RESUMO
Insulin-like growth factor (IGF)-I mediates long-term activities that determine cell fate, including cell proliferation and differentiation. This study aimed to characterize the mechanisms by which IGF-I determines cell fate from the aspect of IGF-I signaling dynamics. In L6 myoblasts, myogenic differentiation proceeded under low IGF-I levels, whereas proliferation was enhanced under high levels. Mathematical and experimental analyses revealed that IGF-I signaling oscillated at low IGF-I levels but remained constant at high levels, suggesting that differences in IGF-I signaling dynamics determine cell fate. We previously reported that differential insulin receptor substrate (IRS)-1 levels generate a driving force for cell competition. Computational simulations and immunofluorescence analyses revealed that asynchronous IRS-1 protein oscillations were synchronized during myogenic processes through cell competition. Disturbances of cell competition impaired signaling synchronization and cell fusion, indicating that synchronization of IGF-I signaling oscillation is critical for myoblast cell fusion to form multinucleate myotubes.
Assuntos
Diferenciação Celular , Fator de Crescimento Insulin-Like I , Mioblastos , Transdução de Sinais , Fator de Crescimento Insulin-Like I/metabolismo , Mioblastos/metabolismo , Mioblastos/citologia , Animais , Linhagem Celular , Proliferação de Células , Desenvolvimento Muscular , Proteínas Substratos do Receptor de Insulina/metabolismo , Ratos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/citologia , Fusão CelularRESUMO
N-acetylaspartate (NAA) is a neuronal metabolite that can be extruded in extracellular fluids and whose blood concentration increases in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Aspartoacylase (ASPA) is the enzyme responsible for NAA breakdown. It is abundantly expressed in skeletal muscle and most other human tissues, but the role of NAA catabolism in the periphery is largely neglected. Here we demonstrate that NAA treatment of differentiated C2C12 muscle cells increases lipid turnover, mitochondrial biogenesis and oxidative metabolism at the expense of glycolysis. These effects were ascribed to NAA catabolism, as CRISPR/Cas9 ASPA KO cells are insensitive to NAA administration. Moreover, the metabolic switch induced by NAA was associated with an augmented resistance to atrophic stimuli. Consistently with in vitro results, SOD1-G93A ALS mice show an increase in ASPA levels in those muscles undergoing the glycolytic to oxidative switch during the disease course. The impact of NAA on the metabolism and resistance capability of myotubes supports a role for this metabolite in the phenotypical adaptations of skeletal muscle in neuromuscular disorders.
Assuntos
Ácido Aspártico , Glicólise , Fibras Musculares Esqueléticas , Animais , Glicólise/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Camundongos , Ácido Aspártico/metabolismo , Ácido Aspártico/análogos & derivados , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/genética , Humanos , Oxirredução , Linhagem Celular , Camundongos TransgênicosRESUMO
Insulin-like growth factor-1-induced activation of ATP citrate lyase (ACLY) improves muscle mitochondrial function through an Akt-dependent mechanism. In this study, we examined whether Akt1 deficiency alters skeletal muscle fiber type and mitochondrial function by regulating ACLY-dependent signaling in male Akt1 knockout (KO) mice (12-16 weeks old). Akt1 KO mice exhibited decreased body weight and muscle wet weight, with reduced cross-sectional areas of slow- and fast-type muscle fibers. Loss of Akt1 did not affect the phosphorylation status of ACLY in skeletal muscle. The skeletal muscle fiber type and expression of mitochondrial oxidative phosphorylation complex proteins were unchanged in Akt1 KO mice compared with the wild-type control. These observations indicate that Akt1 is important for the regulation of skeletal muscle fiber size, whereas the regulation of muscle fiber type and muscle mitochondrial content occurs independently of Akt1 activity.
