RESUMO
In clinical conditions such as diaphragm paralysis or mechanical ventilation, disuse-induced diaphragmatic dysfunction (DIDD) is a condition that poses a threat to life. MuRF1 is a key E3-ligase involved in regulating skeletal muscle mass, function, and metabolism, which contributes to the onset of DIDD. We investigated if the small-molecule mediated inhibition of MuRF1 activity (MyoMed-205) protects against early DIDD after 12 h of unilateral diaphragm denervation. Wistar rats were used in this study to determine the compound's acute toxicity and optimal dosage. For potential DIDD treatment efficacy, diaphragm contractile function and fiber cross-sectional area (CSA) were evaluated. Western blotting investigated potential mechanisms underlying MyoMed-205's effects in early DIDD. Our results indicate 50 mg/kg bw MyoMed-205 as a suitable dosage to prevent early diaphragmatic contractile dysfunction and atrophy following 12 h of denervation without detectable signs of acute toxicity. Mechanistically, treatment did not affect disuse-induced oxidative stress (4-HNE) increase, whereas phosphorylation of (ser632) HDAC4 was normalized. MyoMed-205 also mitigated FoxO1 activation, inhibited MuRF2, and increased phospho (ser473) Akt protein levels. These findings may suggest that MuRF1 activity significantly contributes to early DIDD pathophysiology. Novel strategies targeting MuRF1 (e.g., MyoMed-205) have potential therapeutic applications for treating early DIDD.
Assuntos
Diafragma , Atrofia Muscular , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Animais , Ratos , Diafragma/metabolismo , Diafragma/patologia , Atrofia Muscular/metabolismo , Estresse Oxidativo , Ratos Wistar , Respiração Artificial/efeitos adversos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/metabolismoRESUMO
microRNAs negatively regulate gene expression by blocking translation or increasing mRNA degradation. In skeletal muscle, these molecules play important roles in adaptive responses, and ongoing investigations are necessary to understand the fine-tune regulation of skeletal muscle mass. Herein we showed that skeletal muscle overexpression of miR-29c increased fiber size and force at 7 and 30 days after electrotransfer. At both time points, AKT/mTOR pathway components were downregulated, and, surprisingly, overall protein synthesis was strongly elevated at day 7, which normalized by day 30 after pCMVmiR-29c electrotransfer. These results indicate that miR-29c expression induces skeletal muscle hypertrophy and gain of function, which involves increased overall protein synthesis in spite of the deactivation of the AKT/mTOR pathway.
Assuntos
MicroRNAs , Proteínas Proto-Oncogênicas c-akt , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
One major cause of traumatic injury is firearm-related wounds (i.e., ballistic trauma), common in both civilian and military populations, which is increasing in prevalence and has serious long-term health and socioeconomic consequences worldwide. Common primary injuries of ballistic trauma include soft-tissue damage and loss, haemorrhage, bone fracture, and pain. The majority of injuries are of musculoskeletal origin and located in the extremities, such that skeletal muscle offers a major therapeutic target to aid recovery and return to normal daily activities. However, the underlying pathophysiology of skeletal muscle ballistic trauma remains poorly understood, with limited evidence-based treatment options. As such, this review will address the topic of firearm-related skeletal muscle injury and regeneration. We first introduce trauma ballistics and the immediate injury of skeletal muscle, followed by detailed coverage of the underlying biological mechanisms involved in regulating skeletal muscle dysfunction following injury, with a specific focus on the processes of muscle regeneration, muscle wasting and vascular impairments. Finally, we evaluate novel approaches for minimising muscle damage and enhancing muscle regeneration after ballistic trauma, which may have important relevance for primary care in victims of violence.
