RESUMEN

Dystrophin deficiency leads to the progressive muscle wasting disease Duchenne muscular dystrophy (DMD). Dystrophin-deficient mdx mice are characterized by skeletal muscle weakness and degeneration but they appear outwardly normal in contrast to DMD patients. Mice lacking both dystrophin and the dystrophin homolog utrophin [double knockout (dko)] have muscle degeneration similar to mdx mice, but they display clinical features similar to DMD patients. Dko limb muscles also lack postsynaptic membrane folding and display fiber-type abnormalities including an abundance of phenotypically oxidative muscle fibers. Extraocular muscles, which are spared in mdx mice, show a significant pathology in dko mice. In this study, microarray analysis was used to characterize gene expression differences between mdx and dko tibialis anterior and extraocular skeletal muscles in an effort to understand the phenotypic differences between these two dystrophic mouse models. Analysis of gene expression differences showed that upregulation of slow muscle genes specifically characterizes dko limb muscle and suggests that upregulation of these genes may directly account for the more severe phenotype of dko mice. To investigate whether any upregulation of slow genes is retained in vitro, independent of postsynaptic membrane abnormalities, we derived mdx and dko primary myogenic cultures and analyzed the expression of Myh7 and Myl2. Real-time reverse transcriptase-polymerase chain reaction analysis demonstrates that transcription of these slow genes is also upregulated in dko vs mdx myotubes. This data suggests that at least part of the fiber-type abnormality is due directly to the combined absence of utrophin and dystrophin and is not an indirect effect of the postsynaptic membrane abnormalities.

Asunto(s)

Distrofina/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Músculo Esquelético/fisiología , Utrofina/genética , Animales , Distrofina/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Isoformas de Proteínas/genética , Regulación hacia Arriba , Utrofina/metabolismo

RESUMEN

Skeletal muscles are not created equal. The underutilized concept of muscle allotypes defines distinct muscle groups that differ in their intrinsic capacity to express novel traits when exposed to a facilitating extrinsic environment. Allotype-specific traits may have significance as determinants of the preferential involvement or sparing of muscle groups that is observed in a variety of neuromuscular diseases. Little is known, however, of the developmental mechanisms underlying the distinctive skeletal muscle allotypes. The lack of appropriate in vitro models, to dissociate the cell-autonomous and non-cell-autonomous mechanisms behind allotype diversity, has been a barrier to such studies. Here, we derived novel cell lines from the extraocular and hindlimb muscle allotypes and assessed their similarities and differences during early myogenesis using morphological and gene/protein expression profiling tools. Our data establish that there are fundamental differences in the transcriptional and cellular signaling pathways used by the two myoblast lineages. Taken together, these data show that myoblast lineage plays a significant role in the divergence of the distinctive muscle groups or allotypes.

Asunto(s)

Regulación de la Expresión Génica , Miembro Posterior/metabolismo , Desarrollo de Músculos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Animales , Línea Celular , Perfilación de la Expresión Génica , Miembro Posterior/citología , Miembro Posterior/embriología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Desarrollo de Músculos/genética , Músculo Esquelético/embriología , Mioblastos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal

RESUMEN

To address potential regulatory roles of TGF-beta1 in muscle inflammation and fibrosis associated with dystrophin deficiency, we performed quantitative RT-PCR and in situ hybridization to characterize the temporal and spatial mRNA expression patterns of TGF-beta1 and other TGF-beta subfamily members, TGF-beta2 and TGF-beta3, as well as their receptors, in quadriceps and diaphragm muscles of mdx mice. TGF-beta1 mRNA was markedly upregulated in the endomysial inflammatory cells and regenerating fibers of mdx quadriceps and diaphragm, with the mRNA levels correlated with the degree of endomysial inflammation. Upregulation of TGF-beta2, beta3, and their receptors was also appreciated but to a much lesser degree. While high levels of TGF-beta1 mRNA remained in the aging mdx quadriceps but not the diaphragm, progressive fibrosis only occurred in the diaphragm. Our data support a regulatory role for TGF-beta1 in muscle inflammation in mdx mice. It also suggests different susceptibility of quadriceps and diaphragm muscles to fibrosis induced by TGF-beta1 signaling pathway.

