RESUMO
Curcumin administration attenuates muscle disuse atrophy, but its effectiveness against aging-induced, selective loss of mass or force (presarcopenia or asthenia/dynopenia), or combined loss (sarcopenia), remains controversial. A new systemic curcumin treatment was developed and tested in 18-month-old C57BL6J and C57BL10ScSn male mice. The effects on survival, liver toxicity, loss of muscle mass and force, and satellite cell responsivity and commitment were evaluated after 6-month treatment. Although only 24-month-old C57BL10ScSn mice displayed age-related muscle impairment, curcumin significantly increased survival of both strains (+20-35%), without signs of liver toxicity. Treatment prevented sarcopenia in soleus and presarcopenia in EDL of C57BL10ScSn mice, whereas it did not affect healthy-aged muscles of C57BL6J. Curcumin-treated old C57BL10ScSn soleus preserved type-1 myofiber size and increased type-2A one, whereas EDL maintained adult values of total myofiber number and fiber-type composition. Mechanistically, curcumin only partially prevented the age-related changes in protein level and subcellular distribution of major costamere components and regulators. Conversely, it affected satellite cells, by maintaining adult levels of myofiber maturation in old regenerating soleus and increasing percentage of isolated, MyoD-positive satellite cells from old hindlimb muscles. Therefore, curcumin treatment successfully prevents presarcopenia and sarcopenia development by improving satellite cell commitment and recruitment.
Assuntos
Envelhecimento , Curcumina/farmacologia , Músculo Esquelético , Sarcopenia , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Masculino , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Sarcopenia/tratamento farmacológico , Sarcopenia/metabolismo , Sarcopenia/patologiaRESUMO
NEW FINDINGS: What is the central question of the study? What are the consequences of reducing circulating sphingosine-1-phosphate (S1P) for muscle physiology in the murine model of Duchenne muscular dystrophy (DMD)? What is the main result and its importance? Reduction of the circulating S1P level in mdx mice aggravates the dystrophic phenotype, as seen by an increase in fibre atrophy, fibrosis and loss of specific force, suggesting that S1P signalling is a potential therapeutic target in DMD. Although further studies are needed, plasma S1P levels have the intriguing possibility of being used as a biomarker for disease severity, an important issue in DMD. ABSTRACT: Sphingosine-1-phosphate (S1P) is an important regulator of skeletal muscle properties. The dystrophin-deficient mdx mouse possesses low levels of S1P (â¼50%) compared with wild type. Increased S1P availability was demonstrated to ameliorate the dystrophic phenotype in Drosophila and in mdx mice. Here, we analysed the effects produced by further reduction of S1P availability on the mass, force and regenerative capacity of dystrophic mdx soleus. Circulating S1P was neutralized by a specific anti-S1P antibody (S1P-Ab) known to lower the extracellular concentration of this signalling lipid. The S1P-Ab was administered intraperitoneally in adult mdx mice every 2 days for the duration of experiments. Soleus muscle properties were analysed 7 or 14 days after the first injection. The decreased availability of circulating S1P after the 14 day treatment reduced mdx soleus fibre cross-sectional area (-16%, P < 0.05), an effect that was associated with an increase in markers of proteolytic (MuRF1 and atrogin-1) and autophagic (p62 and LC3-II/LC3-I ratio) pathways. Moreover, an increase of fibrosis was also observed (+26%, P < 0.05). Notably, the treatment also caused a reduction of specific tetanic tension (-29%, P < 0.05). The mdx soleus regenerative capacity was only slightly influenced by reduced S1P. In conclusion, neutralization of circulating S1P reduces the mass and specific force and increases fibrosis of mdx soleus muscle, thus worsening the dystrophic phenotype. The results confirm that active, functional S1P signalling might counteract the progression of soleus mdx pathology and validate the pathway as a potential therapeutic target for muscular dystrophies.
Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Modelos Animais de Doenças , Distrofina/metabolismo , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/uso terapêutico , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Fenótipo , Esfingosina/análogos & derivadosRESUMO
We investigated the effects of S1P3 deficiency on the age-related atrophy, decline in force, and regenerative capacity of soleus muscle from 23-mo-old male (old) mice. Compared with muscle from 5-mo-old (adult) mice, soleus mass and muscle fiber cross-sectional area (CSA) in old wild-type mice were reduced by ~26% and 24%, respectively. By contrast, the mass and fiber CSA of soleus muscle in old S1P3-null mice were comparable to those of adult muscle. Moreover, in soleus muscle of wild-type mice, twitch and tetanic tensions diminished from adulthood to old age. A slowing of contractile properties was also observed in soleus from old wild-type mice. In S1P3-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P3-null mice by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of soleus in old wild-type mice was significantly smaller (-28%) compared with that of regenerated muscle in adult mice. On the contrary, the mean fiber CSA of regenerated soleus in old S1P3-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P3, soleus muscle is protected from the decrease in muscle mass and force, and the attenuation of regenerative capacity, all of which are typical characteristics of aging.
Assuntos
Envelhecimento/genética , Músculo Esquelético/metabolismo , Receptores de Lisoesfingolipídeo/genética , Sarcopenia/genética , Envelhecimento/metabolismo , Animais , Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Contração Muscular/fisiologia , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/patologia , Força Muscular/fisiologia , Músculo Esquelético/fisiopatologia , Receptores de Lisoesfingolipídeo/deficiência , Regeneração/fisiologia , Sarcopenia/metabolismo , Sarcopenia/fisiopatologia , Receptores de Esfingosina-1-FosfatoRESUMO
Antioxidant administration aimed to antagonize the development and progression of disuse muscle atrophy provided controversial results. Here we investigated the effects of curcumin, a vegetal polyphenol with pleiotropic biological activity, because of its ability to upregulate glucose-regulated protein 94 kDa (Grp94) expression in myogenic cells. Grp94 is a sarco-endoplasmic reticulum chaperone, the levels of which decrease significantly in unloaded muscle. Rats were injected intraperitoneally with curcumin and soleus muscle was analysed after 7 days of hindlimb unloading or standard caging. Curcumin administration increased Grp94 protein levels about twofold in muscles of ambulatory rats (P < 0.05) and antagonized its decrease in unloaded ones. Treatment countered loss of soleus mass and myofibre cross-sectional area by approximately 30% (P ≤ 0.02) and maintained a force-frequency relationship closer to ambulatory levels. Indexes of muscle protein and lipid oxidation, such as protein carbonylation, revealed by Oxyblot, and malondialdehyde, measured with HPLC, were significantly blunted in unloaded treated rats compared to untreated ones (P = 0.01). Mechanistic involvement of Grp94 was suggested by the disruption of curcumin-induced attenuation of myofibre atrophy after transfection with antisense grp94 cDNA and by the drug-positive effect on the maintenance of the subsarcolemmal localization of active neuronal nitric oxide synthase molecules, which were displaced to the sarcoplasm by unloading. The absence of additive effects after combined administration of a neuronal nitric oxide synthase inhibitor further supported curcumin interference with this pro-atrophic pathway. In conclusion, curcumin represents an effective and safe tool to upregulate Grp94 muscle levels and to maintain muscle function during unweighting.