Assuntos
Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt , Animais , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Camundongos , Músculo Esquelético/metabolismo , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/deficiência , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , ATP Citrato (pro-S)-Liase/metabolismo , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/deficiênciaRESUMO
Carotenoids are natural pigments utilized as colourants and antioxidants across food, pharmaceutical and cosmetic industries. They exist in carbon chain lengths of C30, C40, C45 and C50, with C40 variants being the most common. Bacterioruberin (BR) and its derivatives are part of the less common C50 carotenoid group, synthesized primarily by halophilic archaea. This study analysed the compositional characteristics of BR extract (BRE) isolated from 'Haloferax marinum' MBLA0078, a halophilic archaeon isolated from seawater near Yeoungheungdo Island in the Republic of Korea, and investigated its antioxidant activity and protective effect on lipopolysaccharide (LPS)-induced C2C12 myotube atrophy. The main components of BRE included all-trans-BR, monoanhydrobacterioruberin, 2-isopentenyl-3,4-dehydrorhodopin and all-trans-bisanhydrobacterioruberin. BRE exhibited higher antioxidant activity and DNA nicking protection activity than other well-known C40 carotenoids, such as ß-carotene, lycopene and astaxanthin. In C2C12 myotubes, LPS treatment led to a reduction in myotube diameter and number, as well as the hypertranscription of the muscle-specific ubiquitin ligase MAFbx and MuRF1. BRE mitigated these changes by activating the Akt/mTOR pathway. Furthermore, BRE abolished the elevated cellular reactive oxygen species levels and the inflammation response induced by LPS. This study demonstrated that 'Hfx. marinum' is an excellent source of natural microbial C50 carotenoids with strong antioxidant capacity and may offer potential protective effects against muscle atrophy.
Assuntos
Antioxidantes , Carotenoides , Lipopolissacarídeos , Fibras Musculares Esqueléticas , Antioxidantes/farmacologia , Animais , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Linhagem Celular , Carotenoides/farmacologia , República da Coreia , Água do Mar/microbiologiaRESUMO
Cannabidiol (CBD) is widely used in sports for recovery, pain management, and sleep improvement, yet its effects on muscle are not well understood. This study aimed to determine the transcriptional response of murine skeletal muscle myotubes to broad-spectrum CBD and synthetic CBD (sCBD). Differentiated C2C12 myotubes were treated with 10 µM CBD, sCBD, or vehicle control (DMSO) for 24 h before RNA extraction. Poly-A tail-enriched mRNA libraries were constructed and sequenced using 2 × 50 bp paired-end sequencing. CBD and sCBD treatment induced 4489 and 1979 differentially expressed genes (DEGs; p < 0.001, FDR step-up <0.05), respectively, with common upregulation of 857 genes and common downregulation of 648 genes. Common upregulated DEGs were associated with "response to unfolded protein," "cell redox homeostasis," "endoplasmic reticulum stress," "oxidative stress," and "cellular response to hypoxia." Common downregulated DEGs were linked to "sarcomere organization," "skeletal muscle tissue development," "regulation of muscle contraction," and "muscle contraction." CBD treatment induced unique DEGs compared to sCBD. The data indicate CBD may induce mild cellular stress, activating pathways associated with altered redox balance, unfolded protein response, and endoplasmic reticulum stress. We hypothesize that CBD interacts with muscle and may elicit a "mitohormetic" effect that warrants further investigation.