Asunto(s)

Expresión Génica/fisiología , Ratones Endogámicos mdx/metabolismo , Músculo Esquelético/metabolismo , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Técnica del Anticuerpo Fluorescente/métodos , Hibridación in Situ/métodos , Masculino , Ratones , Ratones Endogámicos mdx/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factor de Crecimiento Transformador beta/genética

RESUMEN

Remodeling of adherens junction, gap junction, and desmosomal proteins at the intercalated discs of cardiomyocytes in heart characterizes several animal models of cardiomyopathy, especially dilated cardiac myopathy (DCM). In this study, we show that the tight junction protein, claudin-5, is present in cardiac muscle and localizes to the lateral membranes of cardiomyocytes in normal mice. We further examined claudin-5 in utrophin/dystrophin-deficient (double knockout, dko) mice, a mouse model of muscular dystrophy with cardiomyopathy, and found that claudin-5 mRNA and protein levels are decreased in dko hearts as compared with normal. Intercalated disc cell junction proteins, and another tight junction protein, zonula occludens-1 (ZO-1), are not altered in the dko mouse. Ultrastructural data from dko hearts also shows that the lateral membranes of cardiomyocytes exhibit an abnormal wavy appearance. These data demonstrate that claudin-5 is specifically altered in dko hearts, suggesting that alterations of the lateral membranes of cardiomyocytes, rather than intercalated discs, are associated with cardiomyopathy in the dko mouse.

Asunto(s)

Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Membrana Celular/metabolismo , Distrofina/deficiencia , Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Utrofina/deficiencia , Animales , Cardiomiopatías/genética , Claudina-5 , Vasos Coronarios/metabolismo , Regulación hacia Abajo , Distrofina/genética , Distrofina/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Músculos/metabolismo , Miocitos Cardíacos/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/metabolismo , Utrofina/genética , Utrofina/metabolismo , Proteína de la Zonula Occludens-1

RESUMEN

Current models in skeletal muscle biology do not fully account for the breadth, causes, and consequences of phenotypic variation among skeletal muscle groups. The muscle allotype concept arose to explain frank differences between limb, masticatory, and extraocular (EOM) muscles, but there is little understanding of the developmental regulation of the skeletal muscle phenotypic range. Here, we used morphological and DNA microarray analyses to generate a comprehensive temporal profile for rat EOM development. Based upon coordinate regulation of morphologic/gene expression traits with key events in visual, vestibular, and oculomotor system development, we propose a model that the EOM phenotype is a consequence of extrinsic factors that are unique to its local environment and sensory-motor control system, acting upon a novel myoblast lineage. We identified a broad spectrum of differences between the postnatal transcriptional patterns of EOM and limb muscle allotypes, including numerous transcripts not traditionally associated with muscle fiber/group differences. Several transcription factors were differentially regulated and may be responsible for signaling muscle allotype specificity. Significant differences in cellular energetic mechanisms defined the EOM and limb allotypes. The allotypes were divergent in many other functional transcript classes that remain to be further explored. Taken together, we suggest that the EOM allotype is the consequence of tissue-specific mechanisms that direct expression of a limited number of EOM-specific transcripts and broader, incremental differences in transcripts that are conserved by the two allotypes. This represents an important first step in dissecting allotype-specific regulatory mechanisms that may, in turn, explain differential muscle group sensitivity to a variety of metabolic and neuromuscular diseases.

