RESUMO
Altitude camps are used during the preparation of endurance athletes to improve performance based on the stimulation of erythropoiesis by living at high altitude. In addition to such whole-body adaptations, studies have suggested that high-altitude training increases mitochondrial mass, but this has been challenged by later studies. Here, we hypothesized that living and training at high altitude (LHTH) improves mitochondrial efficiency and/or substrate utilization. Female rats were exposed and trained in hypoxia (simulated 3,200 m) for 5 weeks (LHTH) and compared to sedentary rats living in hypoxia (LH) or normoxia (LL) or those that trained in normoxia (LLTL). Maximal aerobic velocity (MAV) improved with training, independently of hypoxia, whereas the time to exhaustion, performed at 65% of MAV, increased both with training (P = 0.009) and hypoxia (P = 0.015), with an additive effect of the two conditions. The distance run was 7.98 ± 0.57 km in LHTH vs. 6.94 ± 0.51 in LLTL (+15%, ns). The hematocrit increased >20% with hypoxia (P < 0.001). The increases in mitochondrial mass and maximal oxidative capacity with endurance training were blunted by combination with hypoxia (-30% for citrate synthase, P < 0.01, and -23% for Vmax glut-succ, P < 0.001 between LHTH and LLTL). A similar reduction between the LHTH and LLTL groups was found for maximal respiration with pyruvate (-29%, P < 0.001), for acceptor-control ratio (-36%, hypoxia effect, P < 0.001), and for creatine kinase efficiency (-48%, P < 0.01). 3-hydroxyl acyl coenzyme A dehydrogenase was not altered by hypoxia, whereas maximal respiration with Palmitoyl-CoA specifically decreased. Overall, our results show that mitochondrial adaptations are not involved in the improvement of submaximal aerobic performance after LHTH, suggesting that the benefits of altitude camps in females relies essentially on other factors, such as the transitory elevation of hematocrit, and should be planned a few weeks before competition and not several months.
RESUMO
INTRODUCTION: As skeletal muscle mass recovery after extensive injury is improved by contractile activity, we explored whether concomitant exercise accelerates recovery of the contractile and metabolic phenotypes after muscle injury. METHODS: After notexin-induced degeneration of a soleus muscle, Wistar rats were assigned to active (running exercise) or sedentary groups. Myosin heavy chains (MHC), metabolic enzymes, and calcineurin were studied during muscle regeneration at different time points. RESULTS: The mature MHC profile recovered earlier in active rats (21 days after injury) than in sedentary rats (42 days). Calcineurin was higher in the active degenerated than in the sedentary degenerated muscles at day 14. Citrate synthase and total lactate dehydrogenase (LDH) activity decreased after injury and were similarly recovered in both active and sedentary groups at 14 or 42 days, respectively. H-LDH isozyme activity recovered earlier in the active rats. CONCLUSIONS: Exercise improved recovery of the slow/oxidative phenotype after soleus muscle injury. Muscle Nerve 55: 91-100, 2017.