Assuntos
Antioxidantes/farmacologia , Curcumina/farmacologia , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Antioxidantes/uso terapêutico , Curcumina/uso terapêutico , Feminino , Elevação dos Membros Posteriores/fisiologia , Glicoproteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/fisiopatologia , Ratos Wistar , Sarcolema/metabolismoRESUMO
Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is characterized by atrophy and weakness of selective muscle groups. FSHD is considered an autosomal dominant disease with incomplete penetrance and unpredictable variability of clinical expression within families. Mice overexpressing FRG1 (FSHD region gene 1), a candidate gene for this disease, develop a progressive myopathy with features of the human disorder. Here, we show that in FRG1-overexpressing mice, fast muscles, which are the most affected by the dystrophic process, display anomalous fast skeletal troponin T (fTnT) isoform, resulting from the aberrant splicing of the Tnnt3 mRNA that precedes the appearance of dystrophic signs. We determine that muscles of FRG1 mice develop less strength due to impaired contractile properties of fast-twitch fibers associated with an anomalous MyHC-actin ratio and a reduced sensitivity to Ca(2+). We demonstrate that the decrease of Ca(2+) sensitivity of fast-twitch fibers depends on the anomalous troponin complex and can be rescued by the substitution with the wild-type proteins. Finally, we find that the presence of aberrant splicing isoforms of TNNT3 characterizes dystrophic muscles in FSHD patients. Collectively, our results suggest that anomalous TNNT3 profile correlates with the muscle impairment in both humans and mice. On the basis of these results, we propose that aberrant fTnT represents a biological marker of muscle phenotype severity and disease progression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Fibras Musculares de Contração Rápida/fisiologia , Debilidade Muscular/metabolismo , Proteínas/metabolismo , Troponina T/metabolismo , Processamento Alternativo/fisiologia , Animais , Biomarcadores , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Troponina T/genéticaRESUMO
The vegetal polyphenol curcumin displays beneficial effects against skeletal muscle derangement induced by oxidative stress, disuse or aging. Since oxidative stress and inflammation are involved in the progression of muscle dystrophy, the effects of curcumin administration were investigated in the diaphragm of mdx mice injected intraperitoneally or subcutaneously with curcumin for 4-12-24 weeks. Curcumin treatment independently of the way and duration of administration (i) ameliorated myofiber maturation index without affecting myofiber necrosis, inflammation and degree of fibrosis; (ii) counteracted the decrease in type 2X and 2B fiber percentage; (iii) increased about 30% both twitch and tetanic tensions of diaphragm strips; (iv) reduced myosin nitrotyrosination and tropomyosin oxidation; (v) acted on two opposite nNOS regulators by decreasing active AMP-Kinase and increasing SERCA1 protein levels, the latter effect being detectable also in myotube cultures from mdx satellite cells. Interestingly, increased contractility, decreased myosin nitrotyrosination and SERCA1 upregulation were also detectable in the mdx diaphragm after a 4-week administration of the NOS inhibitor 7-Nitroindazole, and were not improved further by a combined treatment. In conclusion, curcumin has beneficial effects on the dystrophic muscle, mechanistically acting for the containment of a deregulated nNOS activity.
RESUMO
Sphingosine 1-phosphate (S1P) is a bioactive lipid known to control cell growth that was recently shown to act as a trophic factor for skeletal muscle, reducing the progress of denervation atrophy. The aim of this work was to investigate whether S1P is involved in skeletal muscle fiber recovery (regeneration) after myotoxic injury induced by bupivacaine. The postnatal ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. Immunofluorescence analysis demonstrated that S1P-specific receptors S1P(1) and S1P(3) are expressed by quiescent satellite cells. Soleus muscles undergoing regeneration following injury induced by intramuscular injection of bupivacaine exhibited enhanced expression of S1P(1) receptor, while S1P(3) expression progressively decreased to adult levels. S1P(2) receptor was absent in quiescent cells but was transiently expressed in the early regenerating phases only. Administration of S1P (50 microM) at the moment of myotoxic injury caused a significant increase of the mean cross-sectional area of regenerating fibers in both rat and mouse. In separate experiments designed to test the trophic effects of S1P, neutralization of endogenous circulating S1P by intraperitoneal administration of anti-S1P antibody attenuated fiber growth. Use of selective modulators of S1P receptors indicated that S1P(1) receptor negatively and S1P(3) receptor positively modulate the early phases of regeneration, whereas S1P(2) receptor appears to be less important. The present results show that S1P signaling participates in the regenerative processes of skeletal muscle.
Assuntos
Lisofosfolipídeos/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Animais , Bupivacaína , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Injeções Intramusculares , Lisofosfolipídeos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/fisiopatologia , Ratos , Ratos Wistar , Receptores de Lisoesfingolipídeo/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/metabolismo , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/administração & dosagem , Esfingosina/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle-specific integrin-associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes. METHODS: Muscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail-suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno-associated virus infection. In vivo interference of exogenous melusin with dominant-negative kinases and other atrophy attenuators (Grp94 cDNA; 7-nitroindazole) on size of unloaded rat myofibers was also explored. RESULTS: Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail-suspended rat (P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of ß1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (-31%), compared with controls (-48%, P = 0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin-expressing adeno-associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (-33%) by reducing Atrogin-1 and MurF-1 transcripts (P ≤ 0.002), despite of a two-fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co-transfection with dominant-negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7-nitroindazole treatment or with cDNA transfection of the nNOS-interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy. CONCLUSIONS: Disuse/unloading-induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteína Forkhead Box O3/metabolismo , Elevação dos Membros Posteriores/fisiologia , Proteínas Musculares/metabolismo , Atrofia Muscular/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Feminino , Humanos , Ratos , Ratos Wistar , TransfecçãoRESUMO
Postnatal development of skeletal muscle occurs through the progressive transformation of diverse biochemical, metabolic, morphological, and functional characteristics from the embryonic to the adult phenotype. Since muscle regeneration recapitulates postnatal development of muscle fiber, it offers an appropriate experimental model to investigate the existing relationships between diverse muscle functions and the expression of key protein isoforms, particularly at the single-fiber level. This study was carried out in regenerating soleus muscle 14 days after injury. At this intermediate stage, the regenerating muscle exhibited a recovery of mass greater than its force generation capacity. The lower specific tension of regenerating muscle suggested intrinsic defective excitation-contraction coupling and/or contractility processes. The presence of developmental isoforms of both the voltage-gated Ca(2+) channel (alpha(1)C) and of ryanodine receptor 3, paralleled by an abnormal caffeine contracture development, confirms the immature excitation-contraction coupling of the regenerating muscle. The defective Ca(2+) handling could also be confirmed by the lower sarcoplasmic reticulum caffeine sensitivity of regenerating single fibers. Also, regenerating single fibers revealed a lower maximal specific tension, which was associated with the residual presence of embryonic myosin heavy chains. Moreover, the fibers showed a reduced Ca(2+) sensitivity of myofibrillar proteins, particularly those simultaneously expressing the slow and fast isoforms of troponin C. The present results indicate that the expression of developmental proteins determines the incomplete functional recovery of regenerating soleus.
Assuntos
Contração Isométrica/fisiologia , Proteínas Motores Moleculares/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Miofibrilas/fisiologia , Retículo Sarcoplasmático/fisiologia , Animais , Células Cultivadas , Masculino , Isoformas de Proteínas/metabolismo , Ratos , Ratos WistarRESUMO
Denervation deeply affects muscle structure and function, the alterations being different in slow and fast muscles. Because the effects of denervation on fast muscles are still controversial, and high-throughput studies on gene expression in denervated muscles are lacking, we studied gene expression during atrophy progression following denervation in mouse tibialis anterior (TA). The sciatic nerve was cut close to trochanter in adult CD1 mice. One, three, seven, and fourteen days after denervation, animals were killed and TA muscles were dissected out and utilized for physiological experiments and gene expression studies. Target cDNAs from TA muscles were hybridized on a dedicated cDNA microarray of muscle genes. Seventy-one genes were found differentially expressed. Microarray results were validated, and the expression of relevant genes not probed on our array was monitored by real-time quantitative PCR (RQ-PCR). Nuclear- and mitochondrial-encoded genes implicated in energy metabolism were consistently downregulated. Among genes implicated in muscle contraction (myofibrillar and sarcoplasmic reticulum), genes typical of fast fibers were downregulated, whereas those typical of slow fibers were upregulated. Electrophoresis and Western blot showed less pronounced changes in myofibrillar protein expression, partially confirming changes in gene expression. Isometric tension of skinned fibers was little affected by denervation, whereas calcium sensitivity decreased. Functional studies in mouse extensor digitorum longus muscle showed prolongation in twitch time parameters and shift to the left in force-frequency curves after denervation. We conclude that, if studied at the mRNA level, fast muscles appear not less responsive than slow muscles to the interruption of neural stimulation.