Asunto(s)

Secuencia Conservada/genética , Perfilación de la Expresión Génica/métodos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Animales , Animales Recién Nacidos/genética , Análisis por Conglomerados , Perfilación de la Expresión Génica/estadística & datos numéricos , Regulación de la Expresión Génica/genética , Miembro Posterior/química , Miembro Posterior/crecimiento & desarrollo , Miembro Posterior/metabolismo , Microscopía Electrónica , Morfogénesis/genética , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/ultraestructura , Cadenas Pesadas de Miosina/genética , Unión Neuromuscular/crecimiento & desarrollo , Unión Neuromuscular/ultraestructura , Músculos Oculomotores/química , Músculos Oculomotores/crecimiento & desarrollo , Músculos Oculomotores/metabolismo , Músculos Oculomotores/ultraestructura , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Especificidad de Órganos/genética , Isoformas de Proteínas/genética , Ratas , Ratas Sprague-Dawley

RESUMEN

Mutations in dystrophin are the proximate cause of Duchenne muscular dystrophy (DMD), but pathogenic mechanisms linking the absence of dystrophin from the sarcolemma to myofiber necrosis are not fully known. The muscular dystrophies also have properties not accounted for by current disease models, including the temporal delay to disease onset, broad species differences in severity, and diversity of skeletal muscle responses. To address the mechanisms underlying the differential targeting of muscular dystrophy, we characterized temporal expression profiles of the diaphragm in dystrophin-deficient (mdx) mice between postnatal days 7 and 112 using oligonucleotide microarrays and contrasted these data with published hindlimb muscle data. Although the diaphragm and hindlimb muscle groups differ in severity of response to dystrophin deficiency, and exhibited substantial divergence in some transcript categories including inflammation and muscle-specific genes, our data show that the general mechanisms operative in muscular dystrophy are highly conserved. The two muscle groups principally differed in expression levels of differentially regulated genes, as opposed to the non-conserved induced/repressed transcripts defining fundamentally distinct mechanisms. We also identified a postnatal divergence of the two wild-type muscle group expression profiles that temporally correlated with the onset and progression of the dystrophic process. These findings support the hypothesis that conserved disease mechanisms interacting with baseline differences in muscle group-specific transcriptomes underlie their differential responses to DMD. We further suggest that muscle group-specific transcriptional profiles contribute toward the muscle targeting and sparing patterns observed for a variety of metabolic and neuromuscular diseases.

Asunto(s)

Diafragma/metabolismo , Distrofina/genética , Expresión Génica , Distrofia Muscular de Duchenne/patología , Animales , Diafragma/patología , Distrofina/deficiencia , Distrofina/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Extremidad Inferior/patología , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Factores de Tiempo , Transcripción Genética

RESUMEN

Muscle tissue is an elegant model for biologic integration of structure with function and is frequently affected by a variety of inherited diseases. Traditional muscle classes--skeletal, cardiac, and smooth--share basic aspects of contractile and energetics mechanisms but also have distinctive role-specific adaptations. We used large-scale oligonucleotide microarrays to broaden knowledge of the adaptive expression patterns underlying muscle tissue differences and to identify transcript subsets that are most likely to represent candidate disease genes. Using stringent analysis criteria, we found >or=95 transcripts, which were preferentially expressed by each muscle class and were validated by inclusion of known muscle class-specific and inherited disease-related genes. Differentially expressed transcripts not previously identified as class-specific extend understanding of muscle class transcriptomes and may represent novel muscle-specific disease genes. We also analyzed the expression profile of extraocular muscle, which is divergent from other skeletal muscles, in the broader context of all major muscle classes. Data show that the extraocular muscle phenotype results from the combination of tissue-specific transcripts, novel expression levels of skeletal muscle transcripts, and partial sharing of gene expression patterns with cardiac and smooth muscle. These, and additional proteomic data, establish that extraocular muscle does not constitute a distinctive muscle class but that it does occupy a novel niche within the skeletal muscle class.