Assuntos
Fibras Musculares de Contração Lenta/fisiologia , Doenças Musculares/fisiopatologia , Doenças Musculares/reabilitação , Condicionamento Físico Animal/métodos , Regeneração/fisiologia , Animais , Calcineurina/metabolismo , Citrato (si)-Sintase/metabolismo , Modelos Animais de Doenças , Venenos Elapídicos/toxicidade , Teste de Esforço , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Doenças Musculares/induzido quimicamente , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Regeneração/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The Ubiquitin Proteasome System (UPS) is mainly responsible for the increased protein breakdown observed in muscle wasting. The E3 ligase MuRF1 is so far the only enzyme known to direct the main contractile proteins for degradation (i.e. troponin I, myosin heavy chains and actin). However, MuRF1 does not possess any catalytic activity and thus depends on the presence of a dedicated E2 for catalyzing the covalent binding of polyubiquitin (polyUb) chains on the substrates. The E2 enzymes belonging to the UBE2D family are commonly used for in vitro ubiquitination assays but no experimental data suggesting their physiological role as bona fide MuRF1-interacting E2 enzymes are available. In this work, we first found that the mRNA levels of critical E3 enzymes implicated in the atrophying program (MuRF1, MAFbx, Nedd4 and to a lesser extent Mdm2) are tightly and rapidly controlled during the atrophy (up regulation) and recovery (down regulation) phases in the soleus muscle from hindlimb suspended rats. By contrast, E3 ligases (Ozz, ASB2ß and E4b) implicated in other processes (muscle development or regeneration) poorly responded to atrophy and recovery. UBE2B, an E2 enzyme systematically up regulated in various catabolic situations, was controlled at the mRNA levels like the E3s implicated in the atrophying process. By contrast, UBE2D2 was progressively repressed during atrophy and recovery, which makes it a poor candidate for a role during muscle atrophy. In addition, UBE2D2 did not exhibit any affinity with MuRF1 using either yeast two-hybrid or Surface Plasmon Resonance (SPR) approaches. Finally, UBE2D2 was unable to promote the degradation of the MuRF1 substrate α-actin in HEK293T cells, suggesting that no functional interaction exists between these enzymes within a cellular context. Altogether, our data strongly suggest that UBE2D2 is not the cognate ubiquitinating enzyme for MuRF1 and that peculiar properties of UBE2D enzymes may have biased in vitro ubiquitination assays.
Assuntos
Elevação dos Membros Posteriores/efeitos adversos , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/genética , Ligação Proteica , Ratos , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Hypoxia induces a loss of skeletal muscle mass, but the signaling pathways and molecular mechanisms involved remain poorly understood. We hypothesized that hypoxia could impair skeletal muscle hypertrophy induced by functional overload (Ov). To test this hypothesis, plantaris muscles were overloaded during 5, 12, and 56 days in female rats exposed to hypobaric hypoxia (5,500 m), and then, we examined the responses of specific signaling pathways involved in protein synthesis (Akt/mTOR) and breakdown (atrogenes). Hypoxia minimized the Ov-induced hypertrophy at days 5 and 12 but did not affect the hypertrophic response measured at day 56. Hypoxia early reduced the phosphorylation levels of mTOR and its downstream targets P70(S6K) and rpS6, but it did not affect the phosphorylation levels of Akt and 4E-BP1, in Ov muscles. The role played by specific inhibitors of mTOR, such as AMPK and hypoxia-induced factors (i.e., REDD1 and BNIP-3) was studied. REDD1 protein levels were reduced by overload and were not affected by hypoxia in Ov muscles, whereas AMPK was not activated by hypoxia. Although hypoxia significantly increased BNIP-3 mRNA levels at day 5, protein levels remained unaffected. The mRNA levels of the two atrogenes MURF1 and MAFbx were early increased by hypoxia in Ov muscles. In conclusion, hypoxia induced a transient alteration of muscle growth in this hypertrophic model, at least partly due to a specific impairment of the mTOR/P70(S6K) pathway, independently of Akt, by an undefined mechanism, and increased transcript levels for MURF1 and MAFbx that could contribute to stimulate the proteasomal proteolysis.
Assuntos
Hipóxia/fisiopatologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Transdução de Sinais/fisiologia , Suporte de Carga/fisiologia , Animais , Feminino , Hipertrofia , Hipóxia/metabolismo , Modelos Animais , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Erythropoietin (Epo)-induced polycythemia is the main factor of adaptation to hypoxia. In this study, we analysed the effects of Epo deficiency on intrinsic functional properties of slow and fast twitch muscles in a model of erythropoietin deficient mice (Epo-TAg(h)) exposed to hypoxia. We hypothesised that Epo deficiency would be deleterious for skeletal muscle structure and phenotype, which could change its functional properties and alters the adaptive response to ambient hypoxia. Wild-type (WT) and Epo-TAg(h) mice were left in hypobaric chamber at 420 mm Hg pressure for 14 days. Soleus (SOL) and extensor digitorum longus (EDL) were analysed in vitro by mechanical measurements, immunohistological and biochemical analyses. The results were compared to those obtained in corresponding muscles of age-matched normoxic groups. Our data did not show any difference between the groups whatever the Epo deficiency and/or hypoxic conditions for twitch force, tetanic force, fatigue, typology and myosin heavy chain composition. Normoxic Epo-TAg(h) mice exhibit improved capillary-to-fibre ratio compared to WT mice in both SOL and EDL whereas no angiogenic effects of hypoxia or combined Epo-deficiency/hypoxia were observed. These results suggest that skeletal muscles possess a great capacity of adaptation to Epo deficiency. Then Epo deficiency is not a sufficient factor to modify intrinsic functional properties of skeletal muscles.