Assuntos
Canabidiol , Fibras Musculares Esqueléticas , Transcriptoma , Canabidiol/farmacologia , Animais , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Transcriptoma/efeitos dos fármacos , Linhagem CelularRESUMO
BACKGROUND: Sarcopenic obesity, which is associated with a poorer prognosis than that of sarcopenia alone, may be positively affected by soy isoflavones, known inhibitors of muscle atrophy. Herein, we hypothesize that these compounds may prevent sarcopenic obesity by upregulating the gut metabolites with anti-inflammatory effects. METHODS: To explore the effects of soy isoflavones on sarcopenic obesity and its mechanisms, we employed both in vivo and in vitro experiments. Mice were fed a high-fat, high-sucrose diet with or without soy isoflavone supplementation. Additionally, the mouse C2C12 myotube cells were treated with palmitic acid and daidzein in vitro. RESULTS: The isoflavone considerably reduced muscle atrophy and the expression of the muscle atrophy genes in the treated group compared to the control group (Fbxo32, p = 0.0012; Trim63, p < 0.0001; Foxo1, p < 0.0001; Tnfa, p = 0.1343). Elevated levels of daidzein were found in the muscles and feces of the experimental group compared to the control group (feces, p = 0.0122; muscle, p = 0.0020). The real-time PCR results demonstrated that the daidzein decreased the expression of the palmitate-induced inflammation and muscle atrophy genes in the C2C12 myotube cells (Tnfa, p = 0.0201; Il6, p = 0.0008; Fbxo32, p < 0.0001; Hdac4, p = 0.0002; Trim63, p = 0.0114; Foxo1, p < 0.0001). Additionally, it reduced the palmitate-induced protein expression related to the muscle atrophy in the C2C12 myotube cells (Foxo1, p = 0.0078; MuRF1, p = 0.0119). CONCLUSIONS: The daidzein suppressed inflammatory cytokine- and muscle atrophy-related gene expression in the C2C12 myotubes, thereby inhibiting muscle atrophy.
Assuntos
Citocinas , Isoflavonas , Atrofia Muscular , Isoflavonas/farmacologia , Animais , Camundongos , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Atrofia Muscular/prevenção & controle , Masculino , Citocinas/metabolismo , Citocinas/genética , Linhagem Celular , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sarcopenia/prevenção & controle , Sarcopenia/metabolismo , Sarcopenia/tratamento farmacológico , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Dieta Hiperlipídica/efeitos adversos , Obesidade/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Glycine max/química , Modelos Animais de Doenças , Ácido Palmítico/farmacologiaRESUMO
As older adults tend to reduce their intake of animal-source proteins, plant-source proteins may offer valuable resources for better protein intake. The aim of this study was to assess whether the pea proteins can be used to achieve blood amino acid levels that stimulate muscle protein synthesis. We measured variations in plasma amino acid concentrations in young and older adults given pea (NUTRALYS® S85 Plus) or whey proteins either alone or in a standardized meal. The effect of amino acid concentrations on protein synthesis in C2C12 myotubes was determined. In terms of results, plasma amino acid concentrations reflected the difference between the amino acid contents of whey and pea proteins. Blood leucine showed a greater increase of 91 to 130% with whey protein compared to pea protein, while the opposite was observed for arginine (A greater increase of 147 to 210% with pea compared to whey). Culture media prepared with plasmas from the human study induced age-dependent but not protein-type-dependent changes in myotube protein synthesis. In conclusion, pea and whey proteins have the same qualities in terms of their properties to maintain muscle protein synthesis. Pea proteins can be recommended for older people who do not consume enough animal-source proteins.
Assuntos
Aminoácidos , Fibras Musculares Esqueléticas , Proteínas de Ervilha , Proteínas do Soro do Leite , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Humanos , Masculino , Animais , Idoso , Aminoácidos/sangue , Camundongos , Feminino , Adulto , Adulto Jovem , Biossíntese de Proteínas/efeitos dos fármacos , Linhagem Celular , Proteínas Musculares/biossíntese , Proteínas Musculares/metabolismo , Pisum sativum/químicaRESUMO
Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.
