[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Compartmentalization of the extraocular muscles into well-defined orbital and global layers is highly conserved. Recently, the active pulley hypothesis correlated the anatomic properties of orbital-global muscle layers with layer-specific division of labor. Microarray technology was used to identify muscle-layer-specific transcriptional profiles and, thereby, extend understanding of the structure-function characteristics of extraocular muscle layers.
Laser capture microdissection was used to obtain muscle layer samples from monkey medial rectus muscles. RNA was linearly amplified and hybridized to human U133 series microarrays (Affymetrix, Santa Clara, CA), which have sufficient sequence homology for use in subhuman primates. Data was analyzed using Affymetrix and Robust Multichip Average (RMA) algorithms. Select transcripts were verified by quantitative PCR and in situ hybridization.
A broad spectrum of transcriptional differences (> 181 transcripts) was identified between the two extraocular muscle layers. Patterned differences in the sarcomeric contractile machinery and cytoskeleton were suggestive of key layer differences in contraction speed. Differentially expressed transcript identities, however, extended well beyond those traditionally associated with muscle-fiber-group differences.
Muscle layer transcriptional profiles correlated with the different loads and usage patterns of extraocular muscle layers, as proposed in the active pulley hypothesis. The magnitude and breadth of orbital-global layer expression differences strongly suggests that oculomotor control systems may drive two distinct motor output pathways, each comprising separate motoneurons and muscle fibers, with one output path adapted to determining pulley position and the other to movement of the eye.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Human Molecular Genetics 03/2004; 13(3):257-69. DOI:10.1093/hmg/ddh033 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To address the consequences of visual deprivation paradigms in rat (dark rearing) and monkey (monocular deprivation) on extraocular muscle (EOM) development using genome-wide expression profiling.
Serial analysis of gene expression (SAGE) was used to determine alterations in the EOM transcriptome induced by dark rearing of rats from birth to postnatal day 45. Data were compared with previously published normal EOM SAGE library. DNA microarray similarly assessed changes in gene expression patterns of EOMs of monkeys reared from birth to 4 months of age with monocular deprivation.
Dark rearing produced changes in expression of 280 transcripts in rat EOM. Of these, 71 were known genes representing functional categories that included energy metabolism/mitochondrial-related (21%), protein synthesis and modification (14%), lipid metabolism (13%), and muscle-related (6%) transcripts. Together, the predominant pattern reflected an energetic shift toward fatty acid beta-oxidation and integrated alterations in both myofibers and supportive tissues. The response of monkey rectus muscles to monocular deprivation was considerably less severe.
The visual deprivation paradigms used in this study mimic alterations that are associated with the common disorders of strabismus, congenital cataract, and amblyopia. These data show that postnatal EOM maturation is broadly susceptible to changes in activity patterns that are a consequence of visuomotor maldevelopment. The data extend the concept of an EOM-critical period and establish that activity patterns in developing eye movement systems play vital determinant roles in the novel EOM phenotype.
[Show abstract][Hide abstract] ABSTRACT: 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.
Human Molecular Genetics 09/2003; 12(15):1813-21. DOI:10.1093/hmg/ddg197 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The FASEB Journal 07/2003; 17(10):1370-2. · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The phenotypically novel extraocular muscles (EOMs) exhibit fundamental differences in innervation and neuromuscular junction (NMJ) morphology from other skeletal muscles. In the current study, the morphology and molecular organization of NMJs of EOM singly innervated (SIF) and multiply innervated (MIF) fiber types were evaluated and the distribution of molecules involved in formation and maintenance of NMJs were specifically characterized.
Adult mouse EOM NMJ organization was examined by immunofluorescence and confocal microscopy. Differential cellular localization of components of two established synaptic signaling pathways, (1) neuregulin and erbB receptors 2, 3, and 4 and (2) agrin, MuSK, and rapsyn and select NMJ-associated structural proteins were studied for EOM SIF and MIF populations. Endplate topography and structure were also studied, using both confocal microscopy and transmission electron microscopy, with NMJ morphologic organization correlated with specific EOM fiber types.
Confocal fluorescence microscopy demonstrated that, for NMJs of both EOM SIFs and MIFs, components of neuregulin and agrin pathways and the major components of the junctional dystrophin-glycoprotein complex (DGC) colocalized with acetylcholine receptor (AChR) aggregates. However, EOM exhibited novel fiber-type-specific extrasynaptic localization of two key DGC signaling-related molecules: alpha-dystrobrevin 1 (global MIFs) and syntrophin beta1 (global MIFs and orbital MIFs and SIFs).
The data establish that the molecular organization of EOM SIF and MIF NMJs includes the same signaling and structural molecules previously characterized for other skeletal muscles. By contrast, divergence in other aspects of the synaptic and nonsynaptic sarcolemmal organization of EOM fiber types may underlie the unique responses of these muscles in a variety of neuromuscular disorders.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Human Molecular Genetics 02/2002; 11(3):263-72. · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle fibers are defined by patterned covariation of key traits that determine contractile and metabolic characteristics. Although the functional properties of most skeletal muscles result from their proportional content of a few conserved muscle fiber types, some, typically craniofacial, muscles exhibit fiber types that appear to lie outside the common phenotypic range. We analyzed gene expression profiles of three putative muscle classes, limb, masticatory, and extraocular muscle (EOM), in adult mice by high-density oligonucleotide arrays. Pairwise comparisons using conservative acceptance criteria identified expression differences in 287 genes between EOM and limb and/or masticatory muscles. Use of significance analysis of microarrays methodology identified up to 400 genes as having an EOM-specific expression pattern. Genes differentially expressed in EOM reflect key aspects of muscle biology, including transcriptional regulation, sarcomeric organization, excitation-contraction coupling, intermediary metabolism, and immune response. These patterned differences in gene expression define EOM as a distinct muscle class and may explain the unique response of these muscles in neuromuscular diseases.
Proceedings of the National Academy of Sciences 11/2001; 98(21):12062-7. DOI:10.1073/pnas.211257298 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Extraocular rectus muscle (EOM) pulleys are important determinants of orbital biomechanics in humans. In this study, the authors evaluated orbital connective tissue morphology, specifically characterizing rectus muscle pulleys, in the rat, a species with laterally placed eyes, afoveate vision, and a less complex visuomotor repertoire than primates.
Adult rat orbits were paraffin processed and serially sectioned for histochemical and immunohistochemical staining. Frozen sections of enucleated globes with intact EOMs and associated connective tissue were also studied with myosin immunohistochemistry and histochemistry for the mitochondrial enzyme, nicotinamide adenine dinucleotide (NADH)-tetrazolium reductase, to delineate the orbital layer relationship with the pulley tissue.
Focal condensations of collagenous connective tissue were found in relationship to the rectus muscles in the equatorial Tenon's fascia, similar to those described as human recti muscle pulleys. The fibroelastic pulley rings were coupled to adjacent EOM pulleys by bands containing collagen and elastin. The coupling of pulleys to the orbital walls was significantly less than that previously described in humans. As in humans, there was a dual insertion of rodent rectus muscles, with the orbital layer inserting on the muscle pulley and the global layer attaching to the sclera.
The data support the presence of structures in the rat orbit that are the morphologic equivalent of the human rectus pulley system. Although rodent and human pulleys were similar in many respects, there were species-specific properties that may relate to established differences in orbital anatomy and/or visuomotor behavior. These data extend the rectus muscle pulley concept to rodents and may provide insight into pulley structure-function relationships.