RESUMEN
The reduced release of Ca2+ from sarcoplasmic reticulum (SR) is considered a major determinant of muscle fatigue. In the present study, we investigated whether the presence of dantrolene, an established inhibitor of SR Ca2+ release, or caffeine, a drug facilitating SR Ca2+ release, modifies muscle fatigue development. Accordingly, the effects of Ca2+ release modulators were analyzed in vitro in mouse fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles, fatigued by repeated short tetani (40 Hz for 300 ms, 0.5 s(-1) in soleus and 60 Hz for 300 ms, 0.3 s(-1) in EDL, for 6 min). Caffeine produced a substantial increase of tetanic tension of both EDL and soleus muscles, whereas dantrolene decreased tetanic tension only in EDL muscle. In both EDL and soleus muscles, 5 microM dantrolene did not affect fatigue development, whereas 20 microM dantrolene produced a positive staircase during the first 3 min of stimulation in EDL muscle and a slowing of fatigue development in soleus muscle. The development of the positive staircase was abolished by the addition of 15 microM ML-7, a selective inhibitor of myosin light chain kinase. On the other hand, caffeine caused a larger and faster loss of tension in both EDL and soleus muscles. The results seem to indicate that the changes in fatigue profile induced by caffeine or dantrolene are mainly due to the changes in the initial tetanic tension caused by the drugs, with the resulting changes in the level of contraction-dependent factors of fatigue, rather than to changes in the SR Ca2+ release during fatigue development.
Asunto(s)
Calcio/metabolismo , Dantroleno/farmacología , Fatiga Muscular/fisiología , Relajantes Musculares Centrales/farmacología , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Animales , Cafeína/farmacología , Miosinas Cardíacas/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , Electroforesis en Gel Bidimensional , Ratones , Contracción Muscular/fisiología , Músculo Esquelético/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Retículo Sarcoplasmático/metabolismoRESUMEN
We have examined the influence of innervation on the expression of different isoforms of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in regenerating rat slow twitch muscle. The process of degeneration/regeneration was induced by injection of bupivacaine into rat soleus muscle under four different conditions: (1) in the presence of intact motor nerves, (2) after surgical denervation, (3) with nerve impulse conduction blocked by the Na(+)-channel blocker tetrodotoxin (TTX), and (4) with the axoplasmic flow blocked by vinblastine. Expression of SERCA isoforms was visualized by immunohistochemical and Western blot analysis. In regenerating innervated muscle, SERCA1, the isoform normally expressed in fast twitch fibres, was present after 5 days and was then progressively replaced by SERCA2, the isoform typical of slow twitch fibres. The maximum Ca(2+) uptake, measured in single skinned fibres regenerating for 10-21 days, was similar to that of slow adult fibres and significantly lower than that of fast adult fibres. Denervation or TTX treatment prevented the expression of the SERCA2 isoform. Conversely, vinblastine did not affect the expression of SERCA isoforms. These data indicate that nerve impulses play a determinant role in the expression of the SERCA2 isoform.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Neuronas Motoras/fisiología , Músculo Esquelético/inervación , Músculo Esquelético/fisiología , Regeneración/fisiología , Anestésicos Locales/farmacología , Animales , Antineoplásicos Fitogénicos/farmacología , Transporte Axonal/efectos de los fármacos , Calcio/metabolismo , Desnervación Muscular , Fibras Musculares de Contracción Lenta/enzimología , Músculo Esquelético/citología , Ratas , Ratas Wistar , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Nervio Ciático/efectos de los fármacos , Nervio Ciático/fisiología , Tetrodotoxina/farmacología , Vinblastina/farmacologíaRESUMEN
Bupivacaine-induced regeneration was studied in rat soleus muscle under several conditions, with the focus on type 2A and type 1 myosin heavy chain (MHC) isoform expression. In denervated muscles, type 1 was absent, whereas type 2A was widely expressed, a pattern of regeneration which appeared to be independent of fibrillation activity of the muscle. Both type 1 and type 2A isoforms were absent in muscles regenerated during tetrodotoxin (TTX) block of impulse conduction in the sciatic nerve, but type 2A was still present when the TTX block was associated with the vinblastine block of axoplasmic flow; vinblastine block alone caused the coexpression of type 1 and type 2A isoforms in the majority of fibers. These results suggest that axoplasmic flow carries some chemical factor that inhibits 2A MHC isoform expression. The results are also of clinical interest, contributing to the understanding of factors controlling muscle differentiation and adaptation.
