin during LFS-induced metabolic remodeling of dystrophic muscle and highlight the therapeutic potential of LFS to ameliorate the dystrophic pathology and protect from contraction-induced injury with important implications for DMD and related muscle disorders.
Assuntos
Adaptação Fisiológica/fisiologia , Distrofina/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Utrofina/metabolismo , Animais , Distrofina/genética , Masculino , Engenharia Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Mitocôndrias/metabolismo , Contração Muscular , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Utrofina/genéticaRESUMO
A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.
Assuntos
Sequenciamento por Nanoporos/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento Alternativo , Animais , Éxons , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Splicing de RNA , RNA Mensageiro , TranscriptomaRESUMO
1. In adult mammals, skeletal muscle mass is maintained through a precise balance of protein synthesis and protein degradation, whereas during development cellular (not protein) turnover predominates. When protein balance is shifted towards synthesis, skeletal muscle hypertrophy ensues. In contrast, increased protein degradation leads to skeletal muscle atrophy. Insulin-like growth factor (IGF)-I is among the best documented of the growth factors and regulates skeletal muscle mass by increasing protein synthesis and decreasing protein degradation. However, an IGF-I-independent growth pathway has been identified that involves the activation of beta-adrenoceptors and subsequent skeletal muscle growth, development and hypertrophy. 2. Although the importance of beta-adrenergic signalling in the heart has been well documented and continues to receive significant attention, it is only more recently that we have started to appreciate the importance of this signalling pathway in skeletal muscle structure and function. Studies have identified an important role for beta-adrenoceptors in myogenesis and work from our laboratory has identified a novel role for beta-adrenoceptors in regulating skeletal muscle regeneration after myotoxic injury. In addition, new data suggest that beta-adrenoceptors are markedly upregulated during differentiation of C2C12 cells. 3. It is now clear that beta-adrenoceptors play an important role in regulating skeletal muscle structure and function. Importantly, a clearer understanding of the pathways regulating skeletal muscle mass may lead to the identification of novel therapeutic targets for the treatment of muscle wasting disorders, including sarcopenia, cancer cachexia and the muscular dystrophies.
Assuntos
Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Receptores Adrenérgicos beta/fisiologia , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Músculo Esquelético/crescimento & desenvolvimentoRESUMO
While metabolism was initially thought to play a passive role in cell biology by generating ATP to meet bioenergetic demands, recent studies have identified critical roles for metabolism in the generation of new biomass and provision of obligate substrates for the epigenetic modification of histones and DNA. This review details how metabolites generated through glycolysis and the tricarboxylic acid cycle are utilized by somatic stem cells to support cell proliferation and lineage commitment. Importantly, we also discuss the evolving hypothesis that histones can act as an energy reservoir during times of energy stress. Finally, we discuss how cells integrate both extrinsic metabolic cues and intrinsic metabolic machinery to regulate cell fate.
Assuntos
Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/citologia , Animais , Proliferação de Células , Ciclo do Ácido Cítrico , Glicólise , HumanosRESUMO
In response to injury, skeletal muscle stem cells (MuSCs) undergo myogenesis where they become activated, proliferate rapidly, differentiate and undergo fusion to form multinucleated myotubes. Dramatic changes in cell size, shape, metabolism and motility occur during myogenesis, which cause cellular stress and alter proteostasis. The molecular chaperone heat shock protein 70 (HSP70) maintains proteostasis by regulating protein biosynthesis and folding, facilitating transport of polypeptides across intracellular membranes and preventing stress-induced protein unfolding/aggregation. Although HSP70 overexpression can exert beneficial effects in skeletal muscle diseases and enhance skeletal muscle repair after injury, its effect on myogenesis has not been investigated. Plasmid-mediated overexpression of HSP70 did not affect the rate of C2C12 proliferation or differentiation, but the median number of myonuclei per myotube and median myotube width in differentiated C2C12 myotubes were increased with HSP70 overexpression. These findings reveal that increased HSP70 expression can promote myoblast fusion, identifying a mechanism for its therapeutic potential to enhance muscle repair after injury.This article has an associated First Person interview with the first author of the paper.