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Animais , Camundongos , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Denervação Muscular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/inervação , Atrofia Muscular/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Evidence indicates that extracellular ATP may have relevant functions in skeletal muscle, even though the physiological role and distribution of specific signaling pathway elements are not well known. The present work shows that P2X4 receptor, an extracellular ATP-regulated cell membrane channel permeable to Ca2+, is expressed in several tissues of the rat, including skeletal muscle. A specific antibody detected a protein band of approximately 60 kDa. Immunofluorescence demonstrated that P2X4 has an intracellular localization, and confocal analysis revealed that the receptor colocalizes with the T-tubule membrane DHP receptor. Considering that the natural agonist of P2X4 is ATP, we explored if changes of extracellular ATP levels could occur in contracting skeletal muscle to regulate the channel. In vitro experiments showed that substantial ATP is released and rapidly hydrolyzed after electrical stimulation of rat muscle fibers. Results show that the presence of ATP-degrading enzymes (hexokinase/apyrase), inhibitors of P2X receptors or Ca2+-free conditions, all abolished the progressive twitch tension potentiation produced in soleus muscle by low-frequency (0.05 Hz) stimulation. These data reveal that ATP-mediated Ca2+ entry, most likely through P2X4 receptor, may play an important role in modulating the contractility of skeletal muscle.
Assuntos
Trifosfato de Adenosina/fisiologia , Membranas Intracelulares/fisiologia , Contração Muscular , Músculo Esquelético/fisiologia , Receptores Purinérgicos P2/fisiologia , Animais , Cálcio/metabolismo , Masculino , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores Purinérgicos P2/análise , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X4 , Transdução de SinaisRESUMO
To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P3) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P3-null mice. The body weight of 3-mo-old S1P3-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P3 deficiency enhanced the expression level of S1P1 and S1P2 receptors mRNA in S1P3-null EDL muscle. The contractile properties of S1P3-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P3 appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P3-null muscle. Moreover, in the absence of S1P3, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P3-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P3 makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P3 is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.
Assuntos
Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/fisiologia , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Atrofia/metabolismo , Atrofia/fisiopatologia , Cálcio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos/metabolismo , Camundongos Transgênicos/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Fadiga Muscular/fisiologia , Doenças Musculares/metabolismo , Doenças Musculares/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA Mensageiro/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMO
Skeletal muscle type 2B fibers normally receive a moderate level of motoneuron discharge. As a consequence, we hypothesize that type 2B fiber properties should be less sensitive to the absence of the nerve. Therefore, we have investigated the response of sarcoplasmic reticulum and myofibrillar proteins of type 2B fibers isolated from rat extensor digitorum longus muscle after denervation (2 and 7 days). Single fibers were identified by SDS-PAGE of myosin heavy chain isoforms. Electrophysiological and isometric contractile properties of the whole muscle were also analyzed. The pCa-tension relationship of type 2B single fibers was shifted to the left at 2 days and to right at 7 days after denervation, with significant differences in the Hill coefficients and pCa threshold values in 2- vs. 7-day-denervated fibers. The sarcoplasmic reticulum Ca2+ uptake capacity and rate significantly decreased after 2 days of denervation, whereas both increased at 7 days. Caffeine sensitivity of sarcoplasmic reticulum Ca2+ release was transitory and markedly increased in 2-day-denervated fibers. Our results indicate that type 2B fiber functional properties are highly sensitive to the interruption of nerve supply. Moreover, most of 2-day-denervated changes were reverted at 7 days.
