Human Molecular Genetics, 2002, Vol. 11, No. 3 263-272
© 2002 Oxford University Press
A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice
1Department of Ophthalmology, 2Department of Neurology, 3Department of Neurosciences, 4Departments of Epidemiology and Biostatistics and 5The Comprehensive Cancer Center, Case Western Reserve University and University Hospitals of Cleveland and The Research Institute of University Hospitals of Cleveland, Cleveland, OH 44106-5068, USA
Received October 1, 2001; Revised and Accepted November 19, 2001.
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ABSTRACT |
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 TOP ABSTRACT IntroductionResultsDiscussionMaterials and MethodsSUPPLEMENTARY MATERIALREFERENCES |
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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.
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Introduction |
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TOPABSTRACTIntroductionResultsDiscussionMaterials and MethodsSUPPLEMENTARY MATERIALREFERENCES |
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Absence of dystrophin is the primary defect in Duchenne muscular dystrophy (DMD) (1). Dystrophin is part of the dystrophin–glycoprotein complex (DGC), linking myofiber cytoskeleton to extracellular matrix. Inherited mutations of DGC components either cause muscular dystrophy or are embryo lethal. The most accepted model for muscular dystrophy is that lack of a DGC component breaks a mechanical linkage vital for sarcolemmal integrity. Physical sarcolemmal breaks or calcium leak channel openings then elevate intracellular free calcium, triggering calcium-activated proteases and fiber necrosis (2,3). However, understanding the disease mechanisms in muscular dystrophy is complicated by evidence that the DGC may act as a scaffolding to spatially approximate cell signaling network components necessary for myofiber homeostasis (4). DGC signaling may also play roles in microvascular function (5,6) and muscle fiber type determination (7), each of which represents a potential pathogenic mechanism. The net result is that existing models do not fully explain pathogenic mechanisms in DMD.
While dystrophin deficiency is the proximate cause of DMD, secondary mechanisms may determine whether a muscle group is heavily affected (diaphragm) or spared (extraocular muscle) and whether dystrophinopathy is fatal (DMD) or is transient, but recoverable (mdx mouse). The inflammatory response to myofiber damage is a compelling candidate mechanism for exacerbation of the disease (8–12) [for a review see Spencer and Tidball (13)], but its precise role in pathogenesis of DMD is unclear. Mdx mice also null for a key cytokine, tumor necrosis factor (TNF), exhibit equivocal findings, rather than the expected global amelioration of muscle pathology (14). Immunosuppression therapy improves muscle strength and prolongs ambulation in DMD patients, but, paradoxically, does not appear to act simply by attenuating the mononuclear cell infiltrate (15). A better understanding of the role of inflammation in the disease process may aid DMD therapy in two ways: facilitating direct therapies to delay/block the disease or indirectly, by improving strategies for successful transplantation of dystrophin-competent myoblasts.
Taken together, there is not yet a true consensus on pathogenic mechanisms in DMD; existing models fail to unify the complex primary dystrophin mutation and a constellation of secondary events. Progress may be hindered by experimental strategies that are too focused or biased by existing disease models. To place dystrophin deficiency into the context of other cellular events, we used high-density DNA microarrays to develop a global gene expression signature for skeletal muscle in the mdx mouse. Data present a broad molecular overview of interacting events in dystrophic hindlimb muscle, including a new and comprehensive view of the role of inflammation in DMD. Approximately 75% of the known genes shown here with altered expression in mdx hindlimb have not been previously associated with dystrophinopathy, and thus represent important new information for molecular modeling and many may be potential therapeutic targets.
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Results |
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TOPABSTRACTIntroductionResultsDiscussionMaterials and MethodsSUPPLEMENTARY MATERIALREFERENCES |
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Overview
Skeletal muscles of mdx mice exhibit degeneration and regeneration between 2 and 12 weeks of age. Regardless of the species or nature of the defective DGC protein, early stages of dystrophy show acute plasma membrane fragility and functional ischemia (16). To assess the less well known secondary pathogenic mechanisms in mdx hindlimb, we chose mid-stage (8 week) mdx mice for expression profiling with high-density oligonucleotide microarrays (
10 000 genes/ESTs). Using the Affymetrix algorithm and stringent data acceptance criteria (consistent difference calls across all five pairwise replicates), we initially identified 249 differentially expressed genes. All genes meeting this consistency criterion were reported; an arbitrary fold-change cutoff was not imposed. Changes were highly specific to dystrophic muscle, since there was a nearly identical pattern in affected masticatory muscle, but not in the spared (17,18) extraocular muscles (J.D.Porter, A.P.Merriam, S.Khanna, H.J.Kaminiski, C.R.Richmonds and F.H.Andrade, unpublished data). A separate and independent analysis of our microarray data used significance analysis of microarrays (SAM) (19,20). SAM relies upon the microarray probe set average difference values (i.e. mean of the signal intensity differences between the 20 matched and mismatched probes arrayed for each gene), but ignores other discrimination features designed into the Affymetrix system. SAM avoids the pitfalls that traditional statistical tools encounter in large databases, allowing estimation and management of false positives. Gene expression changes identified as significant by SAM depend upon an investigator-selected threshold with an acceptable false discovery rate (FDR; rough equivalent of P-value). We performed SAM analyses at 20 thresholds, selecting the threshold with the lowest FDR (<0.01). However, the gene list identified by SAM, used in isolation, erroneously includes genes declared as absent from both mdx and control samples by the Affymetrix algorithm. To then take advantage of the strengths of both data analysis strategies, we used the intersection of data sets from the SAM and Affymetrix analyses to detect and reject false positives among the original 249 genes identified by the Affymetrix algorithm. Those genes detected by SAM as potential false positives (n = 7) then were not considered further, leaving 242 differentially expressed genes in mdx leg muscle (Tables 1–3, and see Supplementary Material).