Asunto(s)
Fibras Musculares de Contracción Lenta/metabolismo , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Anestésicos Locales/farmacología , Animales , Antineoplásicos Fitogénicos/farmacología , Transporte Axonal/efectos de los fármacos , Bupivacaína/farmacología , Electroforesis en Gel de Poliacrilamida , Fasciculación/tratamiento farmacológico , Femenino , Inmunohistoquímica , Masculino , Desnervación Muscular , Fibras Musculares de Contracción Lenta/efectos de los fármacos , Fibras Musculares de Contracción Lenta/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Procainamida/farmacología , Isoformas de Proteínas , Ratas , Ratas Wistar , Regeneración/efectos de los fármacos , Regeneración/fisiología , Nervio Ciático/efectos de los fármacos , Nervio Ciático/fisiología , Nervio Ciático/ultraestructura , Tetrodotoxina/farmacología , Vinblastina/farmacologíaRESUMEN
Callipeltin A, a cyclic depsipeptide from the New Caledonian Lithistida sponge Callipelta sp., is a macrocyclic lactone containing four amino acids in the L configuration, Ala, Leu, Thr (2 residues); one (Arg) in the D configuration; two N-methyl amino acids, N-MeAla and N-MeGln; a methoxy tyrosine, a 3, 4-dimethyl-l-glutamine; and a 4-amino-7-guanidino-2,3 dihydroxypentanoic acid (AGDHE), formally derived from L-Arg. In cardiac sarcolemmal vesicles Callipeltin A induces a powerful (IC(50) = 0.85 microM) and selective inhibition of the Na(+)/Ca(2+) exchanger. In electrically driven guinea-pig atria, at concentrations ranging between 0.7 and 2.5 microM, Callipeltin A induces a positive inotropic effect, which at the highest concentrations is accompanied by a rise in resting tension. It is suggested that the positive inotropic effect is linked to the inhibition of the Na(+)/Ca(2+) exchanger and that Callipeltin A may be an useful tool to study the role of the cardiac Na(+)/Ca(2+) exchanger in physiological and pathological conditions.
Asunto(s)
Depsipéptidos , Miocardio/metabolismo , Péptidos Cíclicos/farmacología , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/efectos de los fármacos , Animales , ATPasas Transportadoras de Calcio/efectos de los fármacos , Bovinos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Cobayas , Contracción Miocárdica/efectos de los fármacos , Péptidos Cíclicos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacosRESUMEN
The benzimidazole molecule was modified to synthesize a Ca(2+) sensitizer devoid of additional effects associated with Ca(2+) overload. Newly synthesized compounds, termed 1, 2, 3, 4, and 5, were evaluated in spontaneously beating and electrically driven atria from reserpine-treated guinea pigs. Compound 3 resulted as the most effective positive inotropic agent, and experiments were performed to study its mechanism of action. In spontaneously beating atria, the inotropic effect of 3 was concentration-dependent (3.0 microM-0.3 mM). Compound 3 was more potent and more active than the structurally related Ca(2+) sensitizers sulmazole and caffeine, but unlike them it did not increase the heart rate. In electrically driven atria, the inotropic activity of 3 was well preserved and it was not inhibited by propranolol, prazosin, ranitidine, pyrilamine, carbachol, adenosine deaminase, or ruthenium red. At high concentrations (0.1-1.0 mM) 3 inhibited phosphodiesterase-III, whereas it did not affect Na(+)/K(+)-ATPase, sarcolemmal Ca(2+)-ATPase, Na(+)/Ca(2+) exchange carrier, or sarcoplasmic reticulum Ca(2+) pump activities of guinea pig heart. In skinned fibers obtained from guinea pig papillary muscle and skeletal soleus muscle, compound 3 (0.1 mM, 1 mM) shifted the pCa/tension relation curve to the left, with no effect on maximal tension and no signs of toxicity. Compound 3 did not influence the basal or raised tone of guinea pig isolated aorta rings, whose cells do not contain the contractile protein troponin. The present results indicate that the inotropic effect of compound 3 seems to be primarily sustained by sensitization of the contractile proteins to Ca(2+).