Assuntos
Diferenciação Celular , Proteínas de Choque Térmico HSP70/genética , Desenvolvimento Muscular , Mioblastos/citologia , Mioblastos/metabolismo , Animais , Diferenciação Celular/genética , Fusão Celular , Proliferação de Células , Imunofluorescência , Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismoRESUMO
BACKGROUND: Systemic administration of beta-adrenoceptor (beta-AR) agonists has been found to induce skeletal muscle hypertrophy and significant metabolic changes. In the context of energy homeostasis, the importance of beta-AR signaling has been highlighted by the inability of beta(1-3)-AR-deficient mice to regulate energy expenditure and susceptibility to diet induced obesity. However, the molecular pathways and gene expression changes that initiate and maintain these phenotypic modulations are poorly understood. Therefore, the aim of this study was to identify differential changes in gene expression in murine skeletal muscle associated with systemic (acute and chronic) administration of the beta(2)-AR agonist formoterol. RESULTS: Skeletal muscle gene expression (from murine tibialis anterior) was profiled at both 1 and 4 hours following systemic administration of the beta(2)-AR agonist formoterol, using Illumina 46K mouse BeadArrays. Illumina expression profiling revealed significant expression changes in genes associated with skeletal muscle hypertrophy, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, and oxidative stress. Differentially expressed genes relevant to the regulation of muscle mass and metabolism (in the context of the hypertrophic phenotype) were further validated by quantitative RT-PCR to examine gene expression in response to both acute (1-24 h) and chronic administration (1-28 days) of formoterol at multiple timepoints. In terms of skeletal muscle hypertrophy, attenuation of myostatin signaling (including differential expression of myostatin, activin receptor IIB, phospho-Smad3 etc) was observed following acute and chronic administration of formoterol. Acute (but not chronic) administration of formoterol also significantly induced the expression of genes involved in oxidative metabolism, including hexokinase 2, sorbin and SH3 domain containing 1, and uncoupling protein 3. Interestingly, formoterol administration also appeared to influence some genes associated with the peripheral regulation of circadian rhythm (including nuclear factor interleukin 3 regulated, D site albumin promoter binding protein, and cryptochrome 2). CONCLUSION: This is the first study to utilize gene expression profiling to examine global gene expression in response to acute beta(2)-AR agonist treatment of skeletal muscle. In summary, systemic administration of a beta(2)-AR agonist had a profound effect on global gene expression in skeletal muscle. In terms of hypertrophy, beta(2)-AR agonist treatment altered the expression of several genes associated with myostatin signaling, a previously unreported effect of beta-AR signaling in skeletal muscle. This study also demonstrates a beta(2)-AR agonist regulation of circadian rhythm genes, indicating crosstalk between beta-AR signaling and circadian cycling in skeletal muscle. Gene expression alterations discovered in this study provides insight into many of the underlying changes in gene expression that mediate beta-AR induced skeletal muscle hypertrophy and altered metabolism.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Ritmo Circadiano , Perfilação da Expressão Gênica , Hipertrofia , Músculo Esquelético/metabolismo , Animais , Etanolaminas/farmacologia , Fumarato de Formoterol , Hipertrofia/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
The beta-adrenergic signaling pathway represents a novel therapeutic target for skeletal muscle wasting and weakness due to its role in the mechanisms controlling protein synthesis and degradation and in modulating fiber type. Stimulation of the pathway with beta-adrenoceptor agonists (beta-agonists) has therapeutic potential for muscle wasting disorders including: sarcopenia, cancer cachexia, disuse and inactivity, unloading or microgravity, sepsis and other metabolic disorders, denervation, burns, HIV-AIDS, chronic kidney or heart failure, and neuromuscular diseases. However, there are also pitfalls associated with beta-agonist administration and clinical applications have so far been limited, largely because of cardiovascular side effects. In rats and mice, newer generation beta-agonists (such as formoterol) can elicit an anabolic response in skeletal muscle even at very low doses, with reduced effects on the heart and cardiovascular system compared with older generation beta-agonists (such as fenoterol and clenbuterol). However, the potentially deleterious cardiovascular side effects of beta-agonists have not been obviated completely and so it is important to refine their development and therapeutic approach in order to overcome these obstacles. This review describes the therapeutic potential of stimulating the beta-adrenergic signaling pathway with beta-agonists, highlighting the beneficial effects on skeletal muscle structure and function and identifying some of the pitfalls associated with short- and long-term beta-agonist administration. The review also identifies some important, but as yet unanswered questions, regarding the importance of beta-adrenoceptor signaling in muscle health and disease and the strategies needed to improve the efficacy and safety of beta-agonists for muscle wasting disorders.