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Affymetrix-supplied gene names were updated prior to classification, based upon GenBank accession numbers. Functional classes were assigned to all known genes using information from the Gene Ontology database available at the Jackson Laboratory Mouse Genome Informatics website (http://www.informatics.jax.org/).
Genes that have previously been linked to DMD or any of the animal models of DMD, by any gene or protein assay, are indicated with appropriate literature references in Tables 2 and 3 (see also Supplementary Material). Those genes specifically linked to DMD in prior studies are bolded in each table.
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General program in dystrophic muscle
Gene class expression changes in mdx mice (Table 1) were consistent with the well documented pattern of ongoing muscle degeneration and regeneration. Our data show that the major events at the 8 week stage are inflammation (29.8% of differentially expressed genes), extracellular matrix deposition (9.1%), proteolysis (7.4%), muscle regeneration (7.4%) and energy metabolism (1.7%). The remainder of differentially expressed genes did not fit into categories immediately relevant to pathogenesis of muscular dystrophy (29.3%) or were ESTs (15.3%). Many more genes were overexpressed than underexpressed, a finding previously reported in microarray studies of DMD patients that was attributed to a broad increase in protein turnover associated with the degenerative/regenerative nature of the disease (21). We estimated that
75% of the 205 known genes identified by microarray have not been previously linked to mdx, DMD or any other model of dystrophinopathy (Tables 2 and 3, and see Supplementary Material).
Whereas absence of dystrophin displaces the other DGC components from the sarcolemma, consistent with prior reports (22) we did not find altered dystroglycan and sarcoglycan gene expression. Up-regulation of an 
-dystroglycan binding partner, biglycan, was observed, in agreement with a report that it may be adaptive in dystrophinopathy (23). We also observed up-regulation of the thiol-protease, calpain 2 (Table 2), consistent with well documented calcium-dependent proteolysis and myofiber necrosis (24,25). Increased calpain may produce generalized proteolysis or may simply disrupt normal calpain-influenced regulatory mechanisms (26). Other proteases also were up-regulated in the mdx limb (Table 2, and see Supplementary Material).
The gene expression pattern in mdx limb included a substantial and coordinated inflammatory/repair response. The major participating cellular elements were identified by significant elevations in mRNAs pathognomonic of mast cells (Gp49a, Gp49b), macrophages (Mpeg1, Lgals3), T cells (CD53, CD48), and B cells (Fcgr2b) (Table 3, and see Supplementary Material). Few markers specific for either NK cells or neutrophils were differentially expressed and none specifically identified eosinophils. Up-regulation of BIT suggests that macrophage giant cells are formed in dystrophic muscle. Chronic activation of repair processes was evident in enhanced expression of the fibril forming (types I and III) collagens.
Compromise of genes related to energy metabolism, a prominent feature in 12 week mdx mice and DMD patients (16,27), was not observed in 8 week mdx mice.
Events in dystrophic muscle: muscle regeneration
In dystrophinopathy, muscle stem/satellite cells proliferate and fuse to form replacement muscle fibers. Myogenic markers previously reported to increase in mdx and/or DMD (16,28–31) also were up-regulated in mdx leg, including myogenin, -acetylcholine receptor, insulin-like growth factor 2, cardiac troponin T2 and various ß-tubulins (Table 2, and see Supplementary Material). N-glycan -2,8-sialytransferase, responsible for polysialation of NCAM, a cell surface glycoprotein expressed in regenerated muscle fibers, was also up-regulated. Several additional up-regulated genes linked to myogenesis or muscle regeneration have not been previously reported in DMD.