Asunto(s)
Bencimidazoles/farmacología , Calcio/metabolismo , Animales , Cafeína/farmacología , Relación Dosis-Respuesta a Droga , Cobayas , Imidazoles/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacosRESUMEN
Force decline during fatigue in skeletal muscle is attributed mainly to progressive alterations of the intracellular milieu. Metabolite changes and the decline in free myoplasmic calcium influence the activation and contractile processes. This study was aimed at evaluating whether fatigue also causes persistent modifications of key myofibrillar and sarcoplasmic reticulum (SR) proteins that contribute to tension reduction. The presence of such modifications was investigated in chemically skinned fibers, a procedure that replaces the fatigued cytoplasm from the muscle fiber with a normal medium. Myofibrillar Ca(2+) sensitivity was reduced in slow-twitch muscle (for example, the pCa value corresponding to 50% of maximum tension was 6.23 +/- 0.03 vs. 5.99 + 0.05, P < 0.01, in rested and fatigued fibers) and not modified in fast-twitch muscle. Phosphorylation of the regulatory myosin light chain isoform increased in fast-twitch muscle. The rate of SR Ca(2+) uptake was increased in slow-twitch muscle fibers (14.2 +/- 1.0 vs. 19.6 +/- 2. 5 nmol. min(-1). mg fiber protein(-1), P < 0.05) and not altered in fast-twitch fibers. No persistent modifications of SR Ca(2+) release properties were found. These results indicate that persistent modifications of myofibrillar and SR properties contribute to fatigue-induced muscle force decline only in slow fibers. These alterations may be either enhanced or counteracted, in vivo, by the metabolic changes that normally occur during fatigue development.
Asunto(s)
Fatiga Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Miofibrillas/fisiología , Retículo Sarcoplasmático/fisiología , Animales , Calcio/metabolismo , Técnicas In Vitro , Masculino , Contracción Muscular/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Cadenas Ligeras de Miosina/metabolismo , Concentración Osmolar , Consumo de Oxígeno/fisiología , Fosforilación , Ratas , Ratas WistarRESUMEN
In this review, potential roles for the endogenous sphingolipid, sphingosine, and its derivatives are described for muscle cells. Sphingosine modulates the function of important calcium channels in muscle, including the ryanodine receptor (RyR) calcium release channel of the sarcoplasmic reticulum (SR). Sphingosine blocks calcium release through the SR ryanodine receptor and reduces the activity of single skeletal muscle RyR channels reconstituted into planar lipid bilayers. Sphingosine-blocked calcium release is coincident with the inhibitory effects of sphingosine on [3H]ryanodine binding to the RyR. The sphingomyelin signal transduction pathway has also been identified in both skeletal and cardiac muscle. A neutral form of sphingomyelinase (nSMase) enzyme has been localized to the junctional transverse tubule membrane. The high turnover of the SMase is responsible for the production of ceramide and sphingosine. HPLC analyses indicate that significant resting levels of sphingosine are present in muscle tissue. A model of excitation-contraction coupling is presented suggesting a potential role for this endogenous sphingolipid in normal muscle function. Putative roles for sphingolipid mediators in skeletal muscle dysfunction are also discussed. We hypothesize that sphingosine plays important roles in malignant hyperthermia and during the development of muscle fatigue.
