Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice

Karl Rouger1, Martine Le Cunff1, Marja Steenman1, Marie-Claude Potier2, Nathalie Gibelin2, Claude A. Dechesne3, and Jean J. Leger1

1 Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche (UMR) 533, Faculté de Médecine, 44093 Nantes; 2 Centre National à la Recherche Scientifique UMR 7637, Ecole Supérieure de Physique et de Chimie Industrielle, 75005 Paris; and 3 Centre National à la Recherche Scientifique UMR 6548, 06108 Nice, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mdx mouse is a model for human Duchenne muscular dystrophy (DMD), an X-linked degenerative disease of skeletal muscle tissue characterized by the absence of the dystrophin protein. The mdx mice display a much milder phenotype than DMD patients. After the first week of life when all mdx muscles evolve like muscles of young DMD patients, mdx hindlimb muscles substantially compensate for the lack of dystrophin, whereas mdx diaphragm muscle becomes progressively affected by the disease. We used cDNA microarrays to compare the expression profile of 1,082 genes, previously selected by a subtractive method, in control and mdx hindlimb and diaphragm muscles at 12 time points over the first year of the mouse life. We determined that 1) the dystrophin gene defect induced marked expression remodeling of 112 genes encoding proteins implicated in diverse muscle cell functions and 2) two-thirds of the observed transcriptomal anomalies differed between adult mdx hindlimb and diaphragm muscles. Our results showed that neither mdx diaphram muscle nor mdx hindlimb muscles evolve entirely like the human DMD muscles. This finding should be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.

muscular dystrophy; mdx mice; complementary deoxyribonucleic acid microarray; differential gene expression


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DYSTROPHIN IS A CYTOSKELETAL protein located at the inner face of the cell membrane and is associated with cytosolic membrane glycoproteins (58). Its primary role is to stabilize the muscle plasma membrane (6). Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease occurring in humans that is caused by various mutations or deletions in the dystrophin gene, located on the short arm of the X chromosome (Xp21; see Refs. 23 and 25). This X-linked recessive myopathy affects 1 in 3,500 boys and is characterized by an absence of dystrophin protein expression. Dystrophin deficiency induces a fast, progressive, and severe dystrophy of skeletal and heart muscles, leading to death around the age of 20 yr (18).

Mice of the strain C57BL/10ScSn-Dmdmdx (mdx; X-linked muscular dystrophy) are commonly used as a model for DMD (7). Because of a point-mutation at position 3185 of the murine dystrophin gene (23, 41), mdx muscles lack the subsarcolemmal dystrophin protein, similar to human DMD muscles. However, in mdx mice, the hindlimb muscles differ histologically from diaphragm muscle, which more closely resembles DMD muscles. Hindlimb muscles display an initial period of intense necrosis in 2- to 3-wk-old mice (24). Later on, a sustained regenerative activity allows mdx hindlimb muscles to compensate for the necrosis (8, 12). Despite the lack of dystrophin, mdx hindlimb muscles either do not show obvious functional disability or express only mild weakness in adult mice. In contrast, mdx diaphragm muscle initially exhibits a regeneration process that does not compensate for massive degeneration and fails to restore muscle structure and function. This leads to significant extracellular matrix proliferation (fibrosis) associated with severe functional changes, including a reduction in maximal force production, elasticity, and twitch speed. This dramatic evolution in mdx diaphragm muscle appears similar to that observed in skeletal limb muscles of DMD patients (46). These differences in evolution between mdx and DMD muscle types lead to a questioning of the validity of mdx mice as a model in studies of DMD.

The recent development of cDNA microarrays provided a powerful experimental approach to determine simultaneously the molecular profile of several hundreds to several thousands of genes in defined physiological conditions (5, 14). It offered the possibility to identify molecular or cellular pathways not previously associated with the disease (26). Recently, Chen et al. (10) compared the expression profile of muscle biopsies from patients with DMD or alpha -sarcoglycan deficiency using oligonucleotide arrays. This global analysis of expression profiles revealed that 275/6,000 genes encoding proteins involved in diverse cellular functions like metabolism, immune response, cell growth, or differentiation and signaling were differentially regulated all along the progression of both human diseases. The authors demonstrated a multigenic impact of the unique genetic defect of dystrophin in DMD muscles. Presently, more than a decade after the discovery of the dystrophin gene and its involvement in DMD, no global view is available comparing the transcriptomal states of mdx diaphragm and hindlimb muscles and their evolution with aging.