Events in dystrophic muscle: inflammation
Although immune mediators have been detected in dystrophinopathy (8–12), there has not been a thorough analysis of the inflammatory response. Genome profiling showed that inflammation in the mdx hindlimb was robust. The nature and breadth of immune function markers (30% of differentially expressed genes) indicated a chronic, persistent inflammatory reaction. These data provide considerable resolution of the staging of muscle inflammation in dystrophic muscle.
Activity of cytokines and cytokine receptors. Injury of dystrophin-deficient muscle causes cytokine release by mast cells, fibroblasts and damaged muscle fibers, eliciting coordinated vascular responses and mononuclear cell accumulation. Cytokines (e.g. TNF, IL-1 and IL-4) are key inflammation inducers that are invoked early following tissue damage. TNF expression by injured myofibers in DMD (32) may induce inflammation without the need for a primary response by mononuclear cells. Yet, consistent with prior data in DMD (10), we did not observe altered cytokine gene expression in mdx. Evidence of cytokine-dependent activity did include increases in a TNF receptor (p75 TNF) and Stat6, a transcription factor mediating IL-4 signaling (Table 3). Stat6 up-regulation has many effects, including potent induction of both Vcam1 and the chemokine, Scya2, both of which upregulated in our study (see below).
Mast cells are constitutively present in muscle connective tissues, serve as a cytokine source to initiate inflammation, rapidly accumulate at the injury site after even mild damage (33), and are chronically or transiently present in DMD and mdx muscles, respectively (9,34). Mast cells also store TNF; thus, cytokine release at injury sites is not wholly dependent upon elevated gene transcription. Gene expression patterns (Table 3) showed mast cells were a prominent component of inflammation in the mdx limb, even though reports suggest that their numbers peak by 4 weeks and are in decline by the age studied here (9). Mast cell degranulation increases local blood flow/vascular permeability and causes direct proteolysis of dystrophin-deficient myofibers (35). Gorospe et al. (9) suggested they are a proximate cause of the focal necrosis characteristic of dystrophin deficiency. The severe phenotype of double-mutant mdx mice with exaggerated mast cell activity supports this hypothesis.
Vascular permeability and leukocyte emigration. The vascular endothelium plays a critical role in inflammation. Activation of normally quiescent endothelial cells initiates and maintains the influx and type-specificity of recruited leukocytes. Mdx showed increased expression of two endothelial-specific genes: P2X receptor (P2rx4) and vascular cell adhesion molecule 1 (Vcam1) (Table 3). Endothelial P2rx4 participates in signaling responses mediated by ATP from mast (and other) cells in the inflamed region. Up-regulation of Vcam1 may facilitate leukocyte migration, since it is a cell surface adhesion molecule that recognizes and binds to VLA-4 on leukocytes, particularly monocytes and lymphocytes. Enhanced endothelial expression of Vcam1 has been previously described in both DMD and inflammatory myopathies (36). Increased expression of CD47 also is indicative of leukocyte emigration to the muscle injury site.
Chemokine/chemokine receptors. The nature of the inflammatory response in dystrophic muscle is controlled, at least in part, by the subset of locally expressed chemokines. A total of 28 chemokines, chemokine receptors and chemokine-related genes are represented on the U74 mouse array and expression of members of two major classes of chemokines and chemokine receptors was increased in the mdx limb. These included: a C–X–C class chemokine (Scyb14) and a C–X–C class chemokine receptor (Cmkar4); four C–C class chemokines (Scya2, Scya6, Scya7 and Scya9) and a C–C class receptor (Cmkbr2) (Table 3). Only two chemokines have been previously reported to increase in dystrophinopathy (Scya2 and Cmkbr2) (37). Since muscular dystrophy is chronic, the disease process is largely characterized by infiltration of lymphocytes and macrophages. The C–C chemokines that form the major chemotaxic response in mdx are specific for monocytes, eosinophils, basophils and lymphocytes, but inactive toward neutrophils. Whereas the C–X–C family is chemotaxic for neutrophils and lymphocytes, the up-regulated chemokine in this class, Scyb14, is highly specific for lymphocytes. Chemokines and their receptors then specifically designate the cellular composition of the muscle injury response in dystrophinopathy. Data establish that the precise nature of this response is within the resolution of transcriptional analysis by oligonucleotide microarrays.
Complement system. Twelve soluble plasma proteins interact in three complement pathways—classical, alternative and lectin-mediated—to produce a cytolytic membrane attack complex. The Affymetrix U74A GeneChip surveys 18 complement and complement-related mRNAs. Of these, five complement components or complement-related genes were up-regulated in mdx mice. These included C3, a key component of classical and alternative complement pathways, two C1q subcomponents (C1qa, C1qb) that associate with proenzymes to yield the initial component of the complement system, and properdin, a positive regulator of the alternative pathway (Table 3). The role that complement activation might play in DMD is currently unclear. Complement expression in DMD was interpreted as evidence that sublytic membrane attack complexes may have an additive effect upon an already compromised dystrophin-deficient sarcolemma (38). That membrane attack complexes were found exclusively in association with necrotic myofibers in DMD patient biopsies (39–41) is inconclusive in establishing a direct link between complement activation and the initiation of necrosis in dystrophin-deficient myofibers.
