RESUMEN
INTRODUCTION: α1-syntrophin, a member of the dystrophin complex, recruits membrane molecules, including aquaporin-4, at the sarcolemma. The physiological functions of α1-syntrophin are poorly understood. METHODS: We examined the physiological characteristics of α1-syntrophin-deficient muscles under osmotic stress conditions to test the possibility that mutant muscles are less tolerant of osmotic shock. RESULTS: Isolated muscle bundles from mutant mice showed markedly reduced force production after hypo-osmotic shock. In addition, the mutant muscle bundles showed delayed recovery of specific gravity after being exposed to hypo-osmotic conditions. Two consecutive exercise tests on the treadmill revealed their performance in the second test was significantly lower than for wild-type mice. Furthermore, mutant mice had higher serum lactate concentrations after treadmill exercise. CONCLUSIONS: Although the lack of α1-syntrophin from the sarcolemma does not lead to muscle degeneration, our results suggest that it may be partly involved in the pathophysiology of dystrophin-deficient Duchenne muscular dystrophy.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Proteínas de la Membrana/fisiología , Fatiga Muscular/genética , Fibras Musculares Esqueléticas/fisiología , Proteínas Musculares/fisiología , Fuerza Muscular/fisiología , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Presión Osmótica/fisiología , Animales , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Modelos Animales de Enfermedad , Técnicas In Vitro , Ácido Láctico/sangre , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Fatiga Muscular/fisiología , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Fuerza Muscular/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genéticaRESUMEN
Skeletal-type neuroendocrine-specific protein like 1 (sk-NSPL1) has been demonstrated to be physiologically important in regulating the membrane translocation of glucose transporter 4 (GLUT4) in skeletal muscles. We investigated the levels of phosphorylation in proteins that are thought to be involved in exercise in wild-type and sk-NSPL1-deficient muscles with specific antibodies and phosphate-metal affinity chromatography resin (p-resin). In both normal skeletal muscle and sk-NSPL1-deficient muscle, adenosine monophosphate (AMP)-dependent kinase (AMPK) and acetyl-CoA carboxylase (ACC) were phosphorylated and adsorbed onto p-resin at high levels after exercise. On the other hand, the effect of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), which is an activator of AMPK, in blood glucose was greatly diminished in mutant mice. P-resin adsorbed sk-NSPL1 in the membrane fraction from wild-type muscle after exercise and AICAR administration. Isolated sk-NSPL1 from wild-type also had increased adsorption onto p-resin after treatment with Ca(2+) and adenosine triphosphate (ATP). After long-term incubation of sk-NSPL1-containing membrane without ATP, sk-NSPL1 adsorption onto anion-exchange resin was drastically reduced. These results suggest that the function of sk-NSPL1 is regulated by a [Ca(2+)](i)- and AMPK-mediated pathway under exercise, and support the hypothesis that sk-NSPL1 is an important factor in the downstream of the exercise-dependent pathway in GLUT4 translocation.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiología , Proteínas del Tejido Nervioso/metabolismo , Condicionamiento Físico Animal/fisiología , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Glucemia/metabolismo , Western Blotting , Cromatografía por Intercambio Iónico , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/enzimología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ribonucleótidos/farmacologíaRESUMEN
OBJECTIVE: In skeletal muscles, dantrolene inhibits the exercise-induced membrane translocation of GLUT4. It has been postulated that the inhibitory action of dantrolene on Ca(2+) release from the sarcoplasmic reticulum (SR) causes inhibition of exercise-induced glucose uptake; however, the precise mechanism has not been adequately studied. RESEARCH DESIGN AND METHODS: We discovered that dantrolene can bind to skeletal-type neuroendocrine-specific protein-like 1 (sk-NSPl1) with photoreactive dantrolene derivatives. In sk-NSPl1-deficient muscles, we examined the change in glucose uptake and the membrane translocation of GLUT4. In addition, we examined the change in blood glucose and also measured the glycogen level in both isolated and in situ skeletal muscles after electrical stimulation using our mutant mouse. RESULTS: In sk-NSPl1-deficient muscles, exercise-induced glucose uptake was totally abolished with no change in insulin-induced glucose uptake. The Ca(2+) release mechanism and its inhibition by dantrolene were completely preserved in these muscles. The expression of GLUT4 in the mutant muscles also appeared unchanged. Confocal imaging and results using the membrane isolation method showed that exercise/contraction did not enhance GLUT4 translocation in these sk-NSPl1-deficient muscles under conditions of adequate muscle glycogen consumption. The blood glucose level in normal mice was reduced by electrical stimulation of the hind limbs, but that in mutant mice was unchanged. CONCLUSIONS: sk-NSPl1 is a novel dantrolene receptor that plays an important role in membrane translocation of GLUT4 induced by contraction/exercise. The 23-kDa sk-NSPl1 may also be involved in the regulation of glucose levels in the whole body.