In the present study, we used cDNA microarray technology to determine comparative gene expression profiles between hindlimb and diaphragm muscles of control (C57BL/10) and mdx mice. Previously, we had constructed four subtracted/normalized libraries containing cDNA clones of genes up- or downregulated in hindlimb or diaphragm muscles of 12-wk-old mdx mice (48) using a strategy based on suppression subtractive hybridization (SSH; see Ref. 13). These cDNA clones were printed on microarrays and profiled for gene expression in hindlimb and diaphragm muscles at 12 time points, ranging from 1 to 52 wk of age. We hypothesized that this global approach would provide a large-scale insight into the comparative remodeling of both mdx muscle types over the first year of the mouse life. Our aim was 1) to determine whether these murine muscle types actually represent a realistic animal model of the dramatic evolution observed in human DMD muscles and 2) to identify some of the transcriptomal landmarks and/or related molecular/cellular pathways leading to the distinct evolution between these two muscle types in mdx mice.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mouse strains and RNA isolation. To determine which and how many genes were affected by the primary deficiency of dystrophin during the life span of one year, the gene expression profiles of mdx diaphragm and hindlimb muscles were compared with the control (C57BL/10) counterparts, using cDNA microarrays. Breeding pairs of C57BL/10ScSn mice and of C57BL/10ScSn-Dmdmdx/J mice (41) were purchased from Jackson Laboratories and bred in a conventional animal care facility (veterinarian school). Only male animals were studied to avoid intersexual variation. Animals were killed by cervical dislocation at 12 different ages (1, 2, 3, 4, 5, 7, 9, 12, 16, 24, 36, and 52 wk) representative of the first year of the mouse life. Tissues were rapidly dissected, snap-frozen in liquid nitrogen, and stored at -80°C as samples pooled from at least six animals of the same age to eliminate any interindividual variation in expression levels. Total RNA was extracted using Trizol reagent (Life Technologies) and treated with DNase I. Absence of genomic DNA was systematically assessed in parallel PCR and RT-PCR experiments of purified RNA preparations using actin-specific primers. Poly(A)+ RNA was isolated using the Oligotex mRNA kit (Qiagen).

cDNA clone selection and functional classification. The microarrays used in this study contained 1,082 different cDNA clones; 1,019 out of 1,082 arrayed clones were issued from four subtractive libraries previously prepared by SSH between control and mdx hindlimb muscles and between control and mdx diaphragm muscle of 12-wk-old mice (Table 1; see Refs. 13 and 48). The sequence of 23% of these clones could not be matched to sequences deposited in public databases; therefore, they represented novel genes (C. A. Dechesne and A. V. Tkatchenko, unpublished observations). The remaining 77% represented known genes encoding diverse protein components that were distributed evenly among the six main functional categories (Table 2; see Ref. 1). Roughly one-quarter of the clones is involved in metabolism. The large representation of metabolic genes in our libraries correlates well with the energy-demanding function of the mdx muscles that are composed of numerous regenerated fibers. The next largest functional category is that of cell signaling (containing 15% of the genes), followed by gene/protein expression (11%). A few genes were represented on the microarray by several clones containing sequences from different parts of these genes. This redundancy provided useful repetitive determinations and the possibility to average the expression profiles of the same gene [e.g., parvalbumin, insulin-like growth factor II (IGF-II), titin, etc.]. An additional population of 63 cDNA clones was added to the microarrays. This population consisted of 3'-untranslated region PCR-amplified fragments from components of the multiprotein dystrophin system, nuclear factor-kappa B family proteins, and a few potassium channels (Table 1). Information on all clones (sequence, accession number, copy of the related publication; see Ref. 48) is available as Supplemental Material. Please refer to the Supplemental Material1 for this article (published online at the American Journal of Physiology-Cell Physiology web site).

