RESUMO
MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers, and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs led to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation.
Assuntos
Transdiferenciação Celular , Reprogramação Celular , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Fibroblastos/metabolismo , Desenvolvimento Muscular , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Transdiferenciação Celular/genética , Cromatina/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Conformação de Ácido Nucleico , Fenótipo , Ligação Proteica , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.
Assuntos
Proliferação de Células , Desenvolvimento Muscular , Proteínas Musculares/biossíntese , Distrofia Muscular de Duchenne/metabolismo , Mioblastos Esqueléticos/metabolismo , Biossíntese de Proteínas , RNA/metabolismo , Animais , Genótipo , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Proteínas Musculares/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Mioblastos Esqueléticos/patologia , Fases de Leitura Aberta , Fenótipo , RNA/genética , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Interferência de RNA , Splicing de RNA , RNA Circular , Análise de Sequência de RNA/métodos , Transdução de Sinais , TransfecçãoRESUMO
Autosomal dominant PDGFRß gain-of-function mutations in mice and humans cause a spectrum of wasting and overgrowth disorders afflicting the skeleton and other connective tissues, but the cellular origin of these disorders remains unknown. We demonstrate that skeletal stem cells (SSCs) isolated from mice with a gain-of-function D849V point mutation in PDGFRß exhibit colony formation defects that parallel the wasting or overgrowth phenotypes of the mice. Single-cell RNA transcriptomics with SSC-derived polyclonal colonies demonstrates alterations in osteogenic and chondrogenic precursors caused by PDGFRßD849V. Mutant cells undergo poor osteogenesis in vitro with increased expression of Sox9 and other chondrogenic markers. Mice with PDGFRßD849V exhibit osteopenia. Increased STAT5 phosphorylation and overexpression of Igf1 and Socs2 in PDGFRßD849V cells suggests that overgrowth in mice involves PDGFRßD849V activating the STAT5-IGF1 axis locally in the skeleton. Our study establishes that PDGFRßD849V causes osteopenic skeletal phenotypes that are associated with intrinsic changes in SSCs, promoting chondrogenesis over osteogenesis.
Assuntos
Mutação com Ganho de Função , Mioblastos Esqueléticos/metabolismo , Mutação Puntual , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Substituição de Aminoácidos , Animais , Condrogênese/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Mioblastos Esqueléticos/patologia , Osteogênese/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/genéticaRESUMO
Whether gene repositioning to the nuclear periphery during differentiation adds another layer of regulation to gene expression remains controversial. Here, we resolve this by manipulating gene positions through targeting the nuclear envelope transmembrane proteins (NETs) that direct their normal repositioning during myogenesis. Combining transcriptomics with high-resolution DamID mapping of nuclear envelope-genome contacts, we show that three muscle-specific NETs, NET39, Tmem38A, and WFS1, direct specific myogenic genes to the nuclear periphery to facilitate their repression. Retargeting a NET39 fragment to nucleoli correspondingly repositioned a target gene, indicating a direct tethering mechanism. Being able to manipulate gene position independently of other changes in differentiation revealed that repositioning contributes â to â of a gene's normal repression in myogenesis. Together, these NETs affect 37% of all genes changing expression during myogenesis, and their combined knockdown almost completely blocks myotube formation. This unequivocally demonstrates that NET-directed gene repositioning is critical for developmental gene regulation.
Assuntos
Posicionamento Cromossômico , Regulação da Expressão Gênica no Desenvolvimento , Canais Iônicos/genética , Proteínas de Membrana/genética , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Animais , Diferenciação Celular , Linhagem Celular , Regulação para Baixo , Humanos , Canais Iônicos/metabolismo , Cinética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Interferência de RNA , TransfecçãoRESUMO
Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination of myogenic transcription factors. Myogenic transcription is tightly regulated by various intracellular signaling pathways, which include members of the protein kinase D (PKD) family. PKD is a family of serine-threonine kinases that regulate gene expression, protein secretion, cell proliferation, differentiation and inflammation. PKD is a unique PKC family member that shares distant sequence homology to calcium-regulated kinases and plays an important role in muscle physiology. In this report, we show that class I histone deacetylase (HDAC) inhibition, and in particular HDAC8 inhibition, attenuated PKD phosphorylation in skeletal C2C12 myoblasts in response to phorbol ester, angiotensin II and dexamethasone signaling independent of changes in total PKD protein expression. As class I HDACs and PKD signaling are requisite for myocyte differentiation, these data suggest that HDAC8 functions as a potential feedback regulator of PKD phosphorylation to control myogenic gene expression.