Asunto(s)
Lisofosfolípidos , Músculo Esquelético/fisiología , Esfingolípidos/fisiología , Esfingosina/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/fisiología , Ceramidas/fisiología , Activación del Canal Iónico/fisiología , Transporte Iónico/fisiología , Hipertermia Maligna/fisiopatología , Modelos Biológicos , Contracción Muscular/fisiología , Fatiga Muscular/fisiología , Miocardio/metabolismo , Ratas , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Sistemas de Mensajero Secundario/fisiología , Esfingomielina Fosfodiesterasa/fisiología , Esfingosina/análogos & derivados , PorcinosRESUMEN
The degeneration-regeneration process was induced by bupivacaine injection in innervated, denervated, and nerve-blocked rat soleus muscles. Nerve block was obtained by superfusion of the sciatic nerve with tetrodotoxin (TTX). Two weeks after bupivacaine injection, immunohistochemical and electrophoretical analyses showed the presence of type 1 myosin heavy chain (MHC) only in innervated regenerated muscles, type 2A in innervated and denervated, but not in TTX-paralyzed muscles, and type 2X under all experimental conditions. The presence of type 1 MHC in the innervated, and its absence in both denervated and TTX-paralyzed muscles were also verified immunohistochemically 1 week after bupivacaine injection. It is concluded that the nerve impulses play a determinant role in the expression of 1 and 2A MHC isoforms in the innervated regenerating muscle. The possible causes of the absence of the type 2A MHC isoform in the TTX-paralyzed muscles are discussed.
Asunto(s)
Fibras Musculares de Contracción Lenta/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Bloqueo Nervioso , Regeneración , Animales , Electroforesis en Gel de Poliacrilamida , Histocitoquímica , Immunoblotting , Inmunohistoquímica , Isomerismo , Desnervación Muscular , Fibras Musculares de Contracción Lenta/química , Fibras Musculares de Contracción Lenta/fisiología , Ratas , Ratas Wistar , Regeneración/efectos de los fármacosRESUMEN
The Ca2+ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca2+ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca2+ capacity and the rate of Ca2+ uptake decreased from the control values of 0.384 +/- 0.030 micromol (mg fibre protein)-1 and 19.8 +/- 1.9 nmol min-1 (mg fibre protein)-1, respectively, to 0.210 +/- 0.016 micromol (mg fibre protein)-1 and 13.5 +/- 0.9 nmol min-1 (mg fibre protein)-1; the calculated amount of Ca2+ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 micromol (mg fibre protein)-1. In the 7-day denervated muscle, the SR Ca2+ capacity and the rate of Ca2+ uptake increased to 0.517 +/- 0.06 micromol (mg fibre protein)-1 and 21.6 +/- 2.3 nmol min-1 (mg fibre protein)-1, respectively; the calculated amount of Ca2+ released increased to 0.217 micromol (mg fibre protein)-1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca2+ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation.
Asunto(s)
Desnervación Muscular , Fibras Musculares de Contracción Lenta/fisiología , Retículo Sarcoplasmático/fisiología , Animales , Calcio/metabolismo , Electrofisiología , Pie , Histocitoquímica , Contracción Muscular/fisiología , Músculo Esquelético/inervación , Ratas , Ratas Wistar , Retículo Sarcoplasmático/metabolismoRESUMEN
Calcium release activity of sarcoplasmic reticulum and enzyme-histochemical properties were investigated in extensor digitorum longus (e.d.l.) and soleus muscles in young (4 months and old (24 months) male rats. With age, the caffeine threshold concentration for calcium release from the sarcoplasmic reticulum of soleus skinned muscle fibres showed only minor modifications. On the other hand, in e.d.l. skinned muscle fibres, the caffeine threshold concentration decreased significantly (P < 0.05). The histochemical fibre type composition changed with age both in soleus and in e.d.l. muscles, showing a common transformation toward a more oxidative histochemical profile. In fact, in aged soleus, a significant (P < 0.05) increase was observed of type 1 fibres to represent almost the totality of the muscle fibres (more than 98%), while types 2C and 2A were reduced in proportion. In aged e.d.l. the percentage of type 1 (P < 0.05), 2A and 2X (a recently identified fourth component of the fast-twitch muscle types) fibres increased, with a reduction of type 2B (P < 0.01) fibres. The present results suggest that the changes in contractile properties of aged muscles may be related to the changes not only in fibre composition but also in the mechanism of calcium release from sarcoplasmic reticulum.