Assuntos
Sistemas CRISPR-Cas , Distrofina , Éxons , Duplicação Gênica , Edição de Genes , Distrofia Muscular de Duchenne , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Humanos , Éxons/genética , Distrofina/genética , Edição de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas/genética , Terapia Genética/métodos , Fibras Musculares Esqueléticas/metabolismoRESUMO
The regenerative capacity of muscle, which primarily relies on anabolic processes, diminishes with age, thereby reducing the effectiveness of therapeutic interventions aimed at treating age-related muscle atrophy. In this study, we observed a decline in the expression of methionine adenosine transferase 2A (MAT2A), which synthesizes S-adenosylmethionine (SAM), in the muscle tissues of both aged humans and mice. Considering MAT2A's critical role in anabolism, we hypothesized that its reduced expression contributes to the impaired regenerative capacity of aging skeletal muscle. Mimicking this age-related reduction in the MAT2A level, either by reducing gene expression or inhibiting enzymatic activity, led to inhibiting their differentiation into myotubes. In vivo, inhibiting MAT2A activity aggravated BaCl2-induced skeletal muscle damage and decreased the number of satellite cells, whereas supplementation with SAM improved these effects. RNA-sequencing analysis further revealed that the Fas cell surface death receptor (Fas) gene was upregulated in Mat2a-knockdown C2C12 cells. Suppressing MAT2A expression or activity elevated Fas protein levels and increased the proportion of apoptotic cells. Additionally, inhibition of MAT2A expression or activity increased p53 expression. In conclusion, our findings demonstrated that impaired MAT2A expression or activity compromised the regeneration and repair capabilities of skeletal muscle, partially through p53-Fas-mediated apoptosis.
Assuntos
Metionina Adenosiltransferase , Músculo Esquelético , Animais , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Humanos , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/genética , Masculino , Regeneração , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/farmacologia , Linhagem Celular , Receptor fas/metabolismo , Receptor fas/genética , Apoptose/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Diferenciação Celular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Envelhecimento/metabolismo , Envelhecimento/genética , IdosoRESUMO
It has been demonstrated that filbertone, the principal flavor compound of hazelnuts, exhibits preventive effects against hypothalamic inflammation, obesity, neurodegenerative diseases, and muscle lipid accumulation. However, its influence on muscle aging has yet to be elucidated. The objective of this study was to investigate the effects of filbertone on muscle aging in C2C12 myotubes subjected to senescence induction by either doxorubicin or hydrogen peroxide. To ascertain the mechanisms by which filbertone exerts its effects, we conducted a series of experiments, including Western blot analysis, reverse transcription quantitative polymerase chain reaction (qRT-PCR), and senescence-associated ß-galactosidase (SA-ß-gal) staining. Filbertone was markedly observed to decrease not only the protein levels of p53 (p < 0.01) in senescence-induced skeletal muscle cells, but also the gene expression levels of p21 (p < 0.05), a direct target of p53. The expression of muscle-related genes, including myogenin and muscle RING-finger protein-1 (MuRF1), was found to be significantly enhanced in senescent muscle cells following treatment with filbertone (p < 0.05). In addition, the number of senescent skeletal muscle cells exhibiting ß-galactosidase activity was found to be markedly reduced in the presence of filbertone (p < 0.01). Collectively, these findings suggest that filbertone plays a pivotal role in the regulation of muscle aging.
Assuntos
Senescência Celular , Doxorrubicina , Peróxido de Hidrogênio , Fibras Musculares Esqueléticas , Proteínas Musculares , Miogenina , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Animais , Senescência Celular/efeitos dos fármacos , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Miogenina/metabolismo , Miogenina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Doxorrubicina/farmacologia , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Linhagem Celular , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismoRESUMO
Chronic kidney disease (CKD) is associated with various pathologic changes, including elevations in serum phosphate levels (hyperphosphatemia), vascular calcification, and skeletal muscle atrophy. Elevated phosphate can damage vascular smooth muscle cells and cause vascular calcification. Here, we determined whether high phosphate can also affect skeletal muscle cells and whether hyperphosphatemia, in the context of CKD or by itself, is associated with skeletal muscle atrophy. As models of hyperphosphatemia with CKD, we studied mice receiving an adenine-rich diet for 14 weeks and mice with deletion of Collagen 4a3 (Col4a3-/-). As models of hyperphosphatemia without CKD, we analyzed mice receiving a high-phosphate diet for three and six months as well as a genetic model for klotho deficiency (kl/kl). We found that adenine, Col4a3-/-, and kl/kl mice have reduced skeletal muscle mass and function and develop atrophy. Mice on a high-phosphate diet for six months also had lower skeletal muscle mass and function but no significant signs of atrophy, indicating less severe damage compared with the other three models. To determine the potential direct actions of phosphate on skeletal muscle, we cultured primary mouse myotubes in high phosphate concentrations, and we detected the induction of atrophy. We conclude that in experimental mouse models, hyperphosphatemia is sufficient to induce skeletal muscle atrophy and that, among various other factors, elevated phosphate levels might contribute to skeletal muscle injury in CKD.