RESUMO
In this study we surveyed a rat skeletal muscle RNA-Seq for genes that are induced by hindlimb immobilization and, in turn, become attenuated by leucine supplementation. This approach, in search of leucine-atrophy protection mediating genes, identified histone deacetylase 4 (HDAC4) as highly responsive to both hindlimb immobilization and leucine supplementation. We then examined the impact of leucine on HDAC4 expression, tissue localization, and target genes. A total of 76 male Wistar rats (~280 g) were submitted to hindlimb immobilization and/or leucine supplementation for 3, 7 and 12 days. These animals were euthanized, and soleus muscle was removed for further analysis. RNA-Seq analysis of hindlimb immobilized rats indicated a sharp induction (log2 = 3.4) of HDAC4 expression which was attenuated by leucine supplementation (~50%). Real-time PCR and protein expression analysis by Western blot confirmed increased HDAC4 mRNA after 7 days of hindlimb immobilization and mitigation of induction by leucine supplementation. Regarding the HDAC4 localization, the proportion of positive nuclei was higher in the immobilized group and decreased after leucine supplementation. Also, we found a marked decrease of myogenin and MAFbx-atrogin-1 mRNA levels upon leucine supplementation, while CAMKII and DACH2 mRNA levels were increased by leucine supplementation. Our data suggest that HDAC4 inhibition might be involved in the anti-atrophic effects of leucine.
Assuntos
Suplementos Nutricionais , Membro Posterior/patologia , Histona Desacetilases/metabolismo , Leucina/uso terapêutico , Músculo Esquelético/metabolismo , Animais , Peso Corporal , Membro Posterior/metabolismo , Elevação dos Membros Posteriores , Masculino , Microscopia de Fluorescência , Atrofia Muscular/patologia , RNA-Seq , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
Lack of mechanical load leads to skeletal muscle atrophy, and one major underlying mechanism involves the myostatin pathway that negatively regulates protein synthesis and also activates Atrogin-1/MAFbx and MuRF1 genes. In hindlimb immobilization, leucine was observed to attenuate the upregulation of the referred atrogenes, thereby shortening the impact on fiber cross-sectional area, nonetheless, the possible connection with myostatin is still elusive. This study sought to verify the impact of leucine supplementation on myostatin expression. Male Wistar rats were supplemented with leucine and hindlimb immobilized for 3 and 7 days, after which soleus muscles were removed for morphometric measurements and analyzed for gene and protein expression by real-time PCR and Western blotting, respectively. Muscle wasting was prominent 7 days after immobilization, as expected, leucine feeding mitigated this effect. Atrogin-1/MAFbx gene expression was upregulated only after 3 days of immobilization, and this effect was attenuated by leucine supplementation. Atrogin-1/MAFbx protein levels were elevated after 7 days of immobilization, which leucine supplementation was not able to lessen. On the other hand, myostatin gene expression was upregulated in immobilization for 3 and 7 days, which returned to normal levels after leucine supplementation. Myostatin protein levels followed gene expression at a 3-day time point only. Follistatin gene expression was upregulated during immobilization and accentuated by leucine after 3 days of supplementation. Concerning protein expression, follistatin was not altered neither by immobilization nor in immobilized animals treated with leucine. In conclusion, leucine protects against skeletal muscle mass loss during disuse, and the underlying molecular mechanisms appear to involve myostatin inhibition and Atrogin-1 normalization independently of follistatin signaling.
RESUMO
The high capacity of the skeletal muscle to regenerate is due to the presence of muscle stem cells (MuSCs, or satellite cells). The E3 ubiquitin ligase Parkin is a key regulator of mitophagy and is recruited to mitochondria during differentiation of mouse myoblast cell line. However, the function of mitophagy during regeneration has not been investigated in vivo. Here, we have utilized Parkin deficient (Parkin-/-) mice to investigate the role of Parkin in skeletal muscle regeneration. We found a persistent deficiency in skeletal muscle regeneration in Parkin-/- mice after cardiotoxin (CTX) injury with increased area of fibrosis and decreased cross-sectional area (CSA) of myofibres post-injury. There was also a significant modulation of MuSCs differentiation and mitophagic markers, with altered mitochondrial proteins during skeletal muscle regeneration in Parkin-/- mice. Our data suggest that Parkin-mediated mitophagy plays a key role in skeletal muscle regeneration and is necessary for MuSCs differentiation.