Asunto(s)
Envejecimiento/fisiología , Calcio/metabolismo , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Retículo Sarcoplasmático/fisiología , Animales , Peso Corporal , Cafeína/farmacología , Histocitoquímica , Masculino , Fibras Musculares de Contracción Rápida/química , Fibras Musculares de Contracción Rápida/efectos de los fármacos , Fibras Musculares de Contracción Lenta/química , Fibras Musculares de Contracción Lenta/efectos de los fármacos , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Ratas , Ratas Wistar , Retículo Sarcoplasmático/efectos de los fármacosRESUMEN
The myofibrillar changes of rat denervated soleus muscle were studied in the presence and in the absence of an antifibrillatory drug. After bilateral sciaticotomy, a concentrated solution of procainamide hydrochloride was steadily released, by way of a miniosmotic pump, in the space between the soleus and the gastrocnemius muscles of one leg. Fibrillation activity of soleus muscles was checked electromyografically at 3- to 5-day intervals. On the 21st day following denervation the muscles were excised, stained for adenosine triphosphatase activity and analysed for myosin heavy chain (MHC) isoforms. In the denervated-procainamide-treated muscles fibrillation was consistently (-75% on average) depressed in comparison to the contralateral denervated muscles. Type 1 (slow) fibres and MHC isoform were also significantly reduced, to the advantage of type 2A (fast) fibres and MHC isoform. The results support the view that denervation inactivity, like other kinds of muscle inactivity, favours the expression of fast type myofibrillar isoforms, and that this effect is counteracted, at least partially, by the spontaneous activity of the denervated muscle.
Asunto(s)
Antiarrítmicos/farmacología , Contracción Muscular/fisiología , Animales , Desnervación , Contracción Muscular/efectos de los fármacos , Miosinas/fisiología , Ratas , Ratas EndogámicasRESUMEN
We investigated the calcium sensitivity for tension generation of different fibre types and the possible correlation between calcium sensitivity and the presence of distinct regulatory protein and myosin light chain (MLC) isoforms in rat skinned skeletal muscle fibres. Fibre types 1, 2A and 2B were identified by electrophoretic analysis of myosin heavy chain (MHC) isoforms. Fibres showing more than one MHC isoform were discarded. Type 1 fibres from the soleus showed a higher pCa (-log10 [Ca], where [ ] denotes concentration) threshold and a lower slope of pCa/tension curve than type 2 extensor digitorum longus (EDL) fibres; between type 2 fibres, type 2B showed the higher slope of pCa/tension curve. Type 1 fibres from different muscles showed similar calcium sensitivities when containing only the slow set of regulatory proteins and MLC; when both slow and fast isoforms were present, calcium sensitivity shifted toward fast type fibre values. Type 2A fibres from different muscles showed a similar calcium sensitivity, independently of the set (purely fast or mixed) of regulatory proteins and MLC. It is suggested that when both fast and slow isoforms of regulatory proteins and of MLC are present in a muscle fibre, calcium sensitivity is dictated mainly by the fast isoforms.