Assuntos
Hiperfosfatemia , Músculo Esquelético , Atrofia Muscular , Fosfatos , Animais , Hiperfosfatemia/patologia , Camundongos , Atrofia Muscular/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/etiologia , Músculo Esquelético/patologia , Músculo Esquelético/metabolismo , Fosfatos/sangue , Fosfatos/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/metabolismo , Modelos Animais de Doenças , Camundongos Knockout , Masculino , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/genética , Camundongos Endogâmicos C57BL , Proteínas Klotho/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologiaRESUMO
Skeletal muscle is a highly heterogeneous tissue, and its contractile proteins are composed of different isoforms, forming various types of muscle fiber, each of which has its own metabolic characteristics. It has been demonstrated that endurance exercise induces the transition of muscle fibers from fast-twitch to slow-twitch muscle fiber type. Herein, we discover a novel epigenetic mechanism for muscle contractile property tightly coupled to its metabolic capacity during muscle fiber type transition with exercise training. Our results show that an 8-week endurance exercise induces histone methylation remodeling of PGC-1α and myosin heavy chain (MHC) isoforms in the rat gastrocnemius muscle, accompanied by increased mitochondrial biogenesis and an elevated ratio of slow-twitch to fast-twitch fibers. Furthermore, to verify the roles of reactive oxygen species (ROS) and AMPK in exercise-regulated epigenetic modifications and muscle fiber type transitions, mouse C2C12 myotubes were used. It was shown that rotenone activates ROS/AMPK pathway and histone methylation enzymes, which then promote mitochondrial biogenesis and MHC slow isoform expression. Mitoquinone (MitoQ) partially blocking rotenone-treated model confirms the role of ROS in coupling mitochondrial biogenesis with muscle fiber type. In conclusion, endurance exercise couples mitochondrial biogenesis with MHC slow isoform by remodeling histone methylation, which in turn promotes the transition of fast-twitch to slow-twitch muscle fibers. The ROS/AMPK pathway may be involved in the regulation of histone methylation enzymes by endurance exercise.
Assuntos
Histonas , Cadeias Pesadas de Miosina , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Condicionamento Físico Animal , Espécies Reativas de Oxigênio , Animais , Histonas/metabolismo , Camundongos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Masculino , Cadeias Pesadas de Miosina/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Metilação , Fibras Musculares Esqueléticas/metabolismo , Epigênese Genética , Fibras Musculares de Contração Lenta/metabolismo , Resistência Física/fisiologia , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Linhagem Celular , Proteínas Quinases Ativadas por AMP/metabolismoRESUMO
BACKGROUND: Cell-based strategies are being explored as a therapeutic option for muscular dystrophies, using a variety of cell types from different origin and with different characteristics. Primary pericytes are multifunctional cells found in the capillary bed that exhibit stem cell-like and myogenic regenerative properties. This unique combination allows them to be applied systemically, presenting a promising opportunity for body-wide muscle regeneration. We previously reported the successful isolation of ALP+ pericytes from skeletal muscle of patients with myotonic dystrophy type 1 (DM1). These pericytes maintained normal growth parameters and myogenic characteristics in vitro despite the presence of nuclear (CUG)n RNA foci, the cellular hallmark of DM1. Here, we examined the behaviour of DM1 pericytes during myogenic differentiation. METHODS: DMPK (CTG)n repeat lengths in patient pericytes were assessed using small pool PCR, to be able to relate variation in myogenic properties and disease hallmarks to repeat expansion. Pericytes from unaffected controls and DM1 patients were cultured under differentiating conditions in vitro. In addition, the pericytes were grown in co-cultures with myoblasts to examine their regenerative capacity by forming hybrid myotubes. Finally, the effect of pericyte fusion on DM1 disease hallmarks was investigated. RESULTS: Small pool PCR analysis revealed the presence of somatic mosaicism in pericyte cell pools. Upon differentiation to myotubes, DMPK expression was upregulated, leading to an increase in nuclear foci sequestering MBNL1 protein. Remarkably, despite the manifestation of these disease biomarkers, patient-derived pericytes demonstrated myogenic potential in co-culture experiments comparable to unaffected pericytes and myoblasts. However, only the unaffected pericytes improved the disease hallmarks in hybrid myotubes. From 20% onwards, the fraction of unaffected nuclei in myotubes positively correlated with a reduction of the number of RNA foci and an increase in the amount of free MBNL1. CONCLUSIONS: Fusion of only a limited number of unaffected myogenic precursors to DM1 myotubes already ameliorates cellular disease hallmarks, offering promise for the development of cell transplantation strategies to lower disease burden.