Assuntos
Eritropoetina/genética , Hipóxia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Animais , Eritropoetina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fadiga Muscular/fisiologiaRESUMO
Prolonged intense exercise is challenging for the liver to maintain plasma glucose levels. Hormonal changes cannot fully account for exercise-induced hepatic glucose production (HGP). Contracting skeletal muscles release interleukin-6 (IL-6), a cytokine able to increase endogenous glucose production during exercise. However, whether this is attributable to a direct effect of IL-6 on liver remains unknown. Here, we studied hepatic glycogen, gluconeogenic genes, and IL-6 signaling in response to one bout of exhaustive running exercise in rats. To determine whether IL-6 can modulate gluconeogenic gene mRNA independently of exercise, we injected resting rats with recombinant IL-6. Exhaustive exercise resulted in a profound decrease in liver glycogen and an increase in gluconeogenic gene mRNA levels, phosphoenolpyruvate-carboxykinase (PEPCK), glucose-6-phosphatase (G6P), and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), suggesting a key role for gluconeogenesis in hepatic glucose production. This was associated to an active IL-6 signaling in liver tissue, as shown by signal transducer and activator of transcription and CAAT/enhancer binding protein-beta phosphorylation and IL-6-responsive gene mRNA levels at the end of exercise. Recombinant IL-6 injection resulted in an increase in IL-6-responsive gene mRNA levels in the liver. We found a dose-dependent increase in PEPCK gene mRNA strongly correlated with IL-6-induced gene mRNA levels. No changes in G6P and PGC-1alpha mRNA levels were found. Taken together, our results suggest that, during very demanding exercise, muscle-derived IL-6 could help increase HGP by directly upregulating PEPCK mRNA abundance.
Assuntos
Gluconeogênese/genética , Interleucina-6/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Esforço Físico/fisiologia , Análise de Variância , Animais , Glicemia/metabolismo , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Gluconeogênese/efeitos dos fármacos , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Glicogênio/metabolismo , Interleucina-6/administração & dosagem , Fígado/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologiaRESUMO
PURPOSE: Creatine (Cr) supplementation may improve muscle functional capacity in patients with neuromuscular diseases, disuse atrophy, or muscular dystrophies. Activation of myogenic satellite cells has been reported to be enhanced by Cr both in vitro and in vivo. Therefore, we hypothesized that Cr supplementation may improve the early steps of regeneration after muscle injury and may accelerate the recovery of both muscle mass and phenotype. METHODS: Degeneration of left soleus muscle was induced by notexin injection in rats supplemented or not with Cr. The mass of regenerated muscles was compared with contralateral intact muscles at days 1, 3, 7, 14, 21, 28, 35, and 42 after injury. We also studied protein levels of the proliferator cell nuclear antigen (PCNA) as a marker of cell proliferation, expression of myogenic regulatory factors (MRF) as a marker of differentiation, and the myosin heavy chain (MHC) profile and activities of citrate synthase (CS) and lactate dehydrogenase (LDH) isozymes as markers of muscle phenotype maturation. RESULTS: Cr supplementation accelerated the recovery of muscle Cr content during the regeneration phase. Although there were no other differences between Cr-treated and nontreated rats, we observed that 1) regenerated muscle mass remained lower than that in intact muscle mass 42 d after injury, 2) PCNA and MRF expression strongly increased in regenerated muscles, 3) the MHC profile of regenerated muscles was recovered 28 d after injury, and 4) CS activity was fully recovered from day 14, whereas the specific H isozyme of lactate dehydrogenase activity remained lower than that in intact muscles until 42 d. CONCLUSIONS: In contrast with results from in vitro studies, Cr supplementation had no effects in vivo on the time course of recovery of rat skeletal muscle mass and phenotype after notexin-induced injury.