Asunto(s)

Ojo/anatomía & histología , Perfilación de la Expresión Génica , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Miocardio/metabolismo , Animales , Predisposición Genética a la Enfermedad , Miembro Posterior/anatomía & histología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Enfermedades Musculares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Ratas , Transcripción Genética

RESUMEN

The M-line and its associated creatine kinase (CK) M-isoform (CK-M) are ubiquitous features of skeletal and cardiac muscle. The M-line maintains myosin myofilaments in register, links the contractile apparatus to the cytoskeleton for external force transfer and localizes CK-based energy storage and transfer to the site of highest ATP demand. We establish here that the muscle group responsible for movements of the eye, extraocular muscle (EOM), is divergent from other striated muscles in lacking both an M-line and its associated CK-M. Although an M-line forms during myogenesis, both in vivo and in vitro, it is actively repressed after birth. Transcripts of the major M-line structural proteins, myomesin 1 and myomesin 2, follow the same pattern of postnatal downregulation, while the embryonic heart-specific EH-myomesin 1 transcript is expressed early and retained in adult eye muscle. By immunocytochemistry, myomesin protein is absent from adult EOM sarcomeres. M-line suppression does not occur in organotypic co-culture with oculomotor motoneurons, suggesting that the mechanism for suppression may lie in muscle group-specific activation or workload patterns experienced only in vivo. The M-line is, however, still lost in dark-reared rats, despite the developmental delay this paradigm produces in the visuomotor system and EOMs. EOM was low in all CK isoform transcripts except for the sarcomeric mitochondrial (Ckmt2) isoform. Total CK enzyme activity of EOM was one-third that of hindlimb muscle. These findings are singularly unique among fast-twitch skeletal muscles. Since EOM exhibits isoform diversity for other sarcomeric proteins, the M-line/CK-M divergence probably represents a key physiological adaptation for the unique energetics and functional demands placed on this muscle group in voluntary and reflexive eye movements.

Asunto(s)

Adaptación Fisiológica , Proteínas Musculares/genética , Músculos Oculomotores/anatomía & histología , Ratas/fisiología , Sarcómeros/metabolismo , Secuencia de Aminoácidos , Animales , Conectina , Creatina Quinasa/metabolismo , Forma MM de la Creatina-Quinasa , Cartilla de ADN , Inmunohistoquímica , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Músculos Oculomotores/metabolismo , Estructura Secundaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

Although dystrophin mutations are the proximate cause of Duchenne muscular dystrophy (DMD), interactions among heterogeneous downstream mechanisms may be key phenotypic determinants. Temporal gene expression profiling was used to identify and correlate diverse transcriptional patterns to one another and to the disease course, for both affected and spared muscle groups, in postnatal day 7-112 dystrophin-deficient (mdx) mice. While 719 transcripts were differentially expressed at one or more ages in leg muscle, only 56 genes were altered in the spared extraocular muscles (EOM). Contrasting molecular signatures of affected versus spared muscles provide compelling evidence that the absence of dystrophin alone is necessary but not sufficient to cause the patterned fibrosis, inflammation and failure of muscle regeneration characteristic of dystrophinopathy. Dystrophic and adaptive changes in the microarray profiles were further quantified using an aggregate disease load index (DLI) to measure stage-dependent transcriptional impact in both muscles. DLI analysis highlighted the divergent responses of EOM and leg muscle groups. Cellular process-specific DLIs in leg muscle identified positively correlated temporal expression profiles for some gene classes, and the independence of others, that are linked to major disease components. Data also showed a previously unrecognized transient and selective developmental delay in pre-necrotic mdx skeletal muscle that was confirmed by qPCR. Taken together, validation and targeting of signaling pathways responsible for the coordination of the fibrotic, proteolytic and inflammatory mechanisms shown here for mdx muscle may yield new therapeutic means of mitigating the devastating consequences of DMD.