Mitigation of the inflammatory response. Skeletal muscle in the mdx mouse does not undergo the downward-spiraling degeneration seen in DMD. Thus, the robust up-regulation of inflammation-related genes seen here likely would not be observed in later staged mice. We did see limited evidence for mitigation of the dominant inflammatory response in 8 week mdx. This included increases in IL-1 receptor antagonist, which normally balances IL-1 in modulating the level of inflammation, and IL-10 receptor, a known suppressor of macrophage function.
Events in dystrophic muscle: extracellular matrix
Connective tissue deposition is considered a secondary, or reparative, response that may further compromise muscle function in DMD. In contrast, mdx muscle exhibits a low level of fibrosis, a possible mechanism for the less severe phenotype in the mouse. Using GeneChips, we evaluated expression levels of 26 distinct structural collagen types and found eight collagen mRNAs with elevated expression in mdx hindlimb muscle. Surprisingly, the species-specific phenotypes were not reflected in relative expression patterns of the principal fibril-forming collagens (types I and III), which were up-regulated in the mouse to the same degree as in the highly fibrotic muscles of DMD (16). This discrepancy suggests collagen gene up-regulation per se does not simply translate into a dystrophic phenotype. Additional fibrillar (type V) and non-fibrillar (types VI and VIII) collagens also increased in mdx muscles, as did genes that regulate collagen processing (lumican) and remodeling (matrix metalloproteinase 3) (Table 2, and see Supplementary Material). Resolution of the species-specific relationship between collagen expression/deposition levels and the balance of myofiber protection versus damage may advance understanding of pathogenic mechanisms in dystrophinopathy.
Increases in S100a6 and S100a4 expression (see Supplementary Material) were consistent with fibroblast activation in the mdx limb, as these genes up-regulate when quiescent fibroblasts are stimulated to proliferate. Fibrosis in dystrophin-deficient muscle is directed, at least in part, by the immune system, including T cells (42). Our data revealed a previously unidentified mechanism linking inflammation and repair in dystrophic muscle. Secreted phosphoprotein 1 (Spp1; osteopontin or minopontin), a macrophage product that enhances synthesis and turnover of extracellular matrix, was substantially elevated in mdx limb. Spp1-deficient mice show altered collagen fibrillogenesis in wound healing (43) and support a role for Spp1 in regulating collagen synthesis and accumulation after myocardial infarction (44). Spp1-deficient mice exhibited a dramatic reduction in fibrillar collagen content and blockage of an infarct-induced increase in type I collagen mRNA. Macrophages may contribute toward the repair response in dystrophic leg muscle through up-regulation of Spp1. An observed increase in matrix metalloproteinase 3 (Mmp3) expression may be a related event, as Spp1 is activated by Mmp3 cleavage (45). Finally, a chemotatic role has been suggested for Spp1 (46), suggesting that it may influence both fibrotic and inflammatory responses in DMD.
Chemoattractant RT–PCR and immunoblotting
Taken together, data support the participation of inflammation in general and macrophages in particular in the pathogenesis of dystrophinopathy. Chemoattractant proteins such as the chemokines selectively recruit leukocytes to the site of inflammation and thus are an important mediator of the disease process. To better document the participation of chemoattractants in mdx hindlimb pathology, we further evaluated muscle mRNA and protein content for three C–C class chemokines that were upregulated in mdx and one C–C chemokine seen as absent in both wild-type and mdx, by microarray. Consistent with the pattern of our microarray findings, RT–PCR supported upregulation of Scya2 (MCP-1) and Scya6 (C10), but failed to confirm the increase in Scya9 (MIP-1
) (data not shown). By immunoblot, increases were noted in MCP-1 (Scya2) and C10 (Scya6), consistent with both microarray and RT–PCR data (Fig. 1A). Protein levels for MIP-1 (Scya9) also were elevated in mdx, consistent with the microarray data. Finally, we evaluated expression of a C–C class chemokine that was called absent from mdx muscle by microarray, Scya12 (MCP-5), and found that it also was absent by both RT–PCR and western blot.
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Spp1 (minopontin/osteopontin) may serve a dual role in mdx limb pathology, as a chemoattractant of macrophage origin (46), potentially serving autocrine/paracrine functions, and as an inducer of fibrosis. Immunoblotting indicated that the elevated Spp1 mRNA seen in the microarray analysis translated into increased protein content in mdx muscle (Fig. 1B).