Assuntos
Mioblastos Esqueléticos , Proteína Quinase C , Fosforilação , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Mioblastos Esqueléticos/metabolismoRESUMO
The processes of myogenesis during both development and regeneration share a number of similarities across both amniotes and teleosts. In amniotes, the process of muscle formation is considered largely biphasic, with developmental myogenesis occurring through hyperplastic fibre deposition and postnatal muscle growth driven through hypertrophy of existing fibres. In contrast, teleosts continue generating new muscle fibres during adult myogenesis through a process of eternal hyperplasia using a dedicated stem cell system termed the external cell layer. During developmental and regenerative myogenesis alike, muscle progenitors interact with their niche to receive cues guiding their transition into myoblasts and ultimately mature myofibres. During development, muscle precursors receive input from neighbouring embryological tissues; however, during repair, this role is fulfilled by other injury resident cell types, such as those of the innate immune response. Recent work has focused on the role of macrophages as a pro-regenerative cell type which provides input to muscle satellite cells during regenerative myogenesis. As zebrafish harbour a satellite cell system analogous to that of mammals, the processes of regeneration can be interrogated in vivo with the imaging intensive approaches afforded in the zebrafish system. This review discusses the strengths of zebrafish with a focus on both the similarities and differences to amniote myogenesis during both development and repair.
Assuntos
Desenvolvimento Muscular/fisiologia , Regeneração/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologia , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Macrófagos/fisiologia , Modelos Biológicos , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Regeneração/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation.
Assuntos
Diferenciação Celular , MicroRNAs , RNA Circular , Células Satélites de Músculo Esquelético , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , MicroRNAs/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , RNA Circular/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Suínos , Mioblastos Esqueléticos/metabolismoRESUMO
Skeletal muscle is responsible for the majority of glucose disposal following meals, and this is achieved by insulin-mediated trafficking of glucose transporter type 4 (GLUT4) to the cell membrane. The eight-protein exocyst trafficking complex facilitates targeted docking of membrane-bound vesicles, a process underlying the regulated delivery of fuel transporters. We previously demonstrated the role of exocyst subunit EXOC5 in insulin-stimulated GLUT4 exocytosis and glucose uptake in cultured rat skeletal myoblasts. However, the in vivo role of EXOC5 in skeletal muscle remains unclear. Using mice with inducible, skeletal-muscle-specific knockout of exocyst subunit EXOC5 (Exoc5-SMKO), we examined how muscle-specific disruption of the exocyst would affect glucose homeostasis in vivo. We found that both male and female Exoc5-SMKO mice displayed elevated fasting glucose levels. Additionally, male Exoc5-SMKO mice had impaired glucose tolerance and lower serum insulin levels. Using indirect calorimetry, we observed that male Exoc5-SMKO mice have a reduced respiratory exchange ratio during the light period and lower energy expenditure. Using the hyperinsulinemic-euglycemic clamp method, we further showed that insulin-stimulated skeletal muscle glucose uptake is reduced in Exoc5-SMKO males compared with wild-type controls. Overall, our findings indicate that EXOC5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle and regulate glucose homeostasis in vivo.