Asunto(s)

Modelos Animales de Enfermedad , Distrofina/deficiencia , Expresión Génica , Distrofia Muscular de Duchenne/genética , Transducción de Señal , Animales , Cartilla de ADN , Distrofina/genética , Distrofina/metabolismo , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculos Oculomotores/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Factores de Tiempo

RESUMEN

The M lines are structural landmarks in striated muscles, necessary for sarcomeric stability and as anchoring sites for the M isoform of creatine kinase (CK-M). These structures, especially prominent in fast skeletal muscles, are missing in rodent extraocular muscle, a particularly fast and active muscle group. In this study, we tested the hypotheses that 1). myomesin and M protein (cytoskeletal components of the M lines) and CK-M are downregulated in mouse extraocular muscle compared with the leg muscles, gastrocnemius and soleus; and 2). the expression of other cytosolic and mitochondrial CK isoforms is correspondingly increased. As expected, mouse extraocular muscles expressed lower levels of myomesin, M protein, and CK-M mRNA than the leg muscles. Immunocytochemically, myomesin and M protein were not detected in the banding pattern typically seen in other skeletal muscles. Surprisingly, message abundance for the other known CK isoforms was also lower in the extraocular muscles. Moreover, total CK activity was significantly decreased compared with that in the leg muscles. Based on these data, we reject our second hypothesis and propose that other energy-buffering systems may be more important in the extraocular muscles. The downregulation of major structural and metabolic elements and relative overexpression of two adenylate kinase isoforms suggest that the extraocular muscle group copes with its functional requirements by using strategies not seen in typical skeletal muscles.

Asunto(s)

Creatina Quinasa/metabolismo , Glicoproteínas/metabolismo , Isoenzimas/metabolismo , Proteínas Musculares/metabolismo , Músculos Oculomotores/anatomía & histología , Músculos Oculomotores/metabolismo , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Animales , Conectina , Forma MM de la Creatina-Quinasa , Regulación hacia Abajo , Glicoproteínas/genética , Inmunohistoquímica , Isoenzimas/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Músculos Oculomotores/enzimología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba

RESUMEN

Extraocular muscle (EOM) is spared in Duchenne muscular dystrophy. Here, we tested putative EOM sparing mechanisms predicted from existing dystrophinopathy models. Data show that mdx mouse EOM contains dystrophin-glycoprotein complex (DGC)-competent and DGC-deficient myofibers distributed in a fiber type-specific pattern. Up-regulation of a dystrophin homologue, utrophin, mediates selective DGC retention. Counter to the DGC mechanical hypothesis, an intact DGC is not a precondition for EOM sarcolemmal integrity, and active adaptation at the level of calcium homeostasis is not mechanistic in protection. A partial, fiber type-specific retention of antiischemic nitric oxide to vascular smooth muscle signaling is not a factor in EOM sparing, because mice deficient in dystrophin and alpha-syntrophin, which localizes neuronal nitric oxide synthase to the sarcolemma, have normal EOMs. Moreover, an alternative transmembrane protein, alpha7beta1 integrin, does not appear to substitute for the DGC in EOM. Finally, genomewide expression profiling showed that EOM does not actively adapt to dystrophinopathy but identified candidate genes for the constitutive protection of mdx EOM. Taken together, data emphasize the conditional nature of dystrophinopathy and the potential importance of nonmechanical DGC roles and support the hypothesis that broad, constitutive structural cell signaling, and/or biochemical differences between EOM and other skeletal muscles are determinants of differential disease responsiveness.

Asunto(s)

Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Músculos Oculomotores/metabolismo , Adaptación Fisiológica , Animales , Antígenos CD/genética , Calcio/metabolismo , Proteínas de Unión al Calcio , Análisis por Conglomerados , Proteínas del Citoesqueleto/metabolismo , Distrofina/deficiencia , Distrofina/metabolismo , Expresión Génica , Hemostasis , Inmunohistoquímica , Cadenas alfa de Integrinas/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/deficiencia , Proteínas Musculares/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Músculos Oculomotores/fisiopatología , Análisis de Secuencia por Matrices de Oligonucleótidos , Sarcolema/metabolismo , Utrofina