Assuntos
Creatina/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Regeneração/efeitos dos fármacos , Animais , Creatina/administração & dosagem , Feminino , Músculo Esquelético/lesões , Fenótipo , Proteínas da Gravidez/sangue , Ratos , Ratos WistarRESUMO
In the present study, we tested the hypothesis that chronic ANG I-converting enzyme (ACE) inhibition could improve the training-induced improvement in endurance exercise performance and that this could be related to enhanced skeletal muscle metabolic efficiency. Female Wistar rats were assigned to four groups comprising animals either maintained sedentary or endurance trained (Sed and Tr, respectively), and treated or not for 10 wk with an ACE inhibitor, perindopril (2 mg.kg(-1).day(-1)) (Per and Ct, respectively) (n = 8 each). Trained rats underwent an 8-wk treadmill training protocol that consisted of 2 h/day running at 30 m/min on a 8% decline. Before the start of and 1 wk before the end of experimental conditioning, the running time to exhaustion of rats was measured on a treadmill. The training program led to an increase in endurance time, higher in Tr-Per than in Tr-Ct group (125% in Tr-Ct vs. 183% in Tr-Per groups, P < 0.05). Oxidative capacity, measured in saponin-permeabilized fibers of slow soleus and fast plantaris muscles, increased with training, but less in Tr-Per than in Tr-Ct rats. The training-induced increase in citrate synthase activity also was less in soleus from Tr-Per than Tr-Ct rats. The training-induced increase in the percentage of the type IIa isoform of myosin heavy chain (MHC) (45%, P < 0.05) and type IIx MHC (25%, P < 0.05) associated with decreased type IIb MHC (34%, P < 0.05) was minimized by perindopril administration. These findings demonstrate that the enhancement in physical performance observed in perindopril-treated animals cannot be explained by changes in mitochondrial respiration and/or MHC distribution within muscles involved in running exercise.
Assuntos
Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/enzimologia , Consumo de Oxigênio/fisiologia , Peptidil Dipeptidase A/metabolismo , Condicionamento Físico Animal/fisiologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Citrato (si)-Sintase/metabolismo , Feminino , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/enzimologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Fibras Musculares de Contração Lenta/metabolismo , Cadeias Pesadas de Miosina/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Perindopril/farmacologia , Fenótipo , Esforço Físico/efeitos dos fármacos , Esforço Físico/fisiologia , Ratos , Ratos WistarRESUMO
The present experiment was designed to examine the effects of hypothyroidism and calcineurin inhibition induced by cyclosporin A (CsA) administration on both contractile and metabolic soleus muscle phenotypes, with a novel approach to the signaling pathway controlling mitochondrial biogenesis. Twenty-eight rats were randomly assigned to four groups, normothyroid, hypothyroid, and orally treated with either CsA (25 mg/kg, N-CsA and H-CsA) or vehicle (N-Vh and H-Vh), for 3 wk. Muscle phenotype was estimated by the MHC profile and activities of oxidative and glycolytic enzymes. We measured mRNA levels of the peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), the major regulator of mitochondrial content. We also studied the expression of the catalytic A-subunit of calcineurin (CnA) both at protein and transcript levels and mRNA levels of modulatory calcineurin inhibitor proteins (MCIP)-1 and -2, which are differentially regulated by calcineurin activity and thyroid hormone, respectively. CsA-administration induced a slow-to-fast MHC transition limited to the type IIA isoform, which is associated with increased oxidative capacities. Hypothyroidism strongly decreased both the expression of fast MHC isoforms and oxidative capacities. Effects of CsA administration on muscle phenotype were blocked in conditions of thyroid hormone deficiency. Changes in the oxidative profile were strongly related to PGC-1 alpha changes and associated with phosphorylation of p38 MAPK. Calcineurin and MCIPs mRNA levels were decreased by both hypothyroidism and CsA without additive effects. Taken together, these results suggest that adult muscle phenotype is primarily under the control of thyroid state. Physiological levels of thyroid hormone are required for the effects of calcineurin inhibition on slow oxidative muscle phenotype.