Asunto(s)
Dantroleno/farmacología , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Musculares/fisiología , Relajantes Musculares Centrales/farmacología , Músculo Esquelético/metabolismo , Proteínas del Tejido Nervioso/fisiología , Receptores de Droga/fisiología , Animales , Glucemia/metabolismo , Calcio/metabolismo , Dantroleno/análogos & derivados , Electroforesis en Gel de Poliacrilamida , Fluorodesoxiglucosa F18 , Inmunoprecipitación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Confocal , Contracción Muscular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Condicionamiento Físico Animal , Transporte de Proteínas , Receptores de Droga/genética , Receptores de Droga/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Suzuki-Miyaura coupling of 3-azido-5-(azidomethyl)phenylboronic acid pinacol ester with various aryl bromides affords corresponding diazido-functionalized biaryl compounds in good yields. This approach provides an easy access to radioisotope-free photoaffinity probes possessing biaryl structure. By using this method, we prepared a novel diazido-functionalized dantrolene analog, which showed selective inhibitory effect on physiological Ca(2+) release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal muscle without affecting Ca(2+)-induced Ca(2+) release (CICR).
Asunto(s)
Azidas/química , Etiquetas de Fotoafinidad , Calcio/química , Espectroscopía de Resonancia Magnética , Radioisótopos , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Bifunctional dantrolene derivatives have been synthesized as probes for radioisotope-free photoaffinity labeling with the aim of elucidating the molecular mechanism of skeletal muscle contraction. GIF-0430 and GIF-0665 are aromatic azido-functionalized derivatives that were designed to selectively inhibit physiological Ca2+ release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal muscle without a strong effect on Ca2+-induced Ca2+ release (CICR). These photoaffinity probes consist of either an azidomethyl or an ethynyl group, respectively, which could function as a tag for introduction of an optional detectable marker unit by an appropriate chemoselective ligation method after the photo-cross-linking operation. Actually, the former probe worked to photolabel its target proteins specifically as confirmed by subsequent fluorescent visualization.
Asunto(s)
Calcio/metabolismo , Dantroleno/análogos & derivados , Dantroleno/síntesis química , Músculo Esquelético/metabolismo , Etiquetas de Fotoafinidad/síntesis química , Proteínas/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Dantroleno/química , Dantroleno/farmacología , Diseño de Fármacos , Técnicas In Vitro , Ratones , Estructura Molecular , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Etiquetas de Fotoafinidad/farmacología , Fotoquímica , Proteínas/efectos de los fármacos , Sensibilidad y EspecificidadRESUMEN
A novel method for radioisotope-free photoaffinity labeling was developed, in which a bifunctional ligand is connected to a target protein by activation of a photoreactive group, such as an aromatic azido or 3-trifluoromethyl-3H-diazirin-3-yl group, and identification of the ligated product is achieved by anchoring of a detectable tag through the Staudinger-Bertozzi reaction with an alkyl azido moiety that survives photolysis. The chemical ground of this method was confirmed using model compounds with the bifunctional group under photoirradiation in the presence of trapping agents for reactive intermediates. The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase.
Asunto(s)
Etiquetas de Fotoafinidad/química , Proteínas/química , Azidas/química , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/efectos de la radiación , Ligandos , Luz , Modelos Moleculares , Estructura Molecular , Fotólisis , Conformación Proteica , Proteínas/efectos de la radiación , Sensibilidad y EspecificidadRESUMEN
We analyzed the effect of HMG-CoA reductase inhibitors on Ca(2+) release from the sarcoplasmic reticulum (SR) using chemically skinned skeletal muscle fibers from the mouse and the rat. Cerivastatin (>20 microM) released Ca(2+) from the SR, while pravastatin showed only a little effect. The rates of Ca(2+) release were increased by cerivastatin at all Ca(2+) concentrations tested. Cerivastatin-induced Ca(2+) release in the presence of Ca(2+) was affected by adenosine monophosphate, Mg(2+), and procaine in essentially the same way as for caffeine-induced Ca(2+) release. The Ca(2+)-uptake capacity of the SR was reduced after co-treatment with ryanodine and cerivastatin at pCa 6.0 to a much greater extent than with ryanodine alone. Thus, cerivastatin-induced Ca(2+) release in the presence of Ca(2+) must be a result of the activation of the Ca(2+)-induced Ca(2+) release (CICR) mechanism of the ryanodine receptor. However, even when CICR was maximally inhibited by Mg(2+) and procaine, or in the practical absence of Ca(2+) (pCa >8), cerivastatin still caused Ca(2+) release. These results indicate that cerivastatin causes Ca(2+) release also by activating some other mechanism(s) in addition to the activation of CICR. Either or both of these effects might be related to its adverse effect, rhabdomyolysis.
Asunto(s)
Calcio/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Piridinas/farmacología , Retículo Sarcoplasmático/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos ICR , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Ratas , Ratas Endogámicas F344 , Retículo Sarcoplasmático/metabolismoRESUMEN
The general synthesis of dantrolene analogues with various substituents on its phenyl ring has been developed via palladium-catalyzed cross-coupling reactions, the Stille or Suzuki reaction, as the key step. The effects of synthesized analogues have been evaluated by two kinds of Ca(2+) release modes from sarcoplasmic reticulum (SR) of mouse skeletal muscle fibers based on: (1) the measurement of twitch contraction caused by the physiological Ca(2+) release (PCR) of intact skeletal muscle and (2) the rate of Ca(2+)-induced Ca(2+) release (CICR) in saponin-treated skinned muscle fibers. Although dantrolene, a lead compound, inhibits both twitch contraction and CICR, some structurally modified analogues exhibit one or the other of these effects. The methoxy congener, GIF-0185, potently inhibits the twitch contraction without affecting the CICR, while GIF-0166 and GIF-0248, the ortho-nitro regioisomer and ortho, ortho-dinitro substituted analogues, respectively, doubly potentiate the CICR exclusively.
Asunto(s)
Calcio/metabolismo , Dantroleno/análogos & derivados , Dantroleno/síntesis química , Relajantes Musculares Centrales/síntesis química , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Calcio/farmacología , Dantroleno/farmacología , Técnicas In Vitro , Indicadores y Reactivos , Ratones , Contracción Muscular/efectos de los fármacos , Relajantes Musculares Centrales/farmacología , Músculo Esquelético/efectos de los fármacos , Retículo Sarcoplasmático/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
In order to capture and identify key molecules that regulate the release of Ca(2+) from the sarcoplasmic reticulum (SR) of skeletal muscle, we designed specific photoaffinity probes based on the structural modification of dantrolene. Thus, GIF-0082 and GIF-0276 possessing azido- and trifluoromethyldiazirinyl-benzyl groups, respectively, at the hydantoin moiety were found to have a highly selective inhibitory effect on physiological Ca(2+) release (PCR) without affecting Ca(2+)-induced Ca(2+) release (CICR). Successful realization of the sharp discrimination between PCR and CICR has led to the creation of [(125)I]GIF-0082 and [(125)I]GIF-0276, which were synthesized by substituting a stannyl group with (125)I in the corresponding phenylstannane precursors.
Asunto(s)
Calcio/metabolismo , Dantroleno/análogos & derivados , Relajantes Musculares Centrales/farmacología , Etiquetas de Fotoafinidad/farmacología , Retículo Sarcoplasmático/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Dantroleno/síntesis química , Dantroleno/farmacología , Radioisótopos de Yodo , Ratones , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/fisiología , Músculo Esquelético/ultraestructura , Etiquetas de Fotoafinidad/síntesis químicaRESUMEN
Alpha1-syntrophin is a member of the family of dystrophin-associated proteins; it has been shown to recruit neuronal nitric oxide synthase and the water channel aquaporin-4 to the sarcolemma by its PSD-95/SAP-90, Discs-large, ZO-1 homologous domain. To examine the role of alpha1-syntrophin in muscle regeneration, we injected cardiotoxin into the tibialis anterior muscles of alpha1-syntrophin-null (alpha1syn-/-) mice. After the treatment, alpha1syn-/- muscles displayed remarkable hypertrophy and extensive fiber splitting compared with wild-type regenerating muscles, although the untreated muscles of the mutant mice showed no gross histological change. In the hypertrophied muscles of the mutant mice, the level of insulin-like growth factor-1 transcripts was highly elevated. Interestingly, in an early stage of the regeneration process, alpha1syn-/- mice showed remarkably deranged neuromuscular junctions (NMJs), accompanied by impaired ability to exercise. The contractile forces were reduced in alpha1syn-/- regenerating muscles. Our results suggest that the lack of alpha1-syntrophin might be responsible in part for the muscle hypertrophy, abnormal synapse formation at NMJs, and reduced force generation during regeneration of dystrophin-deficient muscle, all of which are typically observed in the early stages of Duchenne muscular dystrophy patients.
Asunto(s)
Proteínas de la Membrana/fisiología , Proteínas Musculares/fisiología , Músculo Esquelético/fisiología , Unión Neuromuscular/anatomía & histología , Regeneración , Animales , Acuaporina 4 , Acuaporinas/análisis , Proteínas de Unión al Calcio , Proteínas Cardiotóxicas de Elápidos/administración & dosificación , Proteínas Cardiotóxicas de Elápidos/toxicidad , Hipertrofia , Inyecciones Intramusculares , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Fibras Musculares de Contracción Rápida/efectos de los fármacos , Fibras Musculares de Contracción Rápida/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/anatomía & histología , Cadenas Pesadas de Miosina/metabolismo , Esfuerzo Físico/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Somatomedinas/análisis , Factores de TiempoRESUMEN
The adeno-associated virus vector is a good tool for gene transfer into skeletal muscle, but the length of a gene that can be incorporated is limited. To develop a gene therapy for Duchenne muscular dystrophy, we generated a series of rod-truncated micro-dystrophin cDNAs: M3 (one rod repeat, 3.9 kb), AX11 (three rod repeats, 4.4 kb), and CS1 (four rod repeats, 4.9 kb). These micro-dystrophins, driven by a CAG promoter, were used to produce transgenic (Tg) mdx mice and all three micro-dystrophins were shown to localize at the sarcolemma together with the expression of dystrophin-associated proteins. Among them, CS1 greatly improved dystrophic phenotypes of mdx mice and contractile force of the diaphragm in particular was restored to the level of normal C57BL/10 mice. AX11 modestly ameliorated the dystrophic pathology, but, importantly, M3-Tg mdx mice still showed severe dystrophic phenotypes. These data suggest that the rod structure, and its length in particular, is crucial for the function of micro-dystrophin.