Asunto(s)
Calcio/farmacología , Proteínas Musculares/metabolismo , Músculos/fisiología , Miofibrillas/fisiología , Animales , Electroforesis en Gel de Poliacrilamida , Contracción Isométrica/efectos de los fármacos , Proteínas Musculares/análisis , Músculos/química , Músculos/efectos de los fármacos , Miosinas/análisis , Miosinas/metabolismo , RatasRESUMEN
Cordotomy-denervation interactions were studied on contractile and myofibrillar properties of slow (soleus) and fast (extensor digitorum longus) muscles of the rat. The spinal cord was transected midthoracically in neonatal (2-day-old) animals. Two months after birth, a unilateral transection of the sciatic nerve was carried out in both cordotomized and control animals. Five weeks after denervation, contractile properties were tested isometrically in vitro; myofibrillar properties were assessed by histochemical staining of the muscle fibers and by electrophoretic analysis of the myosin heavy chain composition. The following results were obtained: (i) In cordotomized animals the contraction time of the soleus was significantly shorter (-23.3% on average) than that in the control animals and this shortening was accompanied by a proportional slow-to-fast shift in myofibrillar properties. (ii) The extensor digitorum longus properties were not significantly different in the control and cordotomized animals. (iii) Denervation in control animals was followed by a marked increase of contraction and half-relaxation times in the extensor digitorum longus, whereas in the soleus only the half-relaxation time was significantly increased; myofibrillar properties in the soleus showed an appreciable slow-to-fast shift, whereas in the fast muscle the main change was an increase in type 2A fibers to the detriment of type 2B. (iv) In cordotomized animals, denervation caused the soleus contraction time to increase to control values, whereas myofibrillar properties shifted to an even faster pattern; in the extensor digitorum longus denervation caused the same changes seen in the control animals. The results showed that cordotomy at birth caused the soleus to develop as a faster muscle than in the control animals. The concurrent effects of cordotomy and denervation on the myofibrillar properties of the soleus suggest that the slow-to-fast change in these properties is a common consequence of the reduction in the level of motor activity. The opposite effects of the two experimental conditions in the soleus contraction time support the view that the contractile alterations that follow denervation mainly reflect alterations in the muscle activation process.
Asunto(s)
Contracción Muscular , Desnervación Muscular , Miofibrillas/fisiología , Médula Espinal/fisiología , Animales , Miembro Anterior/fisiología , Miembro Posterior/fisiología , Músculos/fisiología , Ratas , Médula Espinal/cirugíaRESUMEN
Electrophoretic analysis in the presence of 33% glycerol of purified myosin from normal human muscle shows three distinct protein bands which are identified as type 1, 2B, and 2A myosin heavy chain (MHC) isoforms by affinity-purified polyclonal antibodies. Analysis of MHC of single human muscle fibres shows that human muscles contain a large population of fibres showing the coexistence of type 2A and 2B MHC.
Asunto(s)
Músculos/análisis , Miosinas/análisis , Adulto , Electroforesis en Gel de Poliacrilamida , Humanos , InmunoelectroforesisRESUMEN
Mammalian skeletal muscles are mixture of three type of fibers: type 1, type 2A, and type 2B fibers. Immunological studies and proteolytic analysis of myosin heavy chains from the three type of fibers have demonstrated the presence of distinct myosin isoforms. By using typed single muscle fibers and improving an electrophoretic method we are able to resolve three distinct polypeptides which are demonstrate to correspond to type 1, 2A and 2B myosin heavy chain isoforms by using specific monoclonal antibodies. The analysis of single muscle fibers shows that different myosin heavy chain isoforms are frequently coexpressed in the same muscle fiber.