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Discussion |
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Although the direct consequences that lack of dystrophin has upon sarcolemmal organization are clear (47), current disease models do not adequately explain the temporal delay in symptom onset, the substantial phenotypic differences in human and animal models, or the total sparing of some muscle groups. DGC integrity is not essential to myofiber survival, at least not at all life stages or in all muscle classes or species. Given the high likelihood that secondary factors play key roles in muscular dystrophy, unbiased genome-wide screens are ideal for dissection of the molecular basis underlying this and other multifactorial diseases. Here, we used large-scale DNA microarrays to study pathogenic mechanisms in dystrophic mice. With a strategy that minimized inter-animal variability, used statistically sufficient replicates, and applied stringent data analysis, we identified 242 differentially expressed genes and ESTs. Most genes were novel to existing models of the dystrophic process. Data analysis was conservative and likely underestimates the extent of differential gene expression in mdx. The paradigm required absolute reproducibility across five independent replicates and used additional validation by SAM as a statistical measure of false positive rates. Finally, grouping differentially expressed genes into disease-relevant functional categories yields new insights into potentially important secondary factors operating in the pathogenesis of the muscular dystrophies.
Prior studies have implicated inflammation in the pathogenesis of muscular dystrophy (12,32,37,48,49), but, as yet, there has been little effort to characterize the full nature of the response (13). Major cellular elements, including early participation of mast cells and neutrophils, and a later role for T cells, have been previously identified in mdx and DMD patients. A major theme in our data was the extent of a highly coordinated response, with 72 identified genes for immune cell signaling or effector steps in the pathogenic cascade, including highly specific mediators of inflammation. Many of these genes globally participate in inflammatory responses, and thus are not specific to dystrophinopathy, but the patterned alterations in these genes must, nonetheless, be considered in comprehensive models of DMD and some may be no less valid therapeutic targets.
None of the prior gene profiling studies in muscular dystrophy (16,27,50) have identified up-regulation of inflammation-related genes of this breadth and resolution, most likely since earlier generation arrays surveying fewer genes were used. Broad transcriptional analysis of inflammation provides further insight into the disease process. The focal nature of lesions in muscular dystrophy is consistent with gradient-based local induction of inflammation by vasoactive/chemoattractant cytokines and chemokines. Knowledge of participating cytokines and chemokines in DMD may allow therapeutic use of specific receptor antagonists now under development. Moreover, the nature of cellular and cell autonomous participants in muscle inflammation also should direct adjuvant treatments to improve the efficacy of myoblast transfer therapies that now are severely hindered by early death of injected donor cells.
Data also yield further insight into the relationship between inflammation and the fibrotic changes that hinder muscle function and interfere with regeneration. Spp1 released by macrophages is a key factor in directing repair subsequent to myocardial infarction (44). Similarly, increased Spp1 mRNA and protein in dystrophic muscle supports a tight linkage between mononuclear cell infiltration and the regulation of muscle fibrosis. The up-regulation of 22 known extracellular matrix genes contrasts with the limited degree of fibrosis observed in the mouse (48). That fibril-forming collagen genes are up-regulated to nearly the same degree in mdx (present results) and DMD (16), suggests that post-transcriptional regulation is responsible for the detrimental fibrosis in humans. Approaches to ameliorate the devastating consequences of DMD might effectively target mRNA processing, translation, assembly or turnover of extracellular matrix components.
Since the discovery of dystrophin in 1987 (1), molecular biology has made tremendous advances in DMD via focused and in-depth, one-gene-at-a-time approaches. Gene profiling can accelerate the pace of discovery and more rapidly identify disease mechanisms, particularly in diseases with tightly interrelated and/or parallel mechanisms. The ability of microarray to point toward unrecognized or neglected mechanisms in disease processes is evident in these data. Similar representative sampling of muscle pathology and disease stages, with adequate replicates, is a difficulty in studies of human disease, particularly when the muscle response is progressive and heterogeneous, as in DMD. Chen et al. (16) used DNA microarray to carefully document altered gene expression in DMD and limb girdle muscular dystrophy. As in our study, these authors observed considerable evidence for muscle regeneration and fibrosis in DMD. In contrast to mdx, DMD patients exhibited downregulation of nuclear-encoded mitochondrial genes, suggestive of a general metabolic crisis in dystrophic muscle. It is likely that this crisis was not observed in mdx hindlimb data since, following a degeneration/regeneration cycle, mdx muscle ultimately recovers whereas DMD does not. Finally, evidence for muscle inflammation was observed both in DMD and in our study; the greater level of inflammatory response in mdx likely reflects the increased representation of these genes on the Affymetrix mouse chip versus the earlier generation human array chip used in the study of Chen et al. (16).