Assuntos
Diferenciação Celular , Fibras Musculares Esqueléticas , Distrofia Miotônica , Miotonina Proteína Quinase , Pericitos , Humanos , Distrofia Miotônica/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/terapia , Distrofia Miotônica/patologia , Fibras Musculares Esqueléticas/metabolismo , Pericitos/metabolismo , Miotonina Proteína Quinase/genética , Miotonina Proteína Quinase/metabolismo , Mioblastos/metabolismo , Mioblastos/citologia , Desenvolvimento Muscular , Células Cultivadas , Masculino , Adulto , Feminino , Técnicas de Cocultura , Pessoa de Meia-Idade , Fusão CelularRESUMO
Elevated circulating branched-chain amino acids (BCAA) have been linked with the severity of insulin resistance across numerous populations, implicating heightened BCAA metabolism as a potential therapy for insulin resistance. Recently, the angiotensin II type 1 receptor (AT1R) inhibitor Valsartan (VAL) was identified as a potent inhibitor of branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), a negative regulator of BCAA metabolism. This work investigated the effect of VAL on myotube metabolism and insulin sensitivity under both insulin sensitive and insulin resistant conditions. C2C12 myotubes were treated with or without VAL at 8 µM for 24 h, both with and without hyperinsulinemic-induced insulin resistance. Oxygen consumption and extracellular acidification were used to measure mitochondrial and glycolytic metabolism, respectively. Gene expression was assessed via qRT-PCR, and insulin sensitivity was assessed via Western blot. Insulin resistance significantly reduced both basal and peak mitochondrial function which were rescued to control levels by concurrent VAL. Changes in mitochondrial function occurred without substantial changes in mitochondrial content or related gene expression. Insulin sensitivity and glycolytic metabolism were unaffected by VAL, as was lipogenic signaling and lipid content. Additionally, both VAL and insulin resistance depressed Bckdha expression. Interestingly, an interaction effect was observed for extracellular isoleucine, valine, and total BCAA (but not leucine), suggesting VAL may alter BCAA utilization in an insulin sensitivity-dependent manner. Insulin resistance appears to suppress mitochondrial function in a myotube model which can be rescued by VAL. Further research will be required to explore the implications of these findings in more complex models.
Assuntos
Resistência à Insulina , Mitocôndrias , Fibras Musculares Esqueléticas , Valsartana , Valsartana/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Animais , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Linhagem Celular , Aminoácidos de Cadeia Ramificada/metabolismo , Aminoácidos de Cadeia Ramificada/farmacologiaRESUMO
Analyses of mitochondrial adaptations in human skeletal muscle have mostly used whole-muscle samples, where results may be confounded by the presence of a mixture of type I and II muscle fibres. Using our adapted mass spectrometry-based proteomics workflow, we provide insights into fibre-specific mitochondrial differences in the human skeletal muscle of men before and after training. Our findings challenge previous conclusions regarding the extent of fibre-type-specific remodelling of the mitochondrial proteome and suggest that most baseline differences in mitochondrial protein abundances between fibre types reported by us, and others, might be due to differences in total mitochondrial content or a consequence of adaptations to habitual physical activity (or inactivity). Most training-induced changes in different mitochondrial functional groups, in both fibre types, were no longer significant in our study when normalised to changes in markers of mitochondrial content.
Assuntos
Exercício Físico , Proteínas Mitocondriais , Humanos , Masculino , Proteínas Mitocondriais/metabolismo , Adulto , Exercício Físico/fisiologia , Proteômica/métodos , Músculo Esquelético/metabolismo , Mitocôndrias Musculares/metabolismo , Adulto Jovem , Fibras Musculares Esqueléticas/metabolismo , Descanso/fisiologia , Mitocôndrias/metabolismo , Proteoma/metabolismo , Adaptação FisiológicaRESUMO
Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in premature death. As a major secondary event, an abnormal elevation of the intracellular calcium concentration in the dystrophin-deficient muscle contributes to disease progression in DMD. In this study, we investigated the specific functional features of induced pluripotent stem cell-derived muscle cells (hiPSC-skMCs) generated from DMD patients to regulate intracellular calcium concentration. As compared to healthy hiPSC-skMCs, DMD hiPSC-skMCs displayed specific spontaneous calcium signatures with high levels of intracellular calcium concentration. Furthermore, stimulations with electrical field or with acetylcholine perfusion induced higher calcium response in DMD hiPSC-skMCs as compared to healthy cells. Finally, Mn2+ quenching experiments demonstrated high levels of constitutive calcium entries in DMD hiPSC-skMCs as compared to healthy cells. Our findings converge on the fact that DMD hiPSC-skMCs display intracellular calcium dysregulation as demonstrated in several other models. Observed calcium disorders associated with RNAseq analysis on these DMD cells highlighted some mechanisms, such as spontaneous and activated sarcoplasmic reticulum (SR) releases or constitutive calcium entries, known to be disturbed in other dystrophin-deficient models. However, store operated calcium entries (SOCEs) were not found to be dysregulated in our DMD hiPSC-skMCs model. These results suggest that all the mechanisms of calcium impairment observed in other animal models may not be as pronounced in humans and could point to a preference for certain mechanisms that could correspond to major molecular targets for DMD therapies.
Assuntos
Cálcio , Células-Tronco Pluripotentes Induzidas , Distrofia Muscular de Duchenne , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Humanos , Cálcio/metabolismo , Sinalização do Cálcio , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Diferenciação Celular , Células Cultivadas , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Retículo Sarcoplasmático/metabolismoRESUMO
We studied the effect of enteral administration of the glucocorticoid deflazacort (DFC, 1.2 mg/kg per day, 28 days) on the state of skeletal muscles and tissue ultrastructure, as well as the composition of the colon microbiota in dystrophin-deficient mdx mice. DFC has been shown to reduce the intensity of degeneration/regeneration cycles in muscle fibers of mdx mice. This effect of DFC was accompanied by normalization of the size of sarcomeres of skeletal muscles of mdx mice, improvement of the ultrastructure of the subsarcolemmal population of mitochondria, and an increase in the number of organelles, as well as normalization of the number of contact interactions between the sarcoplasmic reticulum and mitochondria. In addition, DFC had a corrective effect on the colon microbiota of mdx mice, which manifested in an increase in the number of the Bifidobacterium genus microorganisms and a decrease in the level of E. coli with reduced enzymatic activity.
Assuntos
Colo , Microbioma Gastrointestinal , Glucocorticoides , Camundongos Endogâmicos mdx , Músculo Esquelético , Pregnenodionas , Animais , Camundongos , Colo/efeitos dos fármacos , Colo/microbiologia , Colo/patologia , Colo/ultraestrutura , Pregnenodionas/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/ultraestrutura , Músculo Esquelético/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Masculino , Glucocorticoides/farmacologia , Distrofina/genética , Distrofina/deficiência , Distrofina/metabolismo , Bifidobacterium/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/ultraestrutura , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestruturaRESUMO
Membrane trafficking and actin-remodeling are critical for well-maintained integrity of the cell organization and activity, and they require Arf6 (ADP ribosylation factor 6) activated by GEF (guanine nucleotide exchange factor) including EFA6 (exchange factor for Arf6). In the present immuno-electron microscopic study following previous immunohistochemical study by these authors (Chomphoo et al., 2020) of in situ skeletal myoblasts and myotubes of pre-and perinatal mice, the immunoreactivity for EFA6A was found to be localized at Z-bands and sarcoplasmic reticulum (SR) membranes in I-domains as well as I-domain myofilaments of skeletal myofibers of perinatal mice. Based on the previous finding that EFA6 anchored on the neuronal postsynaptic density via α-actinin which is known to be shared by muscular Z-bands, the present finding suggests that EFA6A is also anchored on Z-bands via α-actinin and involved in the membrane trafficking and actin-remodeling in skeletal myofibers. The localization of EFA6A-immunoreactivity in I-domain SR suggests a differential function in the membrane traffic between the I- and A-domain intracellular membranes in perinatal skeletal myofibers.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Troca do Nucleotídeo Guanina , Retículo Sarcoplasmático , Animais , Camundongos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Retículo Sarcoplasmático/metabolismo , Miofibrilas/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Actinina/metabolismoRESUMO
Muscle wasting is a universal hallmark of aging which is displayed by a wide range of organisms, although the causes and mechanisms of this phenomenon are not fully understood. We used Drosophila to characterize the phenomenon of spontaneous muscle fiber degeneration (SMFD) during aging. We found that SMFD occurs across diverse types of somatic muscles, progresses with chronological age, and positively correlates with functional muscle decline. Data from vital dyes and morphological markers imply that degenerative fibers most likely die by necrosis. Mechanistically, SMFD is driven by the damage resulting from muscle contractions, and the nervous system may play a significant role in this process. Our quantitative model of SMFD assessment can be useful in identifying and validating novel genetic factors that influence aging-related muscle wasting.
Assuntos
Envelhecimento , Estresse Mecânico , Animais , Envelhecimento/genética , Envelhecimento/fisiologia , Drosophila melanogaster/genética , Atrofia Muscular/genética , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Atrofia Muscular/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/metabolismo , Contração MuscularRESUMO
This study investigated the anti-sarcopenic effect of fermented Tenebrio molitor larvae (mealworms) extract (FME) in both dexamethasone (DEX)-treated C2C12 cells and mice. FME (100⯵g/mL) increased the diameter of myotubes and inhibited the gene and protein expression of atrogin-1 compared to DEX- or non-fermented mealworms extract (ME)-treated C2C12 cells. Male C57BL/6N mice were divided into five groups: Normal Control (NC), DEX (10â¯mg/kg, intraperitoneal), and three groups of DEX+FME (100, 200, or 500â¯mg FME/kg/day, oral) for two weeks. FME at doses of 200 and 500â¯mg/kg effectively improved grip strength when compared to the DEX group. Histological analysis of the quadriceps muscle showed a larger muscle fiber size in the DEX+FME groups compared to DEX group. FME (200 and 500â¯mg/kg) significantly increased cross-sectional area of the muscle fiber compared to DEX group. FME (500â¯mg/kg) significantly decreased the ubiquitin, atrogin-1 and MuRF-1 protein levels, and increased levels of MHC and MyoG in DEX-treated mice. The puromycin labeling assay revealed that FME increased protein synthesis in DEX-induced muscle atrophy. The FME treatment demonstrated significant upregulation in phosphorylation levels, including mTOR, FoxO3α, Akt, and PI3K compared to DEX group. In conclusion, FME inhibited the increase in proteins associated with muscle atrophy, including, atrogin-1 and MuRF-1, by regulating the PI3K-Akt-FoxO3α pathway. FME improved the PI3K-Akt-mTOR signaling pathway, which was reduced by DEX. This study suggests that FME has the potential for use in sarcopenia therapy, possibly serving as a natural agent that counteracts the negative effects of DEX on muscle tissue.