Assuntos
Calcineurina/metabolismo , Hipotireoidismo/metabolismo , Músculo Esquelético/enzimologia , Hormônios Tireóideos/metabolismo , Fatores Etários , Animais , Calcineurina/genética , Inibidores de Calcineurina , Domínio Catalítico , Ciclosporina/sangue , Ciclosporina/farmacologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fenótipo , Fosforilação , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The present study was designed to test the hypothesis that increasing physical activity by running exercise could favor the recovery of muscle mass after extensive injury and to determine the main molecular mechanisms involved. Left soleus muscles of female Wistar rats were degenerated by notexin injection before animals were assigned to either a sedentary group or an exercised group. Both regenerating and contralateral intact muscles from active and sedentary rats were removed 5, 7, 14, 21, 28 and 42 days after injury (n = 8 rats/group). Increasing contractile activity through running exercise during muscle regeneration ensured the full recovery of muscle mass and muscle cross-sectional area as soon as 21 days after injury, whereas muscle weight remained lower even 42 days postinjury in sedentary rats. Proliferator cell nuclear antigen and MyoD protein expression went on longer in active rats than in sedentary rats. Myogenin protein expression was higher in active animals than in sedentary animals 21 days postinjury. The Akt-mammalian target of rapamycin (mTOR) pathway was activated early during the regeneration process, with further increases of mTOR phosphorylation and its downstream effectors, eukaryotic initiation factor-4E-binding protein-1 and p70(s6k), in active rats compared with sedentary rats (days 7-14). The exercise-induced increase in mTOR phosphorylation, independently of Akt, was associated with decreased levels of phosphorylated AMP-activated protein kinase. Taken together, these results provided evidence that increasing contractile activity during muscle regeneration ensured early and full recovery of muscle mass and suggested that these beneficial effects may be due to a longer proliferative step of myogenic cells and activation of mTOR signaling, independently of Akt, during the maturation step of muscle regeneration.
Assuntos
Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Recuperação de Função Fisiológica/fisiologia , Regeneração/fisiologia , Animais , Venenos Elapídicos , Fator de Iniciação 4E em Eucariotos/metabolismo , Terapia por Exercício , Feminino , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/imunologia , Músculo Esquelético/fisiologia , Proteína MyoD/metabolismo , Neurotoxinas , Fosforilação , Condicionamento Físico Animal/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR , Regulação para Cima/fisiologiaRESUMO
The present work aimed at determining whether interleukin-6 (IL-6) produced by skeletal muscle during exercise is related, at least partly, to calcineurin activity. Rats were treated with two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, or vehicle (Vhl); they were then subjected to exhaustive treadmill running. Modulatory Calcineurin-Interacting Protein-1 (MCIP-1) mRNA levels, a reliable indicator of calcineurin activity, and IL-6 mRNA levels were measured by real-time RT-PCR in soleus muscles, and IL-6 protein concentration was measured in the plasma. Because low carbohydrates availability enhances IL-6 transcription through p38 Mitogen Activated Protein Kinase (MAPK) pathway, muscle glycogen content and glycaemia were measured and p38 MAPK phosphorylation was determined in skeletal muscle by western blotting. As expected, exercise induced an increase in IL-6 (P < 0.01) and MCIP-1 mRNA (P < 0.01) in soleus muscle of Vhl rats, and enhanced p38 phosphorylation and plasmatic IL-6 protein (P < 0.05). Calcineurin inhibition did not affect running time, glycemia or soleus glycogen content. CsA administration totally inhibited the exercise-induced increase in MCIP-1 mRNA (P < 0.01), blunted the IL-6 gene transcription related to muscle activity, and suppressed the changes in IL-6 protein in plasma. In addition to its inhibition of calcineurin activity, FK506 administration totally suppressed the exercise-induced IL-6 gene transcription, likely by an inhibition of p38 activation. Taken together, these results demonstrate that in addition to p38 MAPK, increased calcineurin activity is one of the signalling events involved in IL-6 gene transcription.