Asunto(s)
Músculos/química , Miosinas/análisis , Fragmentos de Péptidos/análisis , Adenosina Trifosfatasas/análisis , Animales , Electroforesis en Gel de Poliacrilamida , Masculino , Músculos/enzimología , Miofibrillas/enzimología , Subfragmentos de Miosina , Especificidad de Órganos , Ratas , Ratas EndogámicasRESUMEN
We evaluated the isoform composition of heavy and light chains of myosin in single muscle fibers from patients with Duchenne dystrophy, myotonic dystrophy, or polymyositis. In all myopathic muscles, there was an increase in the proportion of intermediate fibers which, by analysis of myosin isoforms, fell into two subpopulations, one that contained both fast and slow myosin and another that contained myosin molecular hybrids. The increased proportion of intermediate (or transitional) fibers suggests changes in the equilibrium between fast and slow motor units. These changes could result from regeneration and subsequent maturation of fibers or from direct transformation of mature fibers of one type into the opposite.
Asunto(s)
Distrofias Musculares/patología , Miosinas/análisis , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miositis/patología , Miotonía/patologíaRESUMEN
Experimental myotonia was induced by feeding rats with 20,25-diazacholesterol for up to 8 months. Histochemical analysis of myotonic extensor digitorum longus (EDL) muscle showed a progressive decrease of type IIB fibres and a concomitant increase of type IIA and type I fibres. A transient hypertrophy of type IIA fibres was observed 6 months after beginning the treatment. Analysis of the pattern of myosin light chains of single fibres from EDL showed that myotonia caused a progressive decrease of fibres showing a pure fast myosin light chain pattern and an increase of fibres showing coexistence of fast and slow myosin light chains (intermediate fibres). Only a small percentage of intermediate fibres showed coexistence of fast and slow myosin heavy chains. Myotonic fibres presented an increased sensitivity to caffeine which approached that of normal soleus fibres. Furthermore, sarcoplasmic reticulum (SR) vesicles isolated from hind limb fast muscles of myotonic rats demonstrated a decrease of Ca2+-dependent ATPase and Ca2+-transport activities as well as a decrease of immunoreactivity with anti-rabbit SR fast Ca2+-ATPase antibody. These results suggest that the increased electrical activity brought about by 20,25-diazacholesterol-induced myotonia, caused a fast to slow transition in the phenotypic expression of myosin and sarcoplasmic reticulum proteins.
Asunto(s)
Músculos/metabolismo , Miotonía/inducido químicamente , Adenosina Trifosfatasas/análisis , Animales , Azacosterol , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Histocitoquímica , Masculino , Músculos/efectos de los fármacos , Miofibrillas/patología , Miosinas/análisis , Miotonía/patología , Ratas , Ratas Endogámicas , Retículo Sarcoplasmático/metabolismoRESUMEN
During several months of denervation, rat mixed muscles lose slow myosin, though with variability among animals. Immunocytochemical studies showed that all the denervated fibers of the hemidiaphragm reacted with anti-fast myosin, while many reacted with anti-slow myosin as well. This has left open the question as to whether multiple forms of myosin co-exist within individual fibers or a unique, possibly embryonic, myosin is present, which shares epitopes with fast and slow myosins. Furthermore, one can ask if the reappearance of embryonic myosin in chronically denervated muscle is related both to its re-expression in the pre-existing fibers and to cell regeneration. To answer these questions we studied the myosin heavy chains from individual fibers of the denervated hemidiaphragm by SDS PAGE and morphologically searched for regenerative events in the long term denervated muscle. 3 mo after denervation the severely atrophic fibers of the hemidiaphragm showed either fast or a mixture of fast and slow myosin heavy chains. Structural analysis of proteins sequentially extracted from muscle cryostat sections showed that slow myosin was still present 16 mo after denervation, in spite of the loss of the selective distribution of fast and slow features. Therefore muscle fibers can express adult fast myosin not only when denervated during their differentiation but also after the slow program has been expressed for a long time. Light and electron microscopy showed that the long-term denervated muscle maintained a steady-state atrophy for the rat's life span. Some of the morphological features indicate that aneural regeneration events continuously occur and significantly contribute to the increasing uniformity of the myosin gene expression in long-term denervated diaphragm.