Assuntos
Diferenciação Celular , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/patologia , Regeneração , Ubiquitina-Proteína Ligases/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitofagia , Músculo Esquelético/metabolismo , Células-Tronco/citologiaRESUMO
AIM: To identify microRNAs (miRs) involved in the regulation of skeletal muscle mass. For that purpose, we have initially utilized an in silico analysis, resulting in the identification of miR-29c as a positive regulator of muscle mass. METHODS: miR-29c was electrotransferred to the tibialis anterior to address its morphometric and functional properties and to determine the level of satellite cell proliferation and differentiation. qPCR was used to investigate the effect of miR-29c overexpression on trophicity-related genes. C2C12 cells were used to determine the impact of miR-29c on myogenesis and a luciferase reporter assay was used to evaluate the ability of miR-29c to bind to the MuRF1 3'UTR. RESULTS: The overexpression of miR-29c in the tibialis anterior increased muscle mass by 40%, with a corresponding increase in fibre cross-sectional area and force and a 30% increase in length. In addition, satellite cell proliferation and differentiation were increased. In C2C12 cells, miR-29c oligonucleotides caused increased levels of differentiation, as evidenced by an increase in eMHC immunostaining and the myotube fusion index. Accordingly, the mRNA levels of myogenic markers were also increased. Mechanistically, the overexpression of miR-29c inhibited the expression of the muscle atrophic factors MuRF1, Atrogin-1 and HDAC4. For the key atrogene MuRF1, we found that miR-29c can bind to its 3'UTR to mediate repression. CONCLUSIONS: The results herein suggest that miR-29c can improve skeletal muscle size and function by stimulating satellite cell proliferation and repressing atrophy-related genes. Taken together, our results indicate that miR-29c might be useful as a future therapeutic device in diseases involving decreased skeletal muscle mass.
Assuntos
MicroRNAs/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Hipertrofia/genética , Hipertrofia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular/genética , Atrofia Muscular/metabolismoRESUMO
We aimed to investigate the mechanisms underlying muscle growth after 12 weeks of resistance training performed with blood flow restriction (RT-BFR) and high-intensity resistance training (HRT) in older individuals. Participants were allocated into the following groups: HRT, RT-BFR, or a control group. High-throughput transcriptome sequencing was performed by the Illumina HiSeq 2500 platform. HRT and RT-BFR presented similar increases in the quadriceps femoris cross-sectional area, and few genes were differently expressed between interventions. The small differences in gene expression between interventions suggest that similar mechanisms may underpin training-induced muscle growth.
Assuntos
Envelhecimento/fisiologia , Músculo Esquelético/metabolismo , Educação Física e Treinamento , Fluxo Sanguíneo Regional/fisiologia , Treinamento Resistido , Transcriptoma/fisiologia , Idoso , DNA/biossíntese , DNA/genética , Dieta , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Perna (Membro)/anatomia & histologia , Perna (Membro)/fisiologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/irrigação sanguínea , Músculo Quadríceps/fisiologia , RNA/biossíntese , RNA/genéticaRESUMO
Our aim is to gain insight into the mechanisms underlying the anti-atrophic effects of leucine, namely, the way that this amino acid can restrain the up-regulation of MuRF1 and Mafbx/Atrogin-1 in muscle atrophy. Male rats received dietary leucine supplementation for 1-3 days, during which time their hind limbs were immobilized. Our results showed that leucine inhibited Forkhead Box O3 (FoxO3a) translocation to cell nuclei. In addition, leucine was able to reverse the expected reduction of FoXO3a ubiquitination caused by immobilization. Unexpectedly, leucine promoted these effects independently of the Class I PI3K/Akt pathway. Vacuolar protein sorting 34 (VPS34; a Class III PI3K) was strongly localized in nuclei after immobilization and leucine supplementation was able to prevent this effect. In experiments on cultured primary myotubes, dexamethasone led to the localization of VPS34 in the nucleus. In addition, the pharmacological inhibition of VPS34 blocked VPS34 nuclear localization and impaired the protective effect of leucine upon myotube trophicity. Finally, the pharmacological inhibition of VPS34 in primary myotubes prevented the protective effects of leucine upon MuRF1 and Mafbx/Atrogin-1 gene expression. Autophagy-related target genes were not responsive to leucine. Thus, we demonstrate that the anti-atrophic effect of leucine is dependent upon FoxO3a suppression and VPS34 activity.
Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Proteína Forkhead Box O3/metabolismo , Leucina/farmacologia , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dexametasona/farmacologia , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , UbiquitinaçãoRESUMO
Recent studies have evidenced the involvement of inflammation-related pathways to the development of cardiac hypertrophy and other consequences on the cardiovascular system, including the calcium-binding protein S100A8. However, this has never been investigated in the thyroid hormone (TH)-prompted cardiac hypertrophy. Thus, we aimed to test whether S100A8 and related signaling molecules, myeloid differentiation factor-88 (MyD88) and nuclear factor kappa B (NF-ÒB), could be associated with the cardiomyocyte hypertrophy induced by TH. Our results demonstrate that the S100A8/MyD88/NF-ÒB signaling pathway is activated in cardiomyocytes following TH stimulation. The knockdown of S100A8 and MyD88 indicates the contribution of those molecules to cardiomyocyte hypertrophy in response to TH, as evaluated by cell surface area, leucine incorporation assay, and gene expression. Furthermore, S100A8 and MyD88 are crucial mediators of NF-ÒB activation, which is also involved in the hypertrophic growth of TH-treated cardiomyocytes. Supporting the in vitro data, the contribution of NF-ÒB for TH-induced cardiac hypertrophy is confirmed in vivo, by using transgenic mice with cardiomyocyte-specific suppression of NF-ÒB. These data identify a novel pathway regulated by TH that mediates cardiomyocyte hypertrophy. However, the potential role of this new pathway in short and long-term cardiac effects of TH remains to be further investigated. KEY MESSAGES: Inflammation-related signaling is activated by T3 in cardiomyocytes. S100A8 and MyD88 have a crucial role in cardiomyocyte hypertrophy by T3. S100A8 and MyD88 mediate NF-ÒB activation by T3. NF-ÒB contributes to T3-induced cardiac hypertrophy in vitro and in vivo.
Assuntos
Calgranulina A/genética , Cardiomegalia/genética , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/genética , Tri-Iodotironina , Animais , Fator Natriurético Atrial/genética , Células Cultivadas , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Encefálico/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos Wistar , Transdução de Sinais , Receptor 4 Toll-Like/genéticaRESUMO
We characterized the metabolic profile of transgenic mice exhibiting enhanced muscle mass driven by increased mIGF-1 expression (MLC/mIGF-1). As expected, 6-month-old MLC/mIGF-1 mice were heavier than age-matched wild type (WT) mice (37.4 ± 0.3 versus 31.8 ± 0.6 g, resp.). MLC/mIGF-1 mice had higher respiratory quotient when compared to WT (0.9 ± 0.03 versus 0.74 ± 0.02, resp.) suggesting a preference for carbohydrate as the major fuel source. MLC/mIGF-1 mice had a higher rate of glucose disposal when compared to WT (3.25 ± 0.14 versus 2.39 ± 0.03%/min, resp.). The higher disposal rate correlated to â¼ 2-fold higher GLUT4 content in the extensor digitorum longus (EDL) muscle. Analysis of mRNA content for the glycolysis-related gene PFK-1 showed â¼ 3-fold upregulation in MLC/mIGF-1 animals. We also found a 50% downregulation of PGC1α mRNA levels in MLC/mIGF-1 mouse EDL muscle, suggesting less abundant mitochondria in this tissue. We found no difference in the expression of PPARα and PPARß/δ, suggesting no modulation of key elements in oxidative metabolism. These data together suggest a shift in metabolism towards higher carbohydrate utilization, and that could explain the increased insulin sensitivity of hypertrophied skeletal muscle in MLC/mIGF-1 mice.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Hipertrofia/metabolismo , Resistência à Insulina/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Animais , Transportador de Glucose Tipo 4/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Musculares/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
INTRODUCTION: We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. METHODS: Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. RESULTS: Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. CONCLUSIONS: Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy.