                              
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Table 1.   Clone origin and expression status


                              
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Table 2.   Functional classification of the gene population

Microarray manufacturing, labeling, hybridizations, and imaging. cDNA microarrays were produced and hybridized as described previously (16, 17, 36). The 1,082 spotted cDNA fragments were cloned in the plasmid vector PCRII-TOPO (Invitrogen) and further amplified using universal sequences flanking the cloning site. The PCR products (0.4-1.5 kb) were precipitated, resuspended in 3× saline-sodium citrate, and robotically arrayed on polylysine-coated glass slides (GeneMachine; Omnigrid). First-strand complex cDNA probes were generated by incorporation of Cy3- or Cy5-dUTP (Amersham and Pharmacia) by reverse transcription of 1 µg of each pooled poly(A)+ RNA using Superscript II (Life Technologies). Each labeled cDNA from control and mdx muscles from mice of the 12 different ages was mixed with an equal amount of the corresponding 12-wk-old C57BL/10 mouse muscles used as control. To avoid unspecific DNA binding, mouse cot-1 DNA (GIBCO-BRL), yeast tRNA, and poly(dT) were added to the mixed Cy3- and Cy5-labeled probes before hybridization. Poly(A)+ RNA samples were alternatively labeled with either Cy3 or Cy5 to minimize the influence of either fluorochrome on the relative gene expression ratios. Hybridized arrays were scanned by fluorescence confocal microscopy (ScanArray 3000; GSI-Lumonics). Measurements were obtained separately for each fluorochrome at 10 µm/pixel resolution.

Analysis of array data. Fluorescence values and ratios for each cDNA array were analyzed using the ScanAlyze software package available at http://rana.stanford.edu/software. Previously described procedures and criteria were used to exclude defective array spots and to correct for differences in DNA labeling efficiency (16). All clones with either a weak hybridization signal (<1,000) or a defective signal (dust, spot deformation, pixel incoherence, etc.) were flagged and excluded from further analysis (see Supplemental Material). All array experiments were (minimally) performed as duplicates; duplicate expression ratios were averaged. Of the arrays used for further analysis, at least 75% of the spots had a regression correlation >0.85. In control experiments, the same poly(A)+ RNA was divided into two parts, each independently labeled with a different fluorochrome before mixing and hybridizing. The median values of the rough differential expression ratios [(signal Cy5)/(signal Cy3) for each gene] remained between -1.5 and 1.5 (Fig. 1A). This was consistent with previous reports on microarray reproducibility and sensitivity (16, 35-36, 38, 40, 45). A normalization procedure was performed to compensate for moderate differences commonly observed between Cy3 and Cy5 signals. Spots with a measured differential expression ratio between 1.5 and 0.66 were selected in each microarray. A linear regression was calculated for both fluorescent signals within this clone subpopulation. The calculated coefficients were then applied to all Cy5 values of the corresponding array to obtain normalized differential expression ratios. In further experiments, performed using different clone preparations, cDNA arrays, and cDNA targets, a differential expression ratio of <0.5 or >2 was observed as the threshold for experimentally reproducible differential expression (Fig. 1B).


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Fig. 1.   Determination of microarray reproducibility and sensitivity. A: normalized fluorescence signals and ratios from one cDNA array hybridized with a mixture of 12 vs. 12-wk-old mouse diaphragm cDNA, labeled with either Cy3 or Cy5. The graphs show that the nonsignificant median values of the differential expression ratios were comprised between 0.67 (1:1.5) and 1.5. B: fluorescence ratios were obtained from two cDNA arrays, both hybridized with a mixture of cDNA probes prepared from 52- and 12-wk-old mdx mice hindlimb muscles. Ratios of 0.5 and 2.0 represented the limits of interexperiment sensitivity.