As a model of muscular dystrophy, the mdx mouse offers the ability to profile muscle pathology at various disease stages with adequate replicates and avoidance of the problem of heterogeneity within and between muscle biopsies that may represent only a small sample of human disease. Since there is not yet a consensus in management of DNA microarray data, analytic approaches must be carefully chosen to avoid the pitfalls of traditional statistics when applied to large databases. Use of a conservative strategy toward data acceptance, and methods such as SAM analysis to ensure that the overall false positive rate is understood and maintained below an acceptable threshold, likely under-represents, but yields considerable confidence in, those genes identified as differentially expressed.
Taken together, the molecular signature of skeletal muscle in mdx mice provides compelling evidence that pathogenic mechanisms in DMD are more complex than previously appreciated. The data draw particular attention to inflammation and the extracellular matrix as active participants in a disease process in which dystrophin deficiency plays a necessary, but not always sufficient, role by making muscle fibers vulnerable to secondary disease mechanisms. The sensitivity and specificity of DNA microarray is seen in the specific and biologically relevant pattern of alterations in gene expression in dystrophic muscles (leg) and its absence in muscles spared from the disease (extraocular; unpublished data). Of the 205 known genes that were differentially expressed in mdx limb, only 29 have been previously reported in DMD (see genes in bold in Tables 2 and 3; and see Supplementary Material). Comparing our findings to the broader literature for DMD and all animal models of dystrophinopathy, 75% of the known genes identified with differential expression patterns in mdx have not been previously reported. Finally, placing these data into the context that the DNA microarrays used here survey, at best estimate, 25–35% of the mouse genome, a conservative estimate is that as many as 1000 genes may be differentially expressed in dystrophic muscle. From this viewpoint, disease gene products can no longer be seen only in simple relationships with their local networks of interacting proteins, particularly when a multidimensional protein like dystrophin is associated with a complex disease like muscular dystrophy.
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Animals
Gastrocnemius and soleus muscles were dissected from 8-week-old male control (C57BL/10SnJ) and mdx (C57BL/10ScSn-Dmdmdx/J) mice (Jackson Laboratory, Bar Harbor, ME). To minimize variability, muscles from 10 mice were pooled for each of five independent replicate analyses of each mouse strain.
Target preparation
Total RNA was extracted using Trizol (GibcoBRL, Rockville, MD). RNA pellets were resuspended at 1 µg RNA/µl DEPC-treated water. Total RNA was prepared for use on Affymetrix (Santa Clara, CA) murine MG-U74A arrays, according to the manufacturer. Briefly, 8 µg of RNA was used in a reverse transcription reaction (SuperScript II; Life Technologies, Rockville, MD) to generate first-strand cDNA. After second-strand synthesis, double-strand cDNA was used in an in vitro transcription (IVT) reaction to generate biotinylated cRNA.
Array hybridization
After purification and fragmentation, 15 µg of cRNA was used in a 300 µl hybridization cocktail containing spiked IVT controls. Approximately 200 µl of cocktail was hybridized onto microarrays for 16 h at 45°C. Standard post-hybridization wash and double-stain protocols used an Affymetrix GeneChip Fluidics Station 400. Arrays were scanned using a Hewlett Packard Gene Array scanner.
Data analysis
Prior to analysis, U74A probe sets identified by Affymetrix as based upon incorrect GenBank sequences (2600) were excluded. Two data analysis methodologies were used. First, we used the Affymetrix Microarray Suite software (version 4.0), which generates fold change data and difference calls (increase/no change/decrease) based upon a proprietary algorithm. Pairwise comparisons were made between independent mdx and control samples run concurrently (i.e. mdx 1 versus control 1, mdx 2 versus control 2, etc.). The fold change (mdx/control ratio) was calculated for each pair of replicates and then an overall average was obtained as the mean of the five ratios. In first-pass data analysis, genes were considered to be differentially expressed in mdx versus control mice if they met the criterion that all five pairwise comparisons yield a consistent difference call (i.e. the five independent replicates for a given gene all had to show a consistent change of increase or decrease). Since this approach ensured confidence in the reproducibility of the results, we chose not to use an arbitrary fold change threshold cutoff (in practice, all genes meeting the absolute reproducibility standard exhibited mean fold change values 
1.5). Secondly, starting again with the full data sets from the five mdx/five control samples, we used a separate method adapted specifically for analysis of microarray data, SAM (19), with modifications as described by Porter et al. (20). Using the SAM results as a tool for exclusion of any false positives generated in the first analysis, we defined and reported only genes in the intersection of the two methods of data analysis as the differentially expressed genes in mdx leg musculature.