Assuntos
Células-Tronco Embrionárias/transplante , Músculo Esquelético/fisiologia , Atrofia Muscular/terapia , Recuperação de Função Fisiológica/fisiologia , Transplante de Células-Tronco/métodos , Animais , Toxinas Botulínicas/toxicidade , Humanos , Masculino , Camundongos , Músculo Esquelético/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/patologiaRESUMO
This study investigated the effect of leucine supplementation on the skeletal muscle regenerative process, focusing on the remodeling of connective tissue of the fast twitch muscle tibialis anterior (TA). Young male Wistar rats were supplemented with leucine (1.35 g/kg per day); then, TA muscles from the left hind limb were cryolesioned and examined after 10 days. Although leucine supplementation induced increased protein synthesis, it was not sufficient to promote an increase in the cross-sectional area (CSA) of regenerating myofibers (p > 0.05) from TA muscles. However, leucine supplementation reduced the amount of collagen and the activation of phosphorylated transforming growth factor-ß receptor type I (TßR-I) and Smad2/3 in regenerating muscles (p < 0.05). Leucine also reduced neonatal myosin heavy chain (MyHC-n) (p < 0.05), increased adult MyHC-II expression (p < 0.05) and prevented the decrease in maximum tetanic strength in regenerating TA muscles (p < 0.05). Our results suggest that leucine supplementation accelerates connective tissue repair and consequent function of regenerating TA through the attenuation of TßR-I and Smad2/3 activation. Therefore, future studies are warranted to investigate leucine supplementation as a nutritional strategy to prevent or attenuate muscle fibrosis in patients with several muscle diseases.
Assuntos
Tecido Conjuntivo/metabolismo , Suplementos Nutricionais , Leucina/farmacologia , Músculo Esquelético/lesões , Tíbia , Animais , Colágeno/efeitos dos fármacos , Tecido Conjuntivo/efeitos dos fármacos , Tecido Conjuntivo/patologia , Leucina/administração & dosagem , Masculino , Músculo Esquelético/metabolismo , Miofibrilas/efeitos dos fármacos , Cadeias Pesadas de Miosina/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Regeneração/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Espasmo/dietoterapiaRESUMO
This study was undertaken in order to provide further insight into the role of leucine supplementation in the skeletal muscle regeneration process, focusing on myofiber size and strength recovery. Young (2-month-old) rats were subjected or not to leucine supplementation (1.35 g/kg per day) started 3 days prior to cryolesion. Then, soleus muscles were cryolesioned and continued receiving leucine supplementation until 1, 3 and 10 days later. Soleus muscles from leucine-supplemented animals displayed an increase in myofiber size and a reduction in collagen type III expression on post-cryolesion day 10. Leucine was also effective in reducing FOXO3a activation and ubiquitinated protein accumulation in muscles at post-cryolesion days 3 and 10. In addition, leucine supplementation minimized the cryolesion-induced decrease in tetanic strength and increase in fatigue in regenerating muscles at post-cryolesion day 10. These beneficial effects of leucine were not accompanied by activation of any elements of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signalling pathway in the regenerating muscles. Our results show that leucine improves myofiber size gain and strength recovery in regenerating soleus muscles through attenuation of protein ubiquitination. In addition, leucine might have therapeutic effects for muscle recovery following injury and in some muscle diseases.
Assuntos
Suplementos Nutricionais , Leucina/administração & dosagem , Músculo Esquelético/efeitos dos fármacos , Distrofias Musculares/dietoterapia , Regeneração/efeitos dos fármacos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Administração Oral , Animais , Temperatura Baixa , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Membro Posterior , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Although it is well known that the thyroid hormone (T3) is an important positive regulator of cardiac function over a short term and that it also promotes deleterious effects over a long term, the molecular mechanisms for such effects are not yet well understood. Because most alterations in cardiac function are associated with changes in sarcomeric machinery, the present work was undertaken to find novel sarcomeric hot spots driven by T3 in the heart. A microarray analysis indicated that the M-band is a major hot spot, and the structural sarcomeric gene coding for the M-protein is severely down-regulated by T3. Real-time quantitative PCR-based measurements confirmed that T3 (1, 5, 50, and 100 physiological doses for 2 days) sharply decreased the M-protein gene and protein expression in vivo in a dose-dependent manner. Furthermore, the M-protein gene expression was elevated 3.4-fold in hypothyroid rats. Accordingly, T3 was able to rapidly and strongly reduce the M-protein gene expression in neonatal cardiomyocytes. Deletions at the M-protein promoter and bioinformatics approach suggested an area responsive to T3, which was confirmed by chromatin immunoprecipitation assay. Functional assays in cultured neonatal cardiomyocytes revealed that depletion of M-protein (by small interfering RNA) drives a severe decrease in speed of contraction. Interestingly, mRNA and protein levels of other M-band components, myomesin and embryonic-heart myomesin, were not altered by T3. We concluded that the M-protein expression is strongly and rapidly repressed by T3 in cardiomyocytes, which represents an important aspect for the basis of T3-dependent sarcomeric deleterious effects in the heart.
Assuntos
Cardiomegalia/genética , Conectina/genética , Regulação para Baixo/genética , Hormônios Tireóideos/farmacologia , Animais , Animais Recém-Nascidos , Sequência de Bases , Cardiomegalia/etiologia , Cardiomegalia/fisiopatologia , Linhagem Celular , Células Cultivadas , Conectina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Hipotireoidismo/genética , Masculino , Camundongos , Dados de Sequência Molecular , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores dos Hormônios Tireóideos/metabolismo , Sarcômeros/metabolismo , Tireotoxicose/complicações , Tri-Iodotironina/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
In the present study we have compared the effects of leucine supplementation and its metabolite ß-hydroxy-ß-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB.
Assuntos
Suplementos Nutricionais , Leucina/administração & dosagem , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sarcopenia/metabolismo , Valeratos/administração & dosagem , Animais , Elevação dos Membros Posteriores/efeitos adversos , Masculino , Músculo Esquelético/patologia , Tamanho do Órgão/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Sarcopenia/tratamento farmacológico , Sarcopenia/patologiaRESUMO
OBJECTIVE: The aim of this study was to assess the mRNA levels of atrogin-1, muscle ring finger 1, and myostatin in rat quadriceps after anterior cruciate ligament (ACL) transection. DESIGN: Wistar rats were randomized into three different groups: ACL (surgery and ACL transection), sham (surgery without ACL transection), and control. Vastus medialis, rectus femoris, and vastus lateralis muscles were harvested at 1, 2, 3, 7, and 15 days after ACL transection. The mRNA levels of atrogin-1, muscle ring finger 1, and myostatin, as well as the ubiquitinated protein content, muscle mass, and cross-sectional area of the muscle fibers, were evaluated. RESULTS: Elevated levels of atrogin-1, muscle ring finger 1, and myostatin mRNA were detected in all tested muscles at most time points. The ubiquitinated protein content was increased at 3 days in the ACL and sham groups. The muscle mass of the ACL group was reduced at 3, 7, and 15 days (vastus lateralis and vastus medialis) and at 7 and 15 days (rectus femoris), whereas it was reduced in the sham group at 3 and 7 days (vastus lateralis and vastus medialis) and at 7 days (rectus femoris). The cross-sectional area of vastus medialis was reduced at 3, 7, and 15 days in the ACL group and at 3 and 7 days in the sham group. The cross-sectional area of the vastus lateralis was reduced at 7 and 15 days in the ACL group and at 7 days in the sham group. Whereas muscle mass and cross-sectional area recovery was noted in the sham group, no recovery was observed in the ACL group. CONCLUSIONS: Quadriceps atrophy after ACL transection involves increased levels of myostatin, atrogin-1, and muscle ring finger 1 mRNA and the accumulation of ubiquitinated protein.
Assuntos
Ligamento Cruzado Anterior/cirurgia , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Miostatina/metabolismo , Músculo Quadríceps/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Análise de Variância , Animais , Ligamento Cruzado Anterior/metabolismo , Biomarcadores/metabolismo , Western Blotting , Modelos Animais de Doenças , Masculino , Proteínas Musculares/genética , Atrofia Muscular/patologia , Miostatina/genética , Músculo Quadríceps/patologia , Domínios RING Finger/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Proteínas Ligases SKP Culina F-Box/genética , Sensibilidade e EspecificidadeRESUMO
In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.
Assuntos
Glutamina/administração & dosagem , Músculo Esquelético , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/dietoterapia , Diabetes Mellitus Experimental/metabolismo , Suplementos Nutricionais , Humanos , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteínas , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
BACKGROUND: Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF. METHODS/PRINCIPAL FINDINGS: Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7(th) month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects' levels. CONCLUSIONS: Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.