Asunto(s)
Desnervación Muscular , Músculo Liso/fisiología , Miosinas/análisis , Animales , Células Cultivadas , Diafragma/fisiología , Diafragma/ultraestructura , Electroforesis en Gel de Poliacrilamida , Masculino , Microscopía Electrónica , Músculo Liso/ultraestructura , Ratas , Ratas EndogámicasRESUMEN
In this study the polymorphism of myofibrillar proteins and the Ca2+-uptake activity of sarcoplasmic reticulum were analysed in single fibres from human skeletal muscles. Two populations of histochemically identified type-I fibres were found differing in the number of light-chain isoforms of the constituent myosin, whereas the pattern of light chains of fast myosin of type-IIA and type-IIB fibres was indistinguishable. Regulatory proteins, troponin and tropomyosin, and other myofibrillar proteins, such as M- and C-proteins, showed specific isoforms in type-I and type-II fibres. Furthermore, tropomyosin presented different stoichiometries of the alpha- and beta-subunits between the two types of fibres. Sarcoplasmic-reticulum volume, as indicated by the maximum capacity for calcium oxalate accumulation, was almost identical in type-I and type-II fibres, whereas the rate of Ca2+ transport was twice as high in type-II as compared with type-I fibres. It is concluded that, in normal human muscle fibres, there is a tight segregation of fast and slow isoforms of myofibrillar proteins that is very well co-ordinated with the relaxing activity of the sarcoplasmic reticulum. These findings may thus represent a molecular correlation with the differences of the twitch-contraction time between fast and slow human motor units. This tight segregation is partially lost in the muscle fibres of elderly individuals.
Asunto(s)
Calcio/metabolismo , Proteínas Musculares/metabolismo , Músculos/metabolismo , Miofibrillas/metabolismo , Retículo Sarcoplasmático/metabolismo , Factores de Edad , Transporte Biológico , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Proteínas Musculares/genética , Miosinas/metabolismo , Polimorfismo GenéticoRESUMEN
Rabbit predominantly fast-twitch-fibre and predominantly slow-twitch-fibre skeletal muscles of the hind limbs, the psoas, the diaphragm and the masseter muscles were fibre-typed by one-dimensional polyacrylamide-gel electrophoresis of the myofibrillar proteins of chemically skinned single fibres. Investigation of the distribution of fast-twitch-fibre and slow-twitch-fibre isoforms of myosin light chains and the type of myosin heavy chains, based on peptide 'maps' published in Cleveland. Fischer, Kirschner & Laemmli [(1977) J. Biol. Chem. 252, 1102-1106], allowed a classification of muscle fibres into four classes, corresponding to histochemical types I, IIA, IIB and IIC. Type I fibres with a pure slow-twitch-type of myosin were found to be characterized by a unique set of isoforms of troponins I, C and T, in agreement with the immunological data of Dhoot & Perry [(1979) Nature (London) 278, 714-718], by predominance of the beta-tropomyosin subunit and by the presence of a small amount of an additional tropomyosin subunit, apparently dissimilar from fast-twitch-fibre alpha-tropomyosin subunit. The myofibrillar composition of type IIB fast-twitch white fibres was the mirror image of that found for slow-twitch fibres in that the fast-twitch-fibre isoforms only of the troponin subunits were present and the alpha-tropomyosin subunit predominated. Type IIA fast-twitch red fibres showed a troponin subunit composition identical with that of type IIB fast-twitch white fibres. On the other hand, a unique type of myosin heavy chains was found to be associated with type IIA fibres. Furthermore, the myosin light-chain composition of these fibres was invariably characterized by a small amount of LC3F light chain and by a pattern that was either a pure fast-twitch-fibre light-chain pattern or a hybrid LC1F/LC2F/LC3F/LC1Sb light-chain pattern. By these criteria type IIA fibres could be distinguished from type IIC intermediate fibres, which showed coexistence of fast-twitch-fibre and slow-twitch-fibre forms of myosin light chains and of troponin subunits.