RESUMEN

Prior studies and the efficacy of immunotherapies provide evidence that inflammation is mechanistic in pathogenesis of Duchenne muscular dystrophy. To identify putative pro-inflammatory mechanisms, we evaluated chemokine gene/protein expression patterns in skeletal muscle of mdx mice. By DNA microarray, reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, and immunoblotting, convergent evidence established the induction of six distinct CC class chemokine ligands in adult MDX: CCL2/MCP-1, CCL5/RANTES, CCL6/mu C10, CCL7/MCP-3, CCL8/MCP-2, and CCL9/MIP-1gamma. CCL receptors, CCR2, CCR1, and CCR5, also showed increased expression in mdx muscle. CCL2 and CCL6 were localized to both monocular cells and muscle fibers, suggesting that dystrophic muscle may contribute toward chemotaxis. Temporal patterns of CCL2 and CCL6 showed early induction and maintained expression in mdx limb muscle. These data raise the possibility that chemokine signaling pathways coordinate a spatially and temporally discrete immune response that may contribute toward muscular dystrophy. The chemokine pro-inflammatory pathways described here in mdx may represent new targets for treatment of Duchenne muscular dystrophy.

Asunto(s)

Quimiocinas CC/metabolismo , Macrófagos/fisiología , Músculo Esquelético/fisiología , Receptores de Quimiocina/metabolismo , Linfocitos T/fisiología , Animales , Animales Recién Nacidos , Western Blotting , Quimiocina CCL5/metabolismo , Quimiocinas CC/clasificación , Análisis por Conglomerados , Cartilla de ADN , Modelos Animales de Enfermedad , Expresión Génica , Miembro Posterior/metabolismo , Inmunohistoquímica , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Proteínas Quimioatrayentes de Monocitos/clasificación , Proteínas Quimioatrayentes de Monocitos/metabolismo , Músculo Esquelético/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/análisis , Receptores de Quimiocina/clasificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

Asunto(s)

Regulación de la Expresión Génica , Músculos Oculomotores/fisiología , Privación Sensorial/fisiología , Animales , Catepsina E/genética , Coenzima A Ligasas/genética , Citocromos b5/genética , Oscuridad , Glutatión Transferasa/genética , Contracción Muscular , Músculos Oculomotores/enzimología , Proteína Quinasa C/genética , Ratas , Ratas Sprague-Dawley , Percepción Visual , Proteínas de Unión al GTP rab/genética

RESUMEN

Mutations in dystrophin cause Duchenne muscular dystrophy (DMD), but absent dystrophin does not invariably cause necrosis in all muscles, life stages and species. Using DNA microarray, we established a molecular signature of dystrophinopathy in the mdx mouse, with evidence that secondary mechanisms are key contributors to pathogenesis. We used variability controls, adequate replicates and stringent analytic tools, including significance analysis of microarrays to estimate and manage false positive rates. In leg muscle, we identified 242 differentially expressed genes, >75% of which have not been previously reported as altered in human or animal dystrophies. Data provide evidence for coordinated activity of numerous components of a chronic inflammatory response, including cytokine and chemokine signaling, leukocyte adhesion and diapedesis, invasive cell type-specific markers, and complement system activation. Selective chemokine upregulation was confirmed by RT-PCR and immunoblot, and may be a key determinant of the nature of the inflammatory response in dystrophic muscle. Up-regulation of secreted phosphoprotein 1 (minopontin, osteopontin) mRNA and protein in dystrophic muscle identified a novel linkage between inflammatory cells and repair processes. Extracellular matrix genes were up-regulated in mdx to levels similar to those in DMD. Since, unlike DMD, mdx exhibits little fibrosis, data suggest that collagen regulation at post-transcriptional stages mediates extensive fibrosis in DMD. Taken together, these data identify a relatively neglected aspect of DMD, suggest new treatment avenues, and highlight the value of genome-wide profiling in study of complex disease processes.

Asunto(s)

Distrofina/deficiencia , Músculo Esquelético/patología , Animales , Factores Quimiotácticos/metabolismo , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/fisiología , Matriz Extracelular/metabolismo , Inflamación/fisiopatología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/química , Distrofia Muscular de Duchenne/etiología , Distrofia Muscular de Duchenne/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Regeneración , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba