RESUMO
Adult skeletal muscle contains populations of satellite cells and muscle-derived stem cells that are capable of forming multinucleate myotubes. The purpose of this study was to determine the phenotype of cells isolated from a common satellite cell isolation and passaging procedure from whole skeletal muscle. To ascertain the characteristics of the cellular phenotype, the myogenic markers MyoD and desmin, the satellite-cell-specific marker Pax7, and the haemopoietic stem cell markers CD34 and CD45 were examined by immunohistochemical analysis. Immediately after isolation, > 90% myogenic marker-positive cells were positive for desmin, MyoD and Pax7. In contrast, approximately 10% of the isolated cells expressed only CD34 or CD45. After three passages, the percentage of cells that were positive for the myogenic markers desmin, MyoD and Pax7 was reduced to approximately 55%, while the population of CD34- or CD45-positive cells increased to approximately 30% after the third passage. Immunohistochemical detection of bromodeoxyuridine demonstrated that the number of proliferating cells decreased progressively after each passaging. Finally, after the third passage the percentage of nuclei in myotubes decreased from 46.7% to 12.5%. Since passaging of muscle progenitor cells is common practice, the results of the current report suggest that characterization of cell heterogeneity needs to be made frequently.
Assuntos
Técnicas de Cultura de Células/métodos , Fibras Musculares Esqueléticas/ultraestrutura , Músculos/citologia , Células Satélites de Músculo Esquelético/citologia , Animais , Antígenos CD34/análise , Biomarcadores/análise , Diferenciação Celular , Divisão Celular , Separação Celular , Células Cultivadas , Desmina/análise , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/análise , Antígenos Comuns de Leucócito/análise , Proteína MyoD/análise , Fator de Transcrição PAX7 , Ratos , Células-Tronco/citologiaRESUMO
The purpose of this review is to present current understanding of cellular and molecular regulation of fibre type expression in skeletal muscle. Published literature seems to conclusively suggest that muscle fibre type expression is regulated by multiple signalling pathways and transcription factors rather than a single 'master' switch or signalling pathway. While the current nomenclature for fibre types is convenient for communication, based upon the evolution of this nomenclature, the prediction that fibre type classifications may change in the future to incorporate post-genomic information is made. It is predicted that future fibre type classifications could be based upon the contractile-activity-induced changes in a common regulatory factor(s) within a subpopulation of genes whose expressions are altered to modify and maintain the new muscle fibre phenotype.
Assuntos
Regulação da Expressão Gênica/genética , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Adaptação Fisiológica/genética , Calcineurina/genética , Glicólise/genética , Humanos , Fibras Musculares Esqueléticas/classificação , Cadeias Pesadas de Miosina/genética , Oxirredução , Fenótipo , Transdução de Sinais/genética , Terminologia como Assunto , Transcrição Gênica/genéticaRESUMO
The proliferation ability of satellite cells (considered the 'stem cells' of mature myofibers) declines with increasing age when cultured under standard cell culture conditions of 21% oxygen. However, actual oxygen levels in the intact myofiber in vivo are an order of magnitude lower. No studies to date have addressed the issue of whether culturing satellite cells from old muscles under more 'physiologic' conditions would enhance their proliferation and/or differentiation ability. Therefore, we analyzed satellite cells derived from 31-month-old rats in standard cultures with 21% O2 and in lowered (approximately 3%) O2. Under the lowered O2 conditions, we noted a remarkable increase in the percentage of large-sized colonies, activation of cell cycle progression factors, phosphorylation of Akt, and downregulation of the cell cycle inhibitor p27Kip1. These data suggest that lower O2 levels provide a milieu that stimulates proliferation by allowing continued cell cycle progression, to result ultimately in the enhanced in vitro replicative life span of the old satellite cells. Such a method therefore provides an improved means for the ex vivo generation of progenitor satellite cell populations for potential therapeutic stem cell transplantation.
Assuntos
Músculo Esquelético/citologia , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases , Animais , Técnicas de Cultura de Células/métodos , Ciclo Celular , Diferenciação Celular , Divisão Celular , Ativação Enzimática , Masculino , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344RESUMO
The purpose of the present study was to investigate the effects of fatiguing muscular activity on glycogen, glycogen phosphorylase (GP), and Ca(2+) uptake associated with the sarcoplasmic reticulum (SR). Tetanic contractions (100 ms, 75 Hz) of the gastrocnemius and plantaris muscles, elicited once per second for 15 min, significantly reduced force to 26.5 +/- 4.0% and whole muscle glycogen to 23% of rested levels. SR glycogen levels were 415.4 +/- 76.6 and 20.4 +/- 2.1 microg/mg SR protein in rested and fatigued samples, respectively. The optical density of GP from SDS-PAGE was reduced to 21% of control, whereas pyridoxal 5'-phosphate concentration, a quantitative indicator of GP content, was significantly reduced to 3% of control. GP activity after exercise, in the direction of glycogen breakdown, was reduced to 4% of control. Maximum SR Ca(2+) uptake rate was also significantly reduced to 81% of control. These data demonstrate that glycogen and GP associated with skeletal muscle SR are reduced after fatiguing activity.