Statistical analyses of the expression data were performed by using the Significance Analysis of Microarrays method (SAM; EXCEL; http://www-stat-class.stanford.edu/SAM/SAMServlet; see Ref. 52). The analyses were performed on the means of the log2-transformed Cy5-to-Cy3 or Cy3-to-Cy5 normalized ratios at each experimental time point. To determine which genes differed either in the control vs. mdx samples, or between both mdx muscle types, arrays were paired according to time point and to experiment. Differences between paired expression ratios of 2.0 (designated as the degree of changes in Ref. 52) were applied to cut off the initial list of significant ratio values deduced from the normalization procedure applied to each cDNA microarray followed by SAM analysis. A false discovery rate, that is an estimate of the percentage of false positives, was obtained after each SAM analysis. The selected genes were the most consistently detected, significantly changed genes and therefore the best candidates for genes modulated by the dystrophin deficiency (see RESULTS). To display the orderly features of the statistically selected data, normalized gene expression ratios were represented according to a color code based on a pseudo-color visualization matrix (Refs. 17 and 35 and see Fig. 5). Both threshold values (1.5 and 2.0) for corrected (normalized) fluorescence ratios were reflected in the presented pseudocolor images.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue and gene collection. To obtain a global and comparative vision of transcriptomal processes involved in control and mdx diaphragm and hindlimb muscles over the first year of mouse life, gene expression profiling was performed at 12 different time points. These time points encompassed two distinct mouse life periods that corresponded to young (1-5 wk old) and adult (7-52 wk old) age (29). Twelve-week-old control mice were initially chosen as the time point reference for the subtractive libraries (48) and were used in the present study as reference for all hybridization reactions. This age was generally considered pivotal for both mdx mice muscles (21, 29, 34, 37). It succeeds to the period of the most intense degeneration/regeneration processes affecting both types of mdx muscles (2-5 wk). It precedes the progressive degeneration and fibrosis that mainly affect adult diaphragm muscle (see additional Fig. 1 in Supplemental Material and Refs. 32 and 34).

The relevance of the 1,082 selected clones for the analysis of differential expression in mdx muscle tissue was enforced by the expression profiles of three different tissues of 12-wk-old control and mdx mice: diaphragm muscle, hindlimb muscles, and kidney. As shown in Fig. 2, a few tens of clones were found to be up- or downregulated in both muscle types, whereas the same clones were expressed equally in control and mdx kidney. These clones corresponded to clones previously identified as differentially expressed in mdx muscle using reverse and direct Northern blot analysis (13).


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Fig. 2.   Comparative expression profiling: hindlimb muscles, diaphragm muscle, and kidney cDNAs from 12 wk-old mdx mice were labeled with Cy5. The corresponding tissues from control mice were labeled with Cy3. cDNAs of the same tissue type were mixed in equal quantities and hybridized to independent arrays. Graphs show the distribution of both fluorescent signals for each clone in each tissue type.

Collection of differentially expressed genes. A complete set of 48 expression ratios in hindlimb and diaphragm muscles from control and mdx mice at 12 different ages was obtained for 843/1,082 clones (Table 1). The remaining 239/1,082 clones were undetectable or only partially detectable. Some of the 843 fully informative clones showed persistent expression changes of >2.0 relative to control muscle at all 12 time points, whereas others were only differentially expressed at certain time points. A first classification consisted of the selection of clones that showed modulation of expression >2.0, in at least two successive time points. This initial classification of temporal gene expression ratios (16, 17, 36) revealed that, at early ages, numerous genes were up- or downregulated in both control and mdx muscles compared with the age of 12 wk. From the ages of 7-9 wk until 1 yr, most of the developmentally regulated genes maintained a persistent differential expression in mdx muscles but no significant expression variations in either control muscle.

The analytical SAM procedure (52) was applied to the collection of the 843 detected clones to identify those that presented statistically significant expression changes in mdx muscles. The SAM analysis was independently applied to comparative time-paired experiments over the (1-5 wk) young and (12-52 wk) adult periods of mouse life (29). On the basis of the degree of significance (52) and an applied global ratio difference of 2.0, SAM analysis produced lists of genes potentially deregulated by the dystrophin deficiency (see SAM-related table in Supplemental Material). Figure 3 summarizes the SAM results obtained from the time-paired comparisons between mdx and control hindlimb (Fig. 3A) and diaphragm (Fig. 3B) muscles, and between both mdx muscle types (Fig. 3C), during the adult life period. No significant gene expression differences were found between control hindlimb and diaphragm muscles in the adult period (Fig. 3D). This suggested that the expression differences detected in and between mdx muscles during the second part of the mouse life span were solely because of dystrophin deficiency. SAM analysis was also performed on the same four muscle pairs during the (1- to 5-wk) young period where numerous genes displayed large differential expression ratios ranging from 2 to 80. Some genes were found to be statistically different between control hindlimb and diaphragm muscles, as well as between control and mdx muscle types and between mdx hindlimb and diaphragm muscles. These observations showed the difficulty to distinguish between the genetic program of normal muscle development and its eventual perturbation because of the dystrophin defect.