Assuntos
Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Animais , Transporte Biológico/fisiologia , Cafeína/farmacologia , Cálcio/metabolismo , Cálcio/farmacocinética , Cálcio/farmacologia , Denervação , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Contração Isométrica/fisiologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/efeitos dos fármacos , Cadeias Pesadas de Miosina/análise , Cadeias Leves de Miosina/análise , Ratos , Ratos Wistar , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Fatores de TempoRESUMO
Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bed-rest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1α expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1α and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5 can be attractive therapeutic targets for pharmacological countermeasures and then deserve further investigations.
Assuntos
Membro Posterior/fisiopatologia , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Esquelético/fisiopatologia , Atrofia Muscular/fisiopatologia , Cadeias Leves de Miosina/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Animais , Comportamento Animal , Fenômenos Bioquímicos , Peso Corporal , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos Transgênicos , Contração Muscular , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Ratos , Descanso , Suporte de CargaRESUMO
Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca(2+) concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.
Assuntos
Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Músculo Esquelético/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Transporte/genética , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Citocinas/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Expressão Gênica , Elevação dos Membros Posteriores , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Musculares Esqueléticas , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Sarcolema/metabolismo , Voo EspacialRESUMO
Sphingosine 1-phosphate is a bioactive lipid that modulates skeletal muscle growth through its interaction with specific receptors localized in the cell membrane of muscle fibers and satellite cells. This study analyzes the role of S1P(2) receptor during in vivo regeneration of soleus muscle in two models of S1P(2) deficiency: the S1P(2)-null mouse and wild-type mice systemically treated with the S1P(2) receptor antagonist JTE-013. To stimulate regeneration, muscle degeneration was induced by injecting into soleus muscle the myotoxic drug notexin. Both ablation of S1P(2) receptor and its functional inactivation delayed regeneration of soleus muscle. The exogenous supplementation of S1P or its removal, by a specific antibody, two conditions known to stimulate or inhibit, respectively, soleus muscle regeneration, were without effects when the S1P(2) receptor was absent or inactive. The delayed regeneration was associated with a lower level of myogenin, a muscle differentiation marker, and reduced phosphorylation of Akt, a key marker of muscle growth. Consistently, silencing of S1P(2) receptor abrogated the pro-myogenic action of S1P in satellite cells, paralleled by low levels of the myogenic transcription factor myogenin. The study indicates that S1P(2) receptor plays a key role in the early phases of muscle regeneration by sustaining differentiation and growth of new-forming myofibers.
Assuntos
Músculo Esquelético/fisiologia , Receptores de Lisoesfingolipídeo/fisiologia , Regeneração/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Regeneração/efeitos dos fármacosRESUMO
The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.
Assuntos
Adaptação Fisiológica , Músculo Esquelético/metabolismo , Ausência de Peso , Animais , Regulação para Baixo , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Pesadas de Miosina/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos , Voo Espacial , Ubiquitina-Proteína Ligases/metabolismo , Regulação para CimaRESUMO
It is commonly accepted that skeletal muscles from dystrophin-deficient mdx mice are more susceptible than those from wild-type mice to damage from eccentric contractions. However, the downstream mechanisms involved in this enhanced force drop remain controversial. We studied the reduction of contractile force induced by eccentric contractions elicited in vivo in the gastrocnemius muscle of wild-type mice and three distinct models of muscle dystrophy: mdx, alpha-sarcoglycan (Sgca)-null, and collagen 6A1 (Col6a1)-null mice. In mdx and Sgca-null mice, force decreased 35% compared with 14% in wild-type mice. Drop of force in Col6a1-null mice was comparable to that in wild-type mice. To identify the determinants of the force drop, we measured force generation in permeabilized fibers dissected from gastrocnemius muscle that had been exposed in vivo to eccentric contractions and from the contralateral unstimulated muscle. A force loss in skinned fibers after in vivo eccentric contractions was detectable in fibers from mdx and Sgca-null, but not wild-type and Col6a1-null, mice. The enhanced force reduction in mdx and Sgca-null mice was observed only when eccentric contractions were elicited in vivo, since eccentric contractions elicited in vitro had identical effects in wild-type and dystrophic skinned fibers. These results suggest that 1) the enhanced force loss is due to a myofibrillar impairment that is present in all fibers, and not to individual fiber degeneration, and 2) the mechanism causing the enhanced force reduction is active in vivo and is lost after fiber permeabilization.