Assuntos
Calcineurina/fisiologia , Interleucina-6/genética , Contração Muscular/fisiologia , Fibras Musculares de Contração Lenta/metabolismo , Transcrição Gênica/fisiologia , Animais , Calcineurina/efeitos dos fármacos , Inibidores de Calcineurina , Ciclosporina/farmacologia , Feminino , Glicogênio/metabolismo , Imunossupressores/farmacologia , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Tacrolimo/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
RATIONALE: Hypoxia-induced pulmonary hypertension involves hypoxia-inducible factor-1alpha (HIF-1alpha) activation as well as elevated resting calcium levels. Cyclosporin A (CsA) inhibits calcium-induced calcineurin activation and blocks the stabilization of HIF-1alpha in cultured cells. OBJECTIVES: We hypothesized that treatment of rats with CsA would prevent HIF-1-dependent gene transcription, lower specific responses to acute hypoxia, and prevent pulmonary hypertension and right ventricle hypertrophy resulting from prolonged exposure to hypoxia. METHODS: Acute and chronic responses to hypoxia were studied in rats treated or not treated with CsA (25 mg x kg(-1) x d(-1)). MEASUREMENTS: Transcript levels of genes encoding the serotonin transporter or four HIF-1 target genes, in rats exposed for 6 h to ambient hypoxia, treated or not by CsA, were measured. In vivo hemodynamics, hematocrit, and heart morphologic characteristics were assessed in rats subjected to hypoxia for 3 wk, treated or not treated with CsA. Changes in mRNA levels of the modulatory calcineurin-interacting protein-1 (MCIP-1) were used as a sensitive indicator of calcineurin activity in lung and heart. MAIN RESULTS: Acute exposure to hypoxia led to a marked increase in mRNA levels of serotonin transporter, modulatory calcineurin-interacting protein-1, and HIF-1 target genes, which was blunted by CsA treatment. Prolonged exposure to hypoxia raised right ventricle pressure, induced right ventricle hypertrophy, and activated cardiac calcineurin, effects that were fully prevented by CsA treatment. CONCLUSIONS: These results suggest that CsA prevents hypoxia-induced pulmonary hypertension and right ventricle hypertrophy, either by inhibiting HIF-1 transcriptional activity in lung, by decreasing calcineurin activity in lung and heart, by direct effects of CsA, or by a combination of these factors.
Assuntos
Ciclosporina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Hipertensão Pulmonar/prevenção & controle , Hipertrofia Ventricular Direita/prevenção & controle , Hipóxia/complicações , Animais , Calcineurina/metabolismo , Inibidores de Calcineurina , Modelos Animais de Doenças , Seguimentos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The responsiveness of mature regenerated soleus (SOL) muscles to cyclosporin A (CsA) administration was studied in rats. Forty-two days after notexin-induced degeneration of left SOL muscles, rats were treated with CsA (25 mg/kg x day) or vehicle daily for 3 weeks. CsA administration decreased by eightfold the level of transcription of MCIP-1, a well-known calcineurin-induced gene, in intact as well as in regenerated muscles (P < 0.001). In response to CsA-administration we observed a slow-to-fast transition in the MHC profile, more marked in regenerated than in intact muscles (P < 0.05), but mainly restricted to MHC-Ibeta toward MHC-IIA. Immunohistochemical analysis showed that MHC-IIA was often co-expressed with MHC-Ibeta within myofibers of intact muscles, whereas it was mainly expressed within pure fast fibers of regenerated muscles. MHC-Ibeta mRNA levels were lower in regenerated than in intact muscles, but did not change in response to CsA-administration. CsA administration induced a significant increase in MHC-IIA mRNA levels (P < 0.001) similar in both intact and regenerated muscles. Present results suggest that in vivo in intact SOL muscles, calcineurin blocks the upregulation of the MHC-IIA isoform at the transcriptional level. On the other hand, the higher response of regenerated muscles to CsA administration cannot be explained by transcriptional events, and may result from either a more rapid turnover of MHC proteins in regenerated muscles than in intact ones, or translational events. This study further suggests that the developmental history of myofibers could play a role in the adaptability of skeletal muscle to variations in neuromuscular activity.