Assuntos
Glicogênio Fosforilase/metabolismo , Glicogênio/metabolismo , Fadiga Muscular/fisiologia , Retículo Sarcoplasmático/metabolismo , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Estimulação Elétrica , Feminino , Contração Muscular/fisiologia , Ratos , Ratos Sprague-Dawley , Retículo Sarcoplasmático/enzimologiaRESUMO
Calcineurin (CaN) has been implicated as a signaling molecule that can transduce physiological stimuli (e.g., contractile activity) into molecular signals that initiate slow-fiber phenotypic gene expression and muscle growth. To determine the influence of muscle phenotype and atrophy on CaN levels in muscle, the levels of soluble CaN in rat muscles of varying phenotype, as assessed by myosin heavy chain (MHC)-isoform proportions, were determined by Western blotting. CaN levels were significantly greater in the plantaris muscle containing predominantly fast (IIx and IIb) MHC isoforms, compared with the soleus (predominantly type I MHC) or vastus intermedius (VI, contains all 4 adult MHC isoforms). Three months after a complete spinal cord transection (ST), the CaN levels in the VI muscle were significantly reduced, despite a significant increase in fast MHC isoforms. Surprisingly, the levels of CaN in the VI were highly correlated with muscle mass but not MHC isoform proportions in ST and control rats. These data demonstrate that CaN levels in skeletal muscle are highly correlated to muscle mass and that the normal relationship with phenotype is lost after ST.
Assuntos
Calcineurina/metabolismo , Músculo Esquelético/fisiologia , Atrofia Muscular/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Adaptação Fisiológica , Animais , Citosol/metabolismo , Feminino , Contração Muscular , Músculo Esquelético/fisiopatologia , Cadeias Pesadas de Miosina/genética , Especificidade de Órgãos , Fenótipo , Isoformas de Proteínas/genética , Ratos , Ratos Sprague-Dawley , Análise de Regressão , Medula Espinal/fisiologiaRESUMO
Approximately 50% of humans older than 85 years have physical frailty due to weak skeletal muscles. This indicates a need for determining mechanisms to combat this problem. A critical cellular factor for postnatal muscle growth is a population of myogenic precursor cells called satellite cells. Given the complex process of sarcopenia, it has been postulated that, at some point in this process, a limited satellite cell proliferation potential could become rate-limiting to the regrowth of old muscles. It is conceivable that if satellite cell proliferative capacity can be maintained or enhanced with advanced age, sarcopenia could potentially be delayed or prevented. Therefore, the purposes of this paper are to describe whether IGF-I can prevent muscular atrophy induced by repeated cycles of hindlimb immobilization, increase the in vitro proliferation in satellite cells from these muscles and, if so, the molecular mechanisms by which IGF-I mediates this increased proliferation. Our results provide evidence that IGF-I can enhance aged muscle regrowth possibly through increased satellite cell proliferation. The results also suggest that IGF-I enhances satellite cell proliferation by decreasing the cell cycle inhibitor, p27Kip1, through the PI3'-K/Akt pathway. These data provide molecular evidence for IGF-I's rescue effect upon aging-associated skeletal muscle atrophy.
Assuntos
Envelhecimento/fisiologia , Fator de Crescimento Insulin-Like I/farmacologia , Músculo Esquelético/citologia , Atrofia Muscular/prevenção & controle , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Elevação dos Membros Posteriores , Cinética , Camundongos , Camundongos Transgênicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologiaRESUMO
This study examined the effects of fatigue on the functional aspects of the contractile apparatus and sarcoplasmic reticulum (SR). Frog semitendinosus muscles were stimulated to fatigue, and skinned fibers or a homogenate fraction was prepared from both fatigued and rested contralateral muscles. In fatigued fibers, maximal Ca2+-activated force of the contractile apparatus was unaltered, whereas maximal actomyosin-ATPase activity was depressed by 20%. The Ca2+ sensitivity of force was increased, whereas that of actomyosin-ATPase was not altered. Also, the rate constant for tension redevelopment was decreased at submaximal Ca2+ concentration. These latter findings suggest that fatigue slows the dissociation of force-generating myosin cross bridges. Ca2+ uptake and Ca2+-ATPase activity of the SR were depressed by 46 and 21%, respectively, in the fatigued muscles. Fatigue also reduced the rates of SR Ca2+ release evoked by AgNO3 and 4-chloro-m-cresol by 38 and 45%, respectively. During fatigue, the contractile apparatus and SR undergo intrinsic functional alterations. These changes likely result in altered force production and energy consumption by the intact muscle.
Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Retículo Sarcoplasmático/fisiologia , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Estimulação Elétrica , Metabolismo Energético/fisiologia , Técnicas In Vitro , Contração Isométrica/fisiologia , Masculino , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/enzimologia , Músculo Esquelético/ultraestrutura , Rana pipiens , Retículo Sarcoplasmático/enzimologiaRESUMO
The purpose of this investigation was to examine changes in sarcoplasmic reticulum (SR) function in muscles subjected to different patterns of muscle activity. Frog sartorius muscles were stimulated with tetanic trains (100 ms, 100 Hz) delivered at rates of 2.0, 0.5, and 0.2 trains/s. In one set of experiments, stimulation was continued until force had declined to approximately 17% of initial (constant fatigue), whereas in the other set, stimulation was continued for 1 min (constant duration). In the constant-fatigue experiments, Ca2+ uptake (1 mM MgATP) and release rates (25 microM AgNO3, 5 mM 4-chloro-m-cresol) were depressed by similar extents following each protocol. This occurred despite 1, 4, and 17 min of stimulation, respectively, used to induce fatigue. In the constant-duration experiments, larger reductions in SR function occurred following the highest frequency stimulation protocol. These data suggest that when muscles are fatigued to similar extents, depressions in SR function are independent of the activity protocol. On the other hand, when a constant duration of activity is imposed, changes in SR function are closely linked to the extent of force reduction.
Assuntos
Cálcio/metabolismo , Fadiga Muscular , Músculo Esquelético/fisiologia , Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cresóis/farmacologia , Ditiotreitol/farmacologia , Estimulação Elétrica , Técnicas In Vitro , Indóis/farmacologia , Masculino , Rana pipiens , Retículo Sarcoplasmático/efeitos dos fármacos , Nitrato de Prata/farmacologia , Azida Sódica/farmacologiaRESUMO
We investigated the effects of glucose 6-phosphate (G6P) on skeletal muscle contractile apparatus and sarcoplasmic reticulum (SR) function. Using rat extensor digitorum longus fibres, the presence of 5 mM G6P decreased the Ca2+ sensitivity of both force production and actomyosin ATPase (AM-ATPase) activity. Conversely, maximal Ca(2+)-activated force was unaffected while maximal AM-ATPase activity was increased by 37%. In SR vesicles isolated from rat gastrocnemius, G6P markedly altered Ca2+ handling. It increased Ca(2+)-stimulated Ca(2+)-ATPase activity but depressed the net rate of Ca2+ uptake. This latter effect appears to be due to G6P-stimulated Ca2+ release. When G6P was added to Ca(2+)-loaded vesicles, a small, transient release of Ca2+ was elicited. In addition, G6P lowered the threshold for Ca(2+)-induced Ca2+ release but depressed the net rates of both AgNO3- and caffeine-induced releases. It is possible that the accumulation of G6P during muscular activity may adversely affect muscle force production and contribute to the fatigue process via its action on the contractile apparatus and SR.
Assuntos
Glucose-6-Fosfato/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/fisiologia , Retículo Sarcoplasmático/efeitos dos fármacos , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Fluorescência , Masculino , Fadiga Muscular/fisiologia , Miosinas/metabolismo , NAD/metabolismo , Oxalatos/farmacologia , Ratos , Ratos Sprague-Dawley , Nitrato de Prata/farmacologia , Tetracaína/farmacologiaRESUMO
Numerous studies suggest that the accumulation of lactate during exercise contributes to the fatigue process. This notion is based on close negative correlations between force and intracellular muscle lactate concentrations during fatigue and recovery. In this investigation, we attempted to determine if lactate directly affects muscle force output. This was accomplished by incubating mouse extensor digitorum longus muscles in extracellular concentrations of 10, 20, 30 and 50 mM L-(+)-lactate at 21 and 37 degrees C and monitoring force output. At 21 degrees C, 30 and 50 mM, extracellular lactate significantly reduced tetanic force (Po 250 ms, 100 Hz) to 95 and 93% of initial, respectively. In addition, the rate of force development (+dP/dt) was reduced to 93 and 89% of initial. At 37 degrees C, the effects of extracellular lactate were augmented as Po was reduced to 73 and 62% of initial and +dP/dt was reduced to 55 and 44% of initial at 30 and 50 mM, respectively. We next sought to determine if the reduction in Po was due to altered sarcoplasmic reticulum (SR) function using a muscle homogenate fraction. The rate of AgNO3-induced SR Ca2+ release was depressed by 31% in the presence of 25 mM lactate. These results suggest that elevated lactate depresses force production by whole muscle and may play some role in the fatigue process. In addition, it appears that lactate depresses force production, in part, by inhibiting Ca2+ release from the SR.