RESUMEN
Duchenne muscular dystrophy (DMD) is an inherited, severe muscle wasting disease caused by the loss of the cytoskeletal protein, dystrophin. Patients usually die in their late teens or early twenties of cardiac or respiratory failure. We have previously demonstrated that the dystrophin related protein, utrophin is able to compensate for the loss of dystrophin in the mdx mouse, the mouse model of the disease. Expression of a utrophin transgene under the control of an HSA promoter results in localization of utrophin to the sarcolemma and prevents the muscle pathology. Here we show that the over-expression of full-length utrophin in a broad range of tissues is not detrimental in the mdx mouse. These findings have important implications for the feasibility of the up-regulation of utrophin in therapy for DMD since they suggest that tissue specific up-regulation may not be necessary.
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Proteínas de la Membrana/biosíntesis , Distrofia Muscular Animal/metabolismo , Envejecimiento , Animales , Western Blotting , Peso Corporal , Creatinina/orina , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Estudios de Factibilidad , Expresión Génica , Terapia Genética , Homocigoto , Inmunohistoquímica , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/terapia , Especificidad de Órganos , Regiones Promotoras Genéticas , Distribución Tisular/genética , Transgenes , Regulación hacia Arriba/genética , UtrofinaRESUMEN
BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy.
Asunto(s)
Proteínas del Citoesqueleto/genética , Distrofina/genética , Terapia Genética , Proteínas de la Membrana/genética , Músculo Esquelético/enzimología , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/enzimología , Óxido Nítrico Sintasa/metabolismo , Adenoviridae/genética , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , NADPH Deshidrogenasa/análisis , NADPH Deshidrogenasa/inmunología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Regiones Promotoras Genéticas , Factores de Tiempo , UtrofinaAsunto(s)
Anticoagulantes/farmacología , Benzamidas/farmacología , Derivados del Benceno/farmacología , Inhibidores del Factor Xa , Fibrinolíticos/farmacología , Animales , Anticoagulantes/química , Anticoagulantes/uso terapéutico , Benzamidas/química , Benzamidas/uso terapéutico , Derivados del Benceno/química , Derivados del Benceno/uso terapéutico , Modelos Animales de Enfermedad , Factor Xa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/uso terapéutico , Humanos , Piperidinas/química , Conejos , Relación Estructura-Actividad , Trombosis/prevención & controlRESUMEN
Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Dedos de Zinc/genética , Animales , Humanos , Ratones , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , UtrofinaRESUMEN
Many cell adhesion-dependent processes are regulated by tyrosine phosphorylation. In order to investigate the role of tyrosine phosphorylation of the utrophin-dystroglycan complex we treated suspended or adherent cultures of HeLa cells with peroxyvanadate and immunoprecipitated (beta)-dystroglycan and utrophin from cell extracts. Western blotting of (&bgr;)-dystroglycan and utrophin revealed adhesion- and peroxyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxy-terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and (beta)-dystroglycan by co-immunoprecipitation with endogenous (beta)-dystroglycan. In extracts from cells treated with peroxyvanadate, where endogenous (beta)-dystroglycan was tyrosine phosphorylated, (beta)-dystroglycan was no longer co-immunoprecipitated with utrophin fusion constructs. Peptide 'SPOTs' assays confirmed that tyrosine phosphorylation of (beta)-dystroglycan regulated the binding of utrophin. The phosphorylated tyrosine was identified as Y(892) in the (beta)-dystroglycan WW domain binding motif PPxY thus demonstrating the physiological regulation of the (beta)-dystroglycan/utrophin interaction by adhesion-dependent tyrosine phosphorylation.
Asunto(s)
Adhesión Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Tirosina/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Adhesión Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/efectos de los fármacos , Distroglicanos , Células HeLa , Humanos , Glicoproteínas de Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Fosforilación , Estructura Terciaria de Proteína/fisiología , Utrofina , Vanadatos/farmacologíaRESUMEN
Spinal muscular atrophy is an autosomal recessive neurodegenerative disease of childhood, resulting from deletion or mutation of the survival motor neuron ( SMN ) gene on chromosome 5q13. SMN exists as part of a 300 kDa multi-protein complex, incorporating several proteins critically required in pre-mRNA splicing. Although SMN mutations render SMN defective in this role, the specific alpha-motor neuron degenerative phenotype seen in the disease remains unexplained. Here we demonstrate the isolation from mouse brain of the murine homologue of a recently identified novel RNA helicase of the DEAD box family, DP103, and its direct and specific binding of SMN. Previous work has shown that DP103 binds viral proteins known to interact with a cellular transcription factor to modulate gene expression. We suggest that the interaction between SMN and DP103 is further evidence for a role for SMN in transcriptional regulation and that SMN may be involved in the regulation of neuron-specific genes essential in neuronal development.
Asunto(s)
Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , ARN Helicasas/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteína 20 DEAD-Box , ARN Helicasas DEAD-box , ADN Complementario/metabolismo , Exones , Genes Reporteros , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Proteínas del Complejo SMN , Homología de Secuencia de Aminoácido , Distribución Tisular , Técnicas del Sistema de Dos HíbridosRESUMEN
High-throughput screening of a combinatorial library of diamidophenols yielded lead compounds with the ability to inhibit human factor Xa (fXa) at micromolar concentrations (e.g. compound 4, fXa apparent K(ass) = 0.64 x 10(6) L/mol). SAR studies in this novel structural series of fXa inhibitors showed that the phenolic hydroxyl group was not essential for activity. The best activity was found in substituted 1,2-dibenzamidobenzenes in which the phenyl group of one benzoyl group (A-ring) was substituted in the 4-position with relatively small lipophilic or polarizable groups such as methoxy, vinyl, or chloro and the phenyl group of the other benzoyl group (B-ring) was substituted in the 4-position with larger lipophilic groups such as tert-butyl or dimethylamino. The central phenyl ring (C-ring) tolerated a wide variety of substituents, but methoxy, methanesulfonamido, hydroxyl, and carboxyl substitution produced slightly higher levels of activity than other substituents when present in combination with favorable B-ring substitution. Methylation of the amide nitrogen atoms was found to greatly decrease activity. Compound 12 is the highest affinity fXa inhibitor in this group of compounds, having fXa apparent K(ass) = 25.5 x 10(6) L/mol, about 40x more active than the original lead. This lead series does not show potent inhibition of human thrombin. A model for the binding of these ligands to the fXa active site is proposed. The model is consistent with the observed SAR and can serve to guide future SAR studies.
Asunto(s)
Anticoagulantes/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores del Factor Xa , Fenilendiaminas/síntesis química , Sulfonamidas/síntesis química , Trombina/antagonistas & inhibidores , Anticoagulantes/química , Anticoagulantes/metabolismo , Sitios de Unión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Factor Xa/química , Factor Xa/metabolismo , Humanos , Modelos Moleculares , Fenilendiaminas/química , Fenilendiaminas/metabolismo , Fenilendiaminas/farmacología , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/metabolismo , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Trombina/metabolismoRESUMEN
Reversal of the A-ring amide link in 1,2-dibenzamidobenzene 1 (fXa K(ass) = 0.81 x 10(6) L/mol) led to a series of human factor Xa (hfXa) inhibitors based on N(2)-aroylanthranilamide 4. Expansion of the SAR around 4 showed that only small planar substituents could be accommodated in the A-ring for binding to the S1 site of hfXa. Bulky groups such as 4-isopropyl, 4-tert-butyl, and 4-dimethylamino were favored in the B-ring to interact with the S4 site of hfXa. The central (C) ring containing a 5-methanesulfonamido group yielded greater activity than carbamoyl groups. Combining the beneficial features from the B- and C-ring SAR, compound 55 represents the most potent hfXa inhibitor in the N(2)-aroylanthranilamide 4 series with hfXa K(ass) = 58 x 10(6) L/mol (K(i) = 11.5 nM).
Asunto(s)
Anticoagulantes/síntesis química , Benzamidas/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores del Factor Xa , Sulfonamidas/síntesis química , Anticoagulantes/química , Anticoagulantes/metabolismo , Benzamidas/química , Benzamidas/metabolismo , Sitios de Unión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Factor Xa/química , Factor Xa/metabolismo , Humanos , Modelos Moleculares , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMEN
To enhance the potency of 1,2-dibenzamidobenzene-derived inhibitors of factor Xa (fXa), an amidine substituent was incorporated on one of the benzoyl side chains to interact with Asp189 in the S1 specificity pocket. Lead molecule 1 was docked into the active site of fXa to facilitate inhibitor design. Subsequently, iterative SAR studies and molecular modeling led to a 1000-fold increase in fXa affinity and a refined model of the new inhibitors in the fXa active site. Strong support for the computational model was achieved through the acquisition of an X-ray crystal structure using thrombin as a surrogate protein. The amidines in this series show high levels of selectivity for the inhibition of fXa relative to other trypsin-like serine proteases. Furthermore, the fXa affinity of compounds in this series (K(ass) = 50-500 x 10(6) L/mol) translates effectively into both anticoagulant activity in vitro and antithrombotic activity in vivo.
Asunto(s)
Amidinas/síntesis química , Anticoagulantes/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores del Factor Xa , Fibrinolíticos/síntesis química , Amidinas/química , Amidinas/metabolismo , Amidinas/farmacología , Animales , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacología , Sitios de Unión , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Factor Xa/química , Factor Xa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/metabolismo , Fibrinolíticos/farmacología , Humanos , Técnicas In Vitro , Masculino , Modelos Moleculares , Tiempo de Protrombina , Conejos , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Trombina/química , Trombina/metabolismo , Trombosis/tratamiento farmacológicoRESUMEN
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by the lack of a subsarcolemmal protein, dystrophin. We have previously shown that the dystrophin-related protein, utrophin is able to compensate for the lack of dystrophin in the mdx mouse, the mouse model for DMD. Here, we explore whether utrophin delivered to the limb muscle of dystrophin/utrophin-deficient double knockout (dko) neonatal mice can protect the muscle from subsequent dystrophic damage. Utrophin delivery may avoid the potential problems of an immune response associated with the delivery of dystrophin to a previously dystrophin-deficient host. Dko muscle (tibialis anterior) was injected with a first generation recombinant adenovirus containing a utrophin minigene. Up to 95% of the fibres continued expressing the minigene 30 days after injection. Expression of utrophin caused a marked reduction from 80% centrally nucleated fibres (CNFs) in the uninjected dko TA to 12% in the injected dko TA. Within the region of the TA expressing the utrophin minigene, a significant decrease in the prevelance of necrosis was noted. These results demonstrate that the utrophin minigene delivered using an adenoviral vector is able to afford protection to the dystrophin/utrophin-deficient muscle of the dko mouse. Gene Therapy (2000) 7, 201-204.
Asunto(s)
Adenoviridae/genética , Proteínas del Citoesqueleto/genética , Proteínas Asociadas a la Distrofina , Distrofina/genética , Técnicas de Transferencia de Gen , Proteínas de la Membrana/genética , Distrofia Muscular de Duchenne/prevención & control , Animales , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/metabolismo , Distroglicanos , Distrofina/deficiencia , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , UtrofinaAsunto(s)
Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Animales , Estudios de Casos y Controles , Proteínas del Citoesqueleto/genética , Distrofina/deficiencia , Distrofina/genética , Metabolismo Energético , Glucosa/metabolismo , Cardiopatías/etiología , Humanos , Técnicas In Vitro , Insulina/farmacología , Espectroscopía de Resonancia Magnética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Distrofia Muscular Animal/complicaciones , Distrofia Muscular de Duchenne/complicaciones , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Utrofina , Función Ventricular IzquierdaRESUMEN
Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5' end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3' to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas de la Membrana/genética , Distrofia Muscular de Duchenne/genética , Regiones Promotoras Genéticas , Regulación hacia Arriba , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Clonación Molecular , Exones , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , ARN Mensajero , Homología de Secuencia de Ácido Nucleico , UtrofinaRESUMEN
Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.
Asunto(s)
Adenoviridae , Proteínas del Citoesqueleto/uso terapéutico , Técnicas de Transferencia de Gen , Vectores Genéticos , Proteínas de la Membrana/uso terapéutico , Distrofias Musculares/terapia , Animales , Animales Recién Nacidos , Proteínas del Citoesqueleto/genética , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos mdx , Músculos/patología , Distrofias Musculares/patología , Fenotipo , UtrofinaRESUMEN
The modulation of utrophin gene expression in muscle by the nerve-derived factor agrin plausibly involves the trophic factor ARIA/heregulin. Here we show that heregulin treatment of mouse and human cultured myotubes caused a approximately 2.5-fold increase in utrophin mRNA levels. Transient transfection experiments with utrophin promoter-reporter gene constructs showed that this increase resulted from an enhanced transcription of the utrophin gene. In the case of the nicotinic acetylcholine receptor delta and epsilon subunit genes, heregulin was previously reported to stimulate transcription via a conserved promoter element, the N-box, which binds the multimeric Ets-related transcription factor GA binding protein (GABP). Accordingly, site-directed mutagenesis of a single N-box motif in the utrophin gene promoter abolished the transcriptional response to heregulin. In addition, overexpression of heregulin, or of the two GABP subunits in cultured myotubes, caused an N-box-dependent increase of the utrophin promoter activity. In vivo, direct gene transfer into muscle confirmed that heregulin regulates utrophin gene expression. Finally, electrophoretic mobility shift assays and supershift experiments performed with muscle extracts revealed that the N-box of the utrophin promoter binds GABP. These findings suggest that the subsynaptic activation of transcription by heregulin via the N-box motif and GABP are conserved among genes expressed at the neuromuscular junction. Because utrophin can functionally compensate for the lack of dystrophin, the elucidation of the molecular mechanisms regulating utrophin gene transcription may ultimately lead to therapies based on utrophin expression throughout the muscle fibers of Duchenne muscular dystrophy patients.
Asunto(s)
Proteínas del Citoesqueleto/genética , Glicoproteínas/farmacología , Proteínas de la Membrana/genética , Músculo Esquelético/fisiología , Factores de Crecimiento Nervioso/farmacología , Receptores Colinérgicos/genética , Animales , Técnicas de Cultivo , Proteínas del Citoesqueleto/biosíntesis , Proteínas de Unión al ADN/genética , Factor de Transcripción de la Proteína de Unión a GA , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas de la Membrana/biosíntesis , Ratones , Neurregulinas , Transducción de Señal/genética , Factores de Transcripción/genética , UtrofinaRESUMEN
31P NMR spectroscopy was used to study the energy metabolism of dystrophin-deficient skeletal muscle of mdx mice, an animal model of Duchenne muscular dystrophy, in which expression of a truncated form of utrophin has been obtained through transgenesis technology. Measurements of ATP, phosphocreatine (PCr), inorganic phosphates (Pi) and intracellular pH (pHi) were made at rest, during a fatigue protocol and during the subsequent recovery. Mechanical fatigue of transgenic muscles was similar to normal muscle, while mdx muscle showed larger force loss. At rest, muscles of all groups had similar values for [ATP], [PCr], [Pi] and pHi. During fatigue, [PCr] decreases mirrored [Pi] increases and were similar in all groups. The major difference between mdx muscles and the group of normal and trc-utrophin muscles concerned the values and evolution of pHi. The mdx muscles showed a more severe intracellular acidosis during exercise and a slower and incomplete post-exercise recovery of normal pHi. In contrast, in trc-utrophin muscles, the kinetics and amplitude of pHi changes were remarkably close to normal behaviour. We conclude that the impaired proton washout which is present in mdx muscles, is corrected to a great extent by the expression of trc-utrophin.
Asunto(s)
Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Hidrógeno/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Actividad Motora/fisiología , Distrofia Muscular Animal/fisiopatología , Animales , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx/metabolismo , Ratones Endogámicos mdx/fisiología , Ratones Transgénicos , Distrofia Muscular Animal/diagnóstico , Distrofia Muscular Animal/metabolismo , Fósforo , UtrofinaRESUMEN
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease usually resulting in death of patients by their early twenties. In contrast, mice lacking dystrophin (Dmd(mdx)), appear physically normal despite their underlying muscle pathology. Mice deficient for both dystrophin and the dystrophin-related protein, utrophin, (Dmd(mdx);Utrn-/- mice) die between 6 and 20 weeks of age suffering from severe muscle weakness with joint contractures, pronounced growth retardation and kyphosis, suggesting that dystrophin and utrophin play complementary roles. The exact cause of death in these mice was not determined. Here we show that expression of a truncated utrophin transgene solely within the skeletal muscle of these mutants prevents premature death and the development of any clinical phenotype. In the absence of full-length dystrophin and utrophin, the presence of truncated utrophin also decreases muscle fibre regeneration, relocalizes the dystrophin protein complex to the sarcolemma and re-establishes a normal expression pattern of developmental muscle proteins. These data suggest that Dmd(mdx);Utrn-/- mice succumb to a skeletal muscle defect and that their reduced lifespan is not due to cardiac or neurogenic components. The phenotypic rescue observed demonstrates that the Dmd(mdx);Utrn-/- mice are an ideal model for testing gene delivery protocols for the expression of utrophin or dystrophin in skeletal muscle. To determine the cause of death of the Dmd(mdx):Utrn-/- mice.
Asunto(s)
Proteínas del Citoesqueleto/genética , Distrofina/deficiencia , Distrofina/genética , Expresión Génica , Proteínas de la Membrana/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/terapia , Transgenes , Animales , Proteínas del Citoesqueleto/deficiencia , Femenino , Terapia Genética , Inmunohistoquímica , Masculino , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , UtrofinaRESUMEN
Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349-353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997) J. Biol. Chem. 272, 8117-8120). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1. 3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24-48 h. Under these conditions, both muscle- and nerve-derived agrin increased the activity of beta-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5' from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding synaptic proteins. Together, these observations are therefore relevant for our basic understanding of the events involved in the assembly and maintenance of the postsynaptic membrane domain of the neuromuscular junction and for the potential use of utrophin as a therapeutic strategy to counteract the effects of Duchenne muscular dystrophy.
Asunto(s)
Agrina/metabolismo , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica , Proteínas de la Membrana/genética , Músculos/metabolismo , Sistema Nervioso/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Ratones , Músculos/citología , Sistema Nervioso/citología , Torpedo , Utrofina , beta-Galactosidasa/genéticaRESUMEN
Utrophin is a homologue of dystrophin, the protein whose absence is responsible for Duchenne muscular dystrophy (DMD). As a first step toward clarifying if adenovirus (AV)-mediated utrophin transfer is a possible option to treat DMD, we have constructed an AV expressing utrophin (AdCMV-Utr) and studied utrophin expression after intramuscular injection of mdx mice, the mouse DMD model. Overexpression of utrophin by AdCMV-Utr was marked and nontoxic. The recombinant utrophin was distributed homogeneously at the surface of the muscle fibers. Its expression was sufficient to restore the normal histochemical pattern of alpha-sarcoglycan and beta-dystroglycan at this site. These two proteins are members of the dystrophin associated protein complex whose distribution is greatly reduced at the surface of the DMD muscle. These data indicate that AV-mediated utrophin transfer is an efficient way of utrophin upregulation in muscle and has the potential of becoming a treatment for DMD.
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Técnicas de Transferencia de Gen , Proteínas de la Membrana/biosíntesis , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Adenoviridae/genética , Animales , Proteínas del Citoesqueleto/genética , Terapia Genética/métodos , Vectores Genéticos , Inyecciones Intramusculares , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos mdx , Distrofia Muscular Animal/terapia , Proteínas Recombinantes/biosíntesis , UtrofinaRESUMEN
The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.