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Fig. 3.   Significance analysis of expression data: significance analysis of microarray (SAM) plots from the expression ratio data from time-paired experiments either between dystrophic and control hindlimb (A) and diaphragm muscles (B) or between both dystrophic (C) and control (D) muscle types of mature mice (age 12-52 wk).

After SAM analysis, 112 clones that were observed as up- or downregulated in mdx hindlimb and/or diaphragm muscles or between both mdx muscle types were finally selected as significantly differential. All cell functions, except cell division, were represented among the 112 differential clones (Table 2). Four functional categories (metabolism, structure/motility, signaling, and gene expression) and the category corresponding to the unknown genes were equivalently perturbed in mdx muscles; each contained roughly one-sixth of the 112 differentially expressed genes. Simultaneous representation of the 112 differential expression profiles, at 12 time points of the mouse life cycle, confirmed that the distribution of gene expression ratios could be separated into two well-defined periods (Fig. 4).


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Fig. 4.   Simultaneous representation of 112 significantly differential expression profiles in control and mdx hindlimb and diaphragm muscles. The Cy5-to-Cy3 fluorescence ratios measured relative gene expression in each of the four muscle types compared with that in the respective 12-wk-old control tissue. Graphs show the different distributions of gene expression ratios over the first year of the mouse life. Fluorescence values in control 12-wk-old mouse hindlimb muscles were arbitrarily set at 1 to stress that all expression measurements referred to that age. Fluorescence ratios in control 12-wk-old mouse diaphragm muscle are experimental values.

Expression profiling in control and mdx muscles. The 112 differential expression profiles were compared and classified into four different groups, as shown in Fig. 5. Within each profile group, we classified the cDNA clones by functional categories (Fig. 5). The first group contained clones with temporal gene expression profiles similar for both mdx muscle types but differing from profiles of their control counterparts (n = 38, subset I). In this group, dystrophin deficiency induced deregulation of numerous genes, associated with membrane disorganization, sarcomere desintegrity, and gene expression. In both adult mdx muscle types, the point mutation in the dystrophin gene clearly had similar multiple effects on diverse transcriptomal activities. The three remaining groups in Fig. 5 were composed of clones that showed differential expression, either only in mdx diaphragm muscle (n = 21, subset IIa) or only in mdx hindlimb muscles (n = 40, subset IIb), or that showed different differential expression in both types of mdx muscle (n = 13, subset IIc).


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Fig. 5.   Clustered gene expression patterns of 112 out of 1,082 clones that display significant and persistent (lifelong) differential expression profiles between mdx and control mice and/or between hindlimb and diaphragm muscles. Each row represents a different cDNA clone, and each column pertains to 1 of the 12 different time points. Normalized data values displayed in shades of red and blue represent increased and decreased expression, respectively, in control and mdx muscles relative to the control 12-wk-old corresponding tissues. The 112 differential clones were clustered into four categories (I, IIa, IIb, and IIc) according to similarities and/or differences in their expression profiles between control and/or mdx muscles. Clones present in each expression category were further grouped according to the functional categories of the gene products (1), and finally they were classified by their increased or decreased expression levels.

Transcriptomal differences observed between both mdx muscle types could be summarized as follows: 1) mdx diaphragm muscle preferentially presented an upregulation of certain genes encoding proteins involved in structure and motility of myofibers and in gene expression, 2) in contrast, mdx hindlimb muscles presented a relative downregulation of other genes encoding proteins involved in structure and motility of myofibers, in association with a general metabolic crisis not observed in diaphragm muscle, 3) both mdx muscles presented interestingly opposite expression levels of parvalbumin and alpha 1-protease inhibitor type 1, 3, and 5 genes, known to be involved in calcium homeostasis of muscle cells. These genes displayed a life-long downregulation in mdx diaphragm muscle and an upregulation in hindlimb muscles. These differences in expression profiles indicated that the same monogenetic defect has distinct life-long transcriptomal consequences in these two functionally related tissues. Thirteen of the 21 differential unknown genes, i.e., with limited or no similarity to known genes, were found in subset IIb; these genes clearly contribute to the genetic differences between both mdx muscle types.