Assuntos
Contração Muscular , Fibras Musculares Esqueléticas , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofias Musculares/fisiopatologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
To define the time course and potential effects of electrical stimulation on permanently denervated muscle, we evaluated excitation-contraction coupling (ECC) of rat leg muscles during progression to long-term denervation by ultrastructural analysis, specific binding to dihydropyridine receptors, ryanodine receptor 1 (RYR-1), Ca channels and extrusion Ca pumps, gene transcription and translation of Ca-handling proteins, and in vitro mechanical properties and electrophysiological analyses of sarcolemmal passive properties and L-type Ca current (ICa) parameters. We found that in response to long-term denervation: 1) isolated muscle that is unable to twitch in vitro by electrical stimulation has very small myofibers but may show a slow caffeine contracture; 2) only roughly half of the muscle fibers with "voltage-dependent Ca channel activity" are able to contract; 3) the ECC mechanisms are still present and, in part, functional; 4)ECC-related gene expression is upregulated; and 5) at any time point, there are muscle fibers that are more resistant than others to denervation atrophy and disorganization of the ECC apparatus. These results support the hypothesis that prolonged "resting" [Ca] may drive progression of muscle atrophy to degeneration and that electrical stimulation-induced [Ca] modulation may mimic the lost nerve influence, playing a key role in modifying the gene expression of denervated muscle. Hence, these data provide a potential molecular explanation for the muscle recovery that occurs in response to rehabilitation strategies developed based on empirical clinical observations.
Assuntos
Contração Muscular/fisiologia , Denervação Muscular/efeitos adversos , Músculo Esquelético/fisiologia , Atrofia Muscular/fisiopatologia , Animais , Canais de Cálcio/fisiologia , Expressão Gênica , Masculino , Potenciais da Membrana/fisiologia , Microscopia Eletrônica de Transmissão , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Sphingosine 1-phosphate (S1P) mediates a number of cellular responses, including growth and proliferation. Skeletal muscle possesses the full enzymatic machinery to generate S1P and expresses the transcripts of S1P receptors. The aim of this work was to localize S1P receptors in rat skeletal muscle and to investigate whether S1P exerts a trophic action on muscle fibers. RT-PCR and Western blot analyses demonstrated the expression of S1P(1) and S1P(3) receptors by soleus muscle. Immunofluorescence revealed that S1P(1) and S1P(3) receptors are localized at the cell membrane of muscle fibers and in the T-tubule membranes. The receptors also decorate the nuclear membrane. S1P(1) receptors were also present at the neuromuscular junction. The possible trophic action of S1P was investigated by utilizing the denervation atrophy model. Rat soleus muscle was analyzed 7 and 14 days after motor nerve cut. During denervation, S1P was continuously delivered to the muscle through a mini osmotic pump. S1P and its precursor, sphingosine (Sph), significantly attenuated the progress of denervation-induced muscle atrophy. The trophic effect of Sph was prevented by N,N-dimethylsphingosine, an inhibitor of Sph kinase, the enzyme that converts Sph into S1P. Neutralization of circulating S1P by a specific antibody further demonstrated that S1P was responsible for the trophic effects of S1P during denervation atrophy. Denervation produced the down regulation of S1P(1) and S1P(3) receptors, regardless of the presence of the receptor agonist. In conclusion, the results suggest that S1P acts as a trophic factor of skeletal muscle.