Assuntos
Inibidores de Calcineurina , Calcineurina/fisiologia , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Animais , Western Blotting , Peso Corporal , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/química , Músculo Esquelético/efeitos dos fármacos , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/genética , Tamanho do Órgão , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Wistar , Regeneração/efeitos dos fármacos , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
In chronic obstructive pulmonary disease, inspiratory muscles face increased resistive and elastic workloads and therefore increased energy requirements. The adaptive response of these muscles to this higher energy demand includes increased oxidative enzymes and changes in contractile protein expression but the consequences on mitochondrial function and energy metabolism have not been assessed so far. We investigated the in situ properties of the mitochondria of costal diaphragm and external intercostal muscles using the skinned fiber technique in 9 emphysematous and 11 age-matched control patients. Biopsies obtained during thoracic surgery were placed in an oxygraphic chamber to measure maximal oxygen uptake. We observed that the maximal oxidative capacity of diaphragm and external intercostal muscles increased significantly in the emphysematous group compared with the control group (+135 and +37%, respectively). Significant correlations were found between the maximal oxidative capacity and patients' pulmonary indexes of obstruction (diaphragm: r = -0.637, intercostal: r = -0.667, p < 0.005) and hyperinflation (diaphragm: r = 0.639, p < 0.003, intercostal: r = 0.634, p < 0.01). Slow myosin heavy chain isoform increased in the diaphragm of the emphysematous group, with significant relationships between indexes of obstruction and hyperinflation and activities of biochemical mitochondrial markers. Thus, severe emphysema was associated with increased mitochondrial capacity and efficiency in the inspiratory muscles, supporting an endurance training-like effect.
Assuntos
Transporte de Elétrons , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Músculos Respiratórios/metabolismo , Adaptação Fisiológica , Biópsia , Estudos de Casos e Controles , Respiração Celular , Creatina Quinase/metabolismo , Metabolismo Energético , Transferência de Energia , Feminino , Volume Expiratório Forçado , Glicólise , Humanos , Masculino , Pessoa de Meia-Idade , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/metabolismo , Oxirredução , Pneumonectomia , Doença Pulmonar Obstrutiva Crônica/cirurgia , Índice de Gravidade de Doença , Capacidade Vital , Trabalho RespiratórioRESUMO
Oxidative capacity of muscles correlates with capillary density and with microcirculation, which in turn depend on various regulatory factors, including NO generated by endothelial nitric oxide synthase (eNOS). To determine the role of eNOS in patterns of regulation of energy metabolism in various muscles, we studied mitochondrial respiration in situ in saponin-permeabilized fibres as well as the energy metabolism enzyme profile in the cardiac, soleus (oxidative) and gastrocnemius (glycolytic) muscles isolated from mice lacking eNOS (eNOS(-/-)). In soleus muscle, the absence of eNOS induced a marked decrease in both basal mitochondrial respiration without ADP (-32%; P <0.05) and maximal respiration in the presence of ADP (-29%; P <0.05). Furthermore, the eNOS(-/-) soleus muscle showed a decrease in total creatine kinase (-29%; P <0.05), citrate synthase (-31%; P <0.01), adenylate kinase (-27%; P <0.05), glyceraldehyde-3-phosphate dehydrogenase (-43%; P <0.01) and pyruvate kinase (-26%; P <0.05) activities. The percentage of myosin heavy chains I (slow isoform) was significantly increased from 24.3+/-1.5% in control to 30.1+/-1.1% in eNOS(-/-) soleus muscle ( P <0.05) at the expense of a slight non-significant decrease in the three other (fast) isoforms. Besides, eNOS(-/-) soleus showed a 28% loss of weight. Interestingly, we did not find differences in any parameters in cardiac and gastrocnemius muscles compared with respective controls. These results show that eNOS knockout has an important effect on muscle oxidative capacity as well on the activities of energy metabolism enzymes in oxidative (soleus) muscle. The absence of such effects in cardiac and glycolytic (gastrocnemius) muscle suggests a specific role for eNOS-produced NO in oxidative skeletal muscle.