Assuntos
Glucose/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Metabolismo dos Carboidratos , Membrana Celular/metabolismo , Citoplasma/metabolismo , Exocitose , Feminino , Intolerância à Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Homeostase , Insulina/análise , Insulina/sangue , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/metabolismo , Transporte Proteico , Proteínas de Transporte Vesicular/fisiologiaRESUMO
BACKGROUND: Although the various pharmacological effects of Corni Fructus are highly correlated with its antioxidant activity, the blocking effect against oxidative stress in muscle cells is not clear. The purpose of this study was to investigate the effect of aqueous extracts of Corni Fructus (CFE) against oxidative stress caused by hydrogen peroxide (H2O2) in murine skeletal C2C12 myoblasts. METHODS AND RESULTS: MTT assay for cell viability, DCF-DA staining for reactive oxygen species (ROS) production, Comet assay for DNA damage, annexin V-FITC and PI double staining for apoptosis, JC-1 staining and caspase assay for monitor mitochondrial integrity, and western blotting for related protein levels were conducted in H2O2 oxidative stressed C2C12 cells. Our results showed that CFE pretreatment significantly ameliorated the loss of cell viability and inhibited apoptosis in H2O2-treated C2C12 cells in a concentration-dependent manner. DNA damage induced by H2O2 was also markedly attenuated in the presence of CFE, which was associated with suppression of ROS generation. In addition, H2O2 reduced mitochondrial membrane potential and caused downregulation of Bcl-2 and upregulation of Bax expression, although these were abrogated by CFE pretreatment. Moreover, CFE blocked H2O2-induced cytosolic release of cytochrome c, activation of caspase-9 and caspase-3, and degradation of poly (ADP-ribose) polymerase. CONCLUSION: Taken together, the present results demonstrate that CFE could protect C2C12 cells from H2O2-induced damage by eliminating ROS generation, thereby blocking mitochondria-mediated apoptosis pathway. These results indicate that CFE has therapeutic potential for the prevention and treatment of oxidative stress-mediated myoblast injury.
Assuntos
Cornus , Mioblastos Esqueléticos , Animais , Apoptose , Sobrevivência Celular , Cornus/metabolismo , Dano ao DNA , Peróxido de Hidrogênio/farmacologia , Camundongos , Mioblastos Esqueléticos/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Muscle progenitor cells (MPCs) in aged muscle exhibit impaired activation into proliferating myoblasts, thereby impairing fusion and changes in secreted factors. The antihyperglycemic drug metformin, currently studied as a candidate antiaging therapy, may have potential to promote function of aged MPCs. We evaluated the impact of 2 wk of metformin ingestion on primary myoblast function measured in vitro after being extracted from muscle biopsies of older adult participants. MPCs were isolated from muscle biopsies of community-dwelling older (4 male/4 female, â¼69 yr) adult participants before (pre) and after (post) the metformin ingestion period and studied in vitro. Cells were extracted from Young participants (4 male/4 female, â¼27 yr) to serve as a "youthful" comparator. MPCs from Old subjects had lower fusion index and myoblast-endothelial cell homing compared with Young, while Old MPCs, extracted after short-term metformin ingestion, performed better at both tasks. Transcriptomic analyses of Old MPCs (vs. Young) revealed decreased histone expression and increased myogenic pathway activity, yet this phenotype was partially restored by metformin. However, metformin ingestion exacerbated pathways related to inflammation signaling. Together, this study demonstrated that 2 wk of metformin ingestion induced persistent effects on Old MPCs that improved function in vitro and altered their transcriptional signature including histone and chromatin remodeling.
Assuntos
Envelhecimento Saudável , Hipoglicemiantes/administração & dosagem , Metformina/administração & dosagem , Mioblastos Esqueléticos/efeitos dos fármacos , Adulto , Fatores Etários , Idoso , Comunicação Celular , Fusão Celular , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Esquema de Medicação , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Mioblastos Esqueléticos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transcriptoma/efeitos dos fármacosRESUMO
Recently, methods for creating three-dimensional (3-D) human skeletal muscle tissues from myogenic cell lines have been reported. Bioengineered muscle tissues are contractile and respond to electrical and chemical stimulation. In this study, we provide an electrophysiological analysis of healthy and dystrophic 3-D bioengineered skeletal muscle tissues, focusing on Duchenne muscular dystrophy (DMD). We enlist the 3-D in vitro model of DMD muscle tissue to evaluate muscle cell electrical properties uncoupled from presynaptic neural inputs, an understudied aspect of DMD. Our data show that previously reported electrophysiological aspects of DMD, including effects on membrane potential and membrane resistance, are replicated in the 3-D muscle tissue model. Furthermore, we test a potential therapeutic compound, poloxamer 188, and demonstrate capacity for improving the membrane potential in DMD muscle. Therefore, this study serves as a baseline for a new in vitro method to examine potential therapies for muscular disorders.