RT–PCR and western analysis
Total RNA was isolated from muscles in Trizol (Gibco BRL), following the manufacturer’s instructions. Reverse transcription was carried out using Superscript II RNase H-Reverse Transcriptase (Gibco BRL), with oligo(dT)12–18 primers. PCR amplification of cDNA used the PCR SuperMix kit (Gibco BRL). Primers used were specific for Scya2 (forward 5'-TCACCTGCTGCTACTCATTCA-3', reverse 5'-CACTGTCACACTGGTCACTCC-3'), Scya6 (forward 5'-CCAAGACTGCCATTTCATTC-3', reverse 5'-AAGCAATGACCTTGTTCCCA-3'), Scya9 (forward 5'-TGGCATATCTGGCTTTGTCA-3', reverse 5'-ATGGCTGTAGCTCAAGATGGT-3'), and Scya12 (forward 5'-TCGAAGTCTTTGACCTCAACA-3', reverse 5'-GGGAACTTCAGGGGGAAATA-3').
Protein was isolated from muscle homogenates (extraction buffer contained 0.3 M KCl, 0.1 M KH2PO4, 50 mM K2HPO4, 10 mM EDTA, 1 mM PMSF, 100 µM soybean trypsin inhibitor, 20 µM leupeptin, 200 µM TPCK and 50 mM DTT). Extracts were centrifuged at 10 000 g to obtain supernatant; equal amounts of protein were loaded on 16.5% Tris–tricine Ready Gels (Bio-Rad, Hercules, CA) and electrophoresed at 100 V for 3 h. Gels were transblotted onto nitrocellulose membranes and membranes were blocked in 5% milk in TBS-Tween-20 overnight at 4°C. Immunoblotting used rabbit anti-C10 (Scya6; CL9169AP; Cedarlane Laboratories, Hornby, Ontario, Canada), anti-MCP-1 (Scya2; 7202; AbCam, Cambridge, UK), anti-MIP-1 (Scya9; CL9185AP; Cedarlane Laboratories), anti-MCP-5 (Scya12; CL9183AP; Cedarlane Laboratories), and mouse anti-osteopontin (Spp1; MPIIIB10; Developmental Studies Hybridoma Bank, Iowa City, IA). Membranes were then incubated in donkey anti-rabbit or sheep anti-mouse Ig-HRP (Amersham, Buckinghamshire, UK). Immunodetection used the ECL Plus system (Amersham) and images were captured with a Kodak Image Station 440CF (Perkin Elmer Life Sciences, Boston, MA).
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Supplementary Material is available at HMG Online.
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Acknowledgements |
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We thank Marty Veigl for DNA microarray assistance, Claire Doerschuk and Eric Pearlman for helpful discussions, and Jonathan Law and Jason Feuerman for additional help with data analysis. This work was supported by grants from The Research Institute of University Hospitals of Cleveland (J.D.P.), Research to Prevent Blindness (Departmental Grant; Senior Scientific Investigator Award to J.D.P.), the Knights-Templar Eye Research Foundation (S.K.), the Muscular Dystrophy Association USA (J.D.P. and F.H.A.), The Evenor Armington Fund (J.D.P. and F.H.A.), and NIH Grants EY09834 (J.D.P.), EY12779 (J.D.P.), EY11998 (H.J.K.), EY12998 (F.H.A.) and P30 EY11373. J.D.P. is also supported by the Carl F.Asseff, M.D. Professorship in Ophthalmology.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Department of Ophthalmology, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106-5068, USA. Tel: +1 216 844 7053; Fax: +1 216 844 4792; Email: jdp7@po.cwru.edu
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TOPABSTRACTIntroductionResultsDiscussionMaterials and MethodsSUPPLEMENTARY MATERIALREFERENCES |
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M. D. GROUNDS and J. TORRISI Anti-TNF{alpha} (Remicade(R)) therapy protects dystrophic skeletal muscle from necrosis FASEB J, April 1, 2004; 18(6): 676 - 682. [Abstract] [Full Text] [PDF]
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J. V. Chakkalakal, M.-A. Harrison, S. Carbonetto, E. Chin, R. N. Michel, and B. J. Jasmin Stimulation of calcineurin signaling attenuates the dystrophic pathology in mdx mice Hum. Mol. Genet., February 15, 2004; 13(4): 379 - 388. [Abstract] [Full Text] [PDF]
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J. D. Porter, A. P. Merriam, P. Leahy, B. Gong, J. Feuerman, G. Cheng, and S. Khanna Temporal gene expression profiling of dystrophin-deficient (mdx) mouse diaphragm identifies conserved and muscle group-specific mechanisms in the pathogenesis of muscular dystrophy Hum. Mol. Genet., February 1, 2004; 13(3): 257 - 269. [Abstract] [Full Text] [PDF]
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I. A. Barash, L. Mathew, A. F. Ryan, J. Chen, and R. L. Lieber Rapid muscle-specific gene expression changes after a single bout of eccentric contractions in the mouse Am J Physiol Cell Physiol, February 1, 2004; 286(2): C355 - C364. [Abstract] [Full Text]
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D. Sanoudou, P. B. Kang, J. N. Haslett, M. Han, L. M. Kunkel, and A. H. Beggs Transcriptional profile of postmortem skeletal muscle Physiol Genomics, January 15, 2004; 16(2): 222 - 228. [Abstract] [Full Text] [PDF]
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 G. Cheng, M. J. Mustari, S. Khanna, and J. D. Porter Comprehensive Evaluation of the Extraocular Muscle Critical Period by Expression Profiling in the Dark-Reared Rat and Monocularly Deprived Monkey Invest. Ophthalmol. Vis. Sci., September 1, 2003; 44(9): 3842 - 3855. [Abstract] [Full Text] [PDF]
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S. C. Goetsch, T. J. Hawke, T. D. Gallardo, J. A. Richardson, and D. J. Garry Transcriptional profiling and regulation of the extracellular matrix during muscle regeneration Physiol Genomics, August 15, 2003; 14(3): 261 - 271. [Abstract] [Full Text] [PDF]
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J. D. Porter, A. P. Merriam, P. Leahy, B. Gong, and S. Khanna Dissection of temporal gene expression signatures of affected and spared muscle groups in dystrophin-deficient (mdx) mice Hum. Mol. Genet., August 1, 2003; 12(15): 1813 - 1821. [Abstract] [Full Text] [PDF]
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A. Hirata, S. Masuda, T. Tamura, K. Kai, K. Ojima, A. Fukase, K. Motoyoshi, K. Kamakura, Y. Miyagoe-Suzuki, and S.'i. Takeda Expression Profiling of Cytokines and Related Genes in Regenerating Skeletal Muscle after Cardiotoxin Injection: A Role for Osteopontin Am. J. Pathol., July 1, 2003; 163(1): 203 - 215. [Abstract] [Full Text] [PDF]
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R. Gilbert, R. W. R. Dudley, A.-B. Liu, B. J. Petrof, J. Nalbantoglu, and G. Karpati Prolonged dystrophin expression and functional correction of mdx mouse muscle following gene transfer with a helper-dependent (gutted) adenovirus-encoding murine dystrophin Hum. Mol. Genet., June 1, 2003; 12(11): 1287 - 1299. [Abstract] [Full Text] [PDF]
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D. Sanoudou, J. N. Haslett, A. T. Kho, S. Guo, H. T. Gazda, S. A. Greenberg, H. G. W. Lidov, I. S. Kohane, L. M. Kunkel, and A. H. Beggs Expression profiling reveals altered satellite cell numbers and glycolytic enzyme transcription in nemaline myopathy muscle PNAS, April 15, 2003; 100(8): 4666 - 4671. [Abstract] [Full Text] [PDF]
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M.C. Monici, M. Aguennouz, A. Mazzeo, C. Messina, and G. Vita Activation of nuclear factor-{kappa}B in inflammatory myopathies and Duchenne muscular dystrophy Neurology, March 25, 2003; 60(6): 993 - 997. [Abstract] [Full Text] [PDF]
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S. Noguchi, T. Tsukahara, M. Fujita, R. Kurokawa, M. Tachikawa, T. Toda, A. Tsujimoto, K. Arahata, and I. Nishino cDNA microarray analysis of individual Duchenne muscular dystrophy patients Hum. Mol. Genet., March 15, 2003; 12(6): 595 - 600. [Abstract] [Full Text] [PDF]
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 A. De Luca, S. Pierno, A. Liantonio, M. Cetrone, C. Camerino, B. Fraysse, M. Mirabella, S. Servidei, U. T. Ruegg, and D. Conte Camerino Enhanced Dystrophic Progression in mdx Mice by Exercise and Beneficial Effects of Taurine and Insulin-Like Growth Factor-1 J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 453 - 463. [Abstract] [Full Text] [PDF]
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J. N. Haslett, D. Sanoudou, A. T. Kho, R. R. Bennett, S. A. Greenberg, I. S. Kohane, A. H. Beggs, and L. M. Kunkel Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle PNAS, November 12, 2002; 99(23): 15000 - 15005. [Abstract] [Full Text] [PDF]
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A. D. Strand, J. M. Olson, and C. Kooperberg Estimating the statistical significance of gene expression changes observed with oligonucleotide arrays Hum. Mol. Genet., September 15, 2002; 11(19): 2207 - 2221. [Abstract] [Full Text] [PDF]
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K. Rouger, M. Le Cunff, M. Steenman, M.-C. Potier, N. Gibelin, C. A. Dechesne, and J. J. Leger Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice Am J Physiol Cell Physiol, September 1, 2002; 283(3): C773 - C784. [Abstract] [Full Text] [PDF]
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