Assuntos
Agonistas Adrenérgicos beta/efeitos adversos , Agonistas Adrenérgicos beta/farmacologia , Agonistas Adrenérgicos beta/uso terapêutico , Atrofia Muscular/tratamento farmacológico , Animais , Sistema Cardiovascular/efeitos dos fármacos , Humanos , Proteínas Musculares/metabolismo , Debilidade Muscular/tratamento farmacológico , Transtornos Musculares Atróficos/tratamento farmacológico , Transdução de SinaisRESUMO
PURPOSE OF REVIEW: The beta-adrenergic signaling pathway represents a novel therapeutic target for skeletal muscle wasting disorders due to its roles in regulating protein synthesis and degradation. beta-Adrenoceptor agonists (beta-agonists) have therapeutic potential for attenuating muscle wasting associated with sarcopenia (age-related muscle wasting), cancer cachexia, sepsis, disuse, burns, HIV-AIDS, chronic kidney or heart failure, and neuromuscular diseases such as the muscular dystrophies. This review describes the role of beta-adrenergic signaling in the mechanisms controlling muscle wasting due to its effects on protein synthesis, protein degradation, and muscle fiber phenotype. RECENT FINDINGS: Stimulation of the beta-adrenergic signaling pathway with beta-agonists has therapeutic potential for muscle wasting since administration can elicit an anabolic response in skeletal muscle. As a consequence of their potent muscle anabolic actions, the effects of beta-agonist administration have been examined in several animal models and human conditions of muscle wasting in the hope of discovering a new therapeutic. The repartitioning characteristics of beta-agonists (increasing muscle mass and decreasing fat mass) have also made them attractive anabolic agents for use in livestock and by some athletes. However, potentially deleterious cardiovascular side-effects of beta-agonists have been identified and these will need to be obviated in order for the therapeutic potential of beta-agonists to be realized. SUMMARY: Multiple studies have identified anticachectic effects of beta-agonists and their therapeutic potential for pathologic states when muscle protein hypercatabolism is indicated. Future studies examining beta-agonist administration for muscle wasting conditions need to separate beneficial effects on skeletal muscle from potentially deleterious effects on the heart and cardiovascular system.
Assuntos
Adrenérgicos/uso terapêutico , Músculo Esquelético/metabolismo , Atrofia Muscular/tratamento farmacológico , Proteínas/metabolismo , Receptores Adrenérgicos beta/metabolismo , Síndrome de Emaciação/metabolismo , Adrenérgicos/efeitos adversos , Animais , Caquexia/complicações , Caquexia/tratamento farmacológico , Caquexia/metabolismo , Humanos , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Transdução de Sinais , Síndrome de Emaciação/tratamento farmacológicoRESUMO
Skeletal muscle has a remarkable capacity to regenerate following injury, a property conferred by a resident population of muscle stem cells (MuSCs). In response to injury, MuSCs must double their cellular content to divide, a process requiring significant new biomass in the form of nucleotides, phospholipids, and amino acids. This new biomass is derived from a series of intracellular metabolic cycles and alternative routing of carbon. In this review, we examine the link between metabolism and skeletal muscle regeneration with particular emphasis on the role of the cellular microenvironment in supporting the production of new biomass and MuSC proliferation.
RESUMO
Metabolism has been shown to alter cell fate in human pluripotent stem cells (hPSC). However, current understanding is almost exclusively based on work performed at 20% oxygen (air), with very few studies reporting on hPSC at physiological oxygen (5%). In this study, we integrated metabolic, transcriptomic, and epigenetic data to elucidate the impact of oxygen on hPSC. Using 13C-glucose labeling, we show that 5% oxygen increased the intracellular levels of glycolytic intermediates, glycogen, and the antioxidant response in hPSC. In contrast, 20% oxygen increased metabolite flux through the TCA cycle, activity of mitochondria, and ATP production. Acetylation of H3K9 and H3K27 was elevated at 5% oxygen while H3K27 trimethylation was decreased, conforming to a more open chromatin structure. RNA-seq analysis of 5% oxygen hPSC also indicated increases in glycolysis, lysine demethylases, and glucose-derived carbon metabolism, while increased methyltransferase and cell cycle activity was indicated at 20% oxygen. Our findings show that oxygen drives metabolite flux and specifies carbon fate in hPSC and, although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity and the global epigenetic landscape.
RESUMO
Skeletal myogenesis is a coordinated sequence of events associated with dramatic changes in cell morphology, motility, and metabolism, which causes cellular stress and alters proteostasis. Chaperones, such as heat-shock proteins (HSPs), play important roles in limiting cellular stresses and maintaining proteostasis, but whether HSPs are specifically involved in myogenesis is not well understood. Here, we characterized gene and protein expression and subcellular localization of various HSPs in proliferating C2C12 myoblasts and differentiating myotubes under control conditions and in response to heat stress. Hsp25, Hsp40, and Hsp60 protein expression declined by 48, 35, and 83%, respectively, during differentiation. In contrast, Hsp70 protein levels doubled during early differentiation. Hsp25 was predominantly localized to the cytoplasm of myoblasts and myotubes but formed distinct aggregates in perinuclear spaces of myoblasts after heat-shock. Hsp40 was distributed diffusely throughout the cytoplasm and nucleus and, after heat-shock, translocated to the nucleus of myoblasts but formed aggregates in myotubes. Hsp60 localized to the perinuclear space in myoblasts but was distributed more diffusely across the cytoplasm in myotubes. Hsp70 was expressed diffusely throughout the cytoplasm and nucleus and translocated to the nucleus after heat-shock in myoblasts, but not in myotubes. Hsp90 was expressed diffusely across the cytoplasm in both myoblasts and myotubes under control conditions and did not change in response to heat-shock. These findings reveal distinct and different roles for HSPs in the regulation of myogenic cell proliferation and differentiation.