Assuntos
Distrofina/metabolismo , Potenciais da Membrana , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mioblastos Esqueléticos/metabolismo , Engenharia Tecidual , Adolescente , Estudos de Casos e Controles , Técnicas de Cultura de Células , Linhagem Celular , Criança , Distrofina/genética , Impedância Elétrica , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/ultraestrutura , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Mutação , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/ultraestrutura , Poloxâmero/farmacologia , Sódio/metabolismoRESUMO
The peroxisome proliferator-activated receptors (PPARs) have been previously implicated in the pathophysiology of skeletal muscle dysfunction in women with breast cancer (BC) and animal models of BC. This study investigated alterations induced in skeletal muscle by BC-derived factors in an in vitro conditioned media (CM) system and tested the hypothesis that BC cells secrete a factor that represses PPAR-γ (PPARG) expression and its transcriptional activity, leading to downregulation of PPARG target genes involved in mitochondrial function and other metabolic pathways. We found that BC-derived factors repress PPAR-mediated transcriptional activity without altering protein expression of PPARG. Furthermore, we show that BC-derived factors induce significant alterations in skeletal muscle mitochondrial function and lipid accumulation, which are rescued with exogenous expression of PPARG. The PPARG agonist drug rosiglitazone was able to rescue BC-induced lipid accumulation but did not rescue effects of BC-derived factors on PPAR-mediated transcription or mitochondrial function. These data suggest that BC-derived factors alter lipid accumulation and mitochondrial function via different mechanisms that are both related to PPARG signaling, with mitochondrial dysfunction likely being altered via repression of PPAR-mediated transcription, and lipid accumulation being altered via transcription-independent functions of PPARG.
Assuntos
Neoplasias da Mama/metabolismo , Caquexia/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias Musculares/metabolismo , Mioblastos Esqueléticos/metabolismo , PPAR gama/metabolismo , Comunicação Parácrina , Animais , Neoplasias da Mama/complicações , Neoplasias da Mama/patologia , Caquexia/etiologia , Caquexia/genética , Caquexia/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Feminino , Células HEK293 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/patologia , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/patologia , PPAR gama/agonistas , PPAR gama/genética , Rosiglitazona/farmacologia , Transdução de Sinais , Transcrição GênicaRESUMO
Skeletal muscle development is a complex biological process involving multiple key genes, signaling pathways and noncoding RNAs, including microRNAs and circular RNAs (circRNAs). However, the regulatory relationship among them is so complicated that it has not yet been fully elucidated. In this study, we found that miR-7 inhibited C2C12 cell proliferation and differentiation by targeting transcription factor 12 (TCF12). circHIPK3 acted as a competing endogenous RNA, and its overexpression effectively reversed the regulation of miR-7 on C2C12 cell proliferation and differentiation by increasing TCF12 expression. Taken together, our findings provide evidence that circHIPK3 regulates skeletal muscle development through the miR-7/TCF12 pathway. This study provides a scientific basis for further research on skeletal muscle development at the circRNA level.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Proliferação de Células , MicroRNAs/metabolismo , Desenvolvimento Muscular , Mioblastos Esqueléticos/metabolismo , RNA Circular/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Regulação da Expressão Gênica , Camundongos , MicroRNAs/genética , RNA Circular/genética , Transdução de SinaisRESUMO
Fat infiltration in skeletal muscle is observed in several myopathies, is associated with muscular dysfunction, and is strongly correlated with insulin resistance, diabetes, obesity, and aging. In animal production, skeletal muscle fat (also known as intermuscular and intramuscular fat) is positively related to meat quality including tenderness, flavor, and juiciness. Thus, understanding the cell origin and regulation mechanism of skeletal muscle fat infiltration is important for developing therapies against human myopathies as well as for improving meat quality. Notably, age, sarcopenia, oxidative stress, injury, and regeneration can activate adipogenic differentiation potential in myoblasts and affect fat accumulation in skeletal muscle. In addition, several transcriptional and nutritional factors can directly induce transdifferentiation of myoblasts into adipocytes. In this review, we focused on the recent progress in understanding the muscle-to-adipocyte differentiation and summarized and discussed the genetic, nutritional, and physiological factors that can induce transdifferentiation of myoblasts into adipocytes. Moreover, the regulatory roles and mechanisms of these factors during the transdifferentiation process were also discussed.
Assuntos
Adipócitos/metabolismo , Adipogenia , Transdiferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Adipócitos/patologia , Fatores Etários , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Composição Corporal , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/patologia , Mioblastos Esqueléticos/patologia , Estado Nutricional , Estresse Oxidativo , Fenótipo , Transdução de SinaisRESUMO
Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA-free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α-linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.
Assuntos
Ácidos Graxos/toxicidade , Gotículas Lipídicas/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Insulina/farmacologia , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Camundongos , Força Muscular/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Mioblastos Esqueléticos/metabolismo , Mioblastos Esqueléticos/patologia , Engenharia TecidualRESUMO
Earth's gravity is essential for maintaining skeletal muscle mass and function in the body. The role of gravity in the myogenic mechanism has been studied with animal experiments in the International Space Station. Recently, gravity-control devices allow to study the effects of gravity on cultured cells on the ground. This study demonstrated that simulated microgravity accelerated aging of human skeletal muscle myoblasts in an in-vitro culture. The microgravity culture induced a significant decrease in cell proliferation and an enlargement of the cytoskeleton and nucleus of cells. Similar changes are often observed in aged myoblasts following several passages. In fact, by the microgravity culture the expression of senescence associated ß-Gal was significantly enhanced, and some muscle-specific proteins decreased in the enlarged cells. Importantly, these microgravity effects remained with the cells even after a return to normal gravity conditions. Consequently, the microgravity-affected myoblasts demonstrated a reduced capability of differentiation into myotubes. In the body, it is difficult to interpret the disability of microgravity-affected myoblasts, since muscle regeneration is linked to the supply of new myogenic cells. Therefore, our in-vitro cell culture study will be advantageous to better understand the role of each type of myogenic cell in human muscle without gravitational stress at the single cell level.
Assuntos
Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Mioblastos Esqueléticos/patologia , Análise de Célula Única/métodos , Simulação de Ausência de Peso/métodos , Envelhecimento/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Citoesqueleto/metabolismo , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismoRESUMO
At-risk alcohol use is prevalent and increases dysglycemia among people living with human immunodeficiency virus (PLWH). Skeletal muscle (SKM) bioenergetic dysregulation is implicated in dysglycemia and type 2 diabetes. The objective of this study was to determine the relationship between at-risk alcohol, glucose tolerance, and SKM bioenergetic function in PLWH. Thirty-five PLWH (11 females, 24 males, age: 53 ± 9 yr, body mass index: 29.0 ± 6.6 kg/m2) with elevated fasting glucose enrolled in the ALIVE-Ex study provided medical history and alcohol use information [Alcohol Use Disorders Identification Test (AUDIT)], then underwent an oral glucose tolerance test (OGTT) and SKM biopsy. Bioenergetic health and function and mitochondrial volume were measured in isolated myoblasts. Mitochondrial gene expression was measured in SKM. Linear regression adjusting for age, sex, and smoking was performed to examine the relationship between glucose tolerance (2-h glucose post-OGTT), AUDIT, and their interaction with each outcome measure. Negative indicators of bioenergetic health were significantly (P < 0.05) greater with higher 2-h glucose (proton leak) and AUDIT (proton leak, nonmitochondrial oxygen consumption, and bioenergetic health index). Mitochondrial volume was increased with the interaction of higher 2-h glucose and AUDIT. Mitochondrial gene expression decreased with higher 2-h glucose (TFAM, PGC1B, PPARG, MFN1), AUDIT (MFN1, DRP1, MFF), and their interaction (PPARG, PPARD, MFF). Decreased expression of mitochondrial genes were coupled with increased mitochondrial volume and decreased bioenergetic health in SKM of PLWH with higher AUDIT and 2-h glucose. We hypothesize these mechanisms reflect poorer mitochondrial health and may precede overt SKM bioenergetic dysregulation observed in type 2 diabetes.
Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Metabolismo Energético , Infecções por HIV/metabolismo , Sobreviventes de Longo Prazo ao HIV , Mitocôndrias Musculares/metabolismo , Mioblastos Esqueléticos/metabolismo , Músculo Quadríceps/metabolismo , Adulto , Consumo de Bebidas Alcoólicas/sangue , Consumo de Bebidas Alcoólicas/epidemiologia , Consumo de Bebidas Alcoólicas/fisiopatologia , Biomarcadores/sangue , Células Cultivadas , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/fisiopatologia , Humanos , Resistência à Insulina , Louisiana/epidemiologia , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio , Músculo Quadríceps/fisiopatologia , Medição de Risco , Fatores de Risco , Adulto JovemRESUMO
Hyperoxic conditions are known to accelerate skeletal muscle regeneration after injuries. In the early phase of regeneration, macrophages invade the injured area and subsequently secrete various growth factors, which regulate myoblast proliferation and differentiation. Although hyperoxic conditions accelerate muscle regeneration, it is unknown whether this effect is indirectly mediated by macrophages. Here, using C2C12 cells, we show that not only hyperoxia but also hypoxia enhance myoblast proliferation directly, without accelerating differentiation into myotubes. Under hyperoxic conditions (95% O2 + 5% CO2), the cell membrane was damaged because of lipid oxidization, and a disrupted cytoskeletal structure, resulting in suppressed cell proliferation. However, a culture medium containing vitamin C (VC), an antioxidant, prevented this lipid oxidization and cytoskeletal disruption, resulting in enhanced proliferation in response to hyperoxia exposure of ≤4 h/day. In contrast, exposure to hypoxic conditions (95% N2 + 5% CO2) for ≤8 h/day enhanced cell proliferation. Hyperoxia did not promote cell differentiation into myotubes, regardless of whether the culture medium contained VC. Similarly, hypoxia did not accelerate cell differentiation. These results suggest that regardless of hyperoxia or hypoxia, changes in oxygen tension can enhance cell proliferation directly, but do not influence differentiation efficiency in C2C12 cells. Moreover, excess oxidative stress abrogated the enhancement of myoblast proliferation induced by hyperoxia. This research will contribute to basic data for applying the effects of hyperoxia or hypoxia to muscle regeneration therapy.
Assuntos
Diferenciação Celular , Proliferação de Células , Desenvolvimento Muscular , Mioblastos Esqueléticos/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Regeneração , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Cinética , Metabolismo dos Lipídeos , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/patologia , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/toxicidade , Regeneração/efeitos dos fármacosRESUMO
Over the past 20 years, various identifiers of cellular senescence have been used to quantify the abundance of these cells in different tissues. These include classic markers such as p16, senescence-associated ß-gal, and γH2AX, in addition to more recent markers (Sudan Black B and HMGB1). In vivo data on the usefulness of these markers in skeletal muscle are very limited and inconsistent. In the present study, we attempted to identify senescent cells in frozen human skeletal muscle biopsies using these markers to determine the effects of age and obesity on senescent cell burden; however, we were only able to assess the abundance of DNA-damaged nuclei using γH2AX immunohistochemistry. The abundance of γH2AX+ cells, including satellite cells, was not higher in muscle from old compared to young individuals; however, γH2AX+ cells were higher with obesity. Additionally, terminally differentiated, postmitotic myofiber nuclei from obese individuals had elevated γH2AX abundance compared to muscle from lean individuals. Analyses of gene expression support the conclusion that the elevated DNA damage and the senescence-associated secretory phenotype are preferentially associated with obesity in skeletal muscle. These data implicate obesity as a larger contributor to DNA damage in skeletal muscle than aging; however, more sensitive senescence markers for human skeletal muscle are needed to determine if these cells are in fact senescent.
Assuntos
Envelhecimento/metabolismo , Histonas/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Biomarcadores/metabolismo , Diferenciação Celular , Senescência Celular , Dano ao DNA , Reparo do DNA/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Obesidade/patologia , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Adulto JovemRESUMO
In skeletal muscle fibers, mitochondria are densely packed adjacent to myofibrils because adenosine triphosphate (ATP) is needed to fuel sarcomere shortening. However, despite this close physical and biochemical relationship, the effects of mitochondrial dynamics on skeletal muscle contractility are poorly understood. In this study, we analyzed the effects of Mitochondrial Division Inhibitor 1 (mdivi-1), an inhibitor of mitochondrial fission, on the structure and function of both mitochondria and myofibrils in skeletal muscle tissues engineered on micromolded gelatin hydrogels. Treatment with mdivi-1 did not alter myotube morphology, but did increase the mitochondrial turbidity and oxidative capacity, consistent with reduced mitochondrial fission. Mdivi-1 also significantly increased basal, twitch, and tetanus stresses, as measured using the Muscular Thin Film (MTF) assay. Finally, mdivi-1 increased sarcomere length, potentially due to mdivi-1-induced changes in mitochondrial volume and compression of myofibrils. Together, these results suggest that mdivi-1 increases contractile stress generation, which may be caused by an increase in maximal respiration and/or sarcomere length due to increased volume of individual mitochondria. These data reinforce that mitochondria have both biochemical and biomechanical roles in skeletal muscle and that mitochondrial dynamics can be manipulated to alter muscle contractility.