In addition, we observed that more than two-thirds of the 112 differentially expressed genes were upregulated (Fig. 4). Such an imbalance in the quantity of up- and downregulated genes in each mdx muscle could simply be because of the limited ability of the microarray method to detect genes with low expression. Most likely, no biological meaning should be attached to this finding. A selection of 55 genes showing the most differential expression is presented in Table 3. A complete report of expression data, gene identification, and some examples of individual expression profiles is available on-line for public access (see Supplemental Material).

                              
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Table 3.   Main genes up- or downregulated in hindlimb and/or diaphragm muscles over the first year of the mdx life


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first report of large-scale temporal gene expression profiling in two mdx muscles. The data were obtained using arrays containing 1,082 cDNA clones selected from four substractive libraries prepared from control and mdx diaphragm and hindlimb muscles (48). The application of the stringent analytical method SAM (52) to the numerous expression data drastically limited the number of informative genes. Roughly 10% of all the clones previously isolated by SSH (48) were finally detected as being differentially expressed in our microarray experiments. Different factors [the redundancy observed in the SSH procedure applied on 12-wk-old muscles only (48), the cellular complexity of the tissue samples used for the targets, the insensitivity of the array method when analyzing weakly expressed genes, etc.] could explain the discrepancy observed between the two systems of gene selection and their limits of application.

The present differential gene set of 112 out of 1,082 in mdx mice is parallel to the set of 275 out of 6,000 human DMD differential genes identified in a recent study analyzing expression profiles of muscle biopsies of patients with DMD and patients with alpha -sarcoglycan deficiency, using oligonucleotide arrays (see Table 3, Ref. 10, and http://microarray.NMCTesearch.org/resources.htm). Both murine and human gene sets constitute the largest (but obviously not exhaustive) collection of transcriptomal landmarks in dystrophin-deficient muscles. A flow chart, indicating the pathological cascades initiated by dystrophin deficiency, was proposed to interpret the oligonucleotide microarray expression profiling study of human DMD muscles (10). It listed the main molecular circuits or pathways that are perturbed in dystrophic cells, their possible progressive interactions, and the relationship between cell autonomous and microenvironmental changes. With regard to the proposed flow chart and the related profiles in human dystrophic muscles, two main conclusions can be drawn from our expression results in mdx muscles.

Conclusion 1: Dystrophin deficiency initiates globally identical pathological cascades in murine and human dystrophic muscles. The most central feature in all dystrophic muscles of both animal species clearly was the occurrence of perturbations in calcium homeostasis (21). This is obviously connected to the primary function of dystrophin, which is to stabilize the sarcolemmal membrane of myofibers. In DMD (4) and mdx myofibers (30, 51), dystrophin deficiency results in membrane lesions, leading to chronic leakage of intracellular constituents and increased intracellular calcium concentration (24). As previously documented in mdx hindlimb myofibers, this abnormal calcium accumulation causes most of the multiple progressive downstream consequences of the dystrophin defect by mediating an elevated rate of protein degradation (15, 44; Fig. 5, subset I).

In addition, numerous other (secondary) markers involved in proliferation/dedifferentiation and inflammation processes were identified in both murine and human dystrophic muscles, indicating that the absence of dystrophin initiates the same perturbations in the genetic machinery of both animal species. Examples were osteoblast-specific factor 2 (Osf2), IGF-II, lyzozyme M and P, etc., which were upregulated in DMD and both mdx muscles. Osf2, a chondrocyte-related transcript, was one of the strongest upregulated genes in both muscle types in adult mdx mice (expression ratios of 8 and 4 in diaphragm and hindlimb muscles, respectively). Its persistent upregulation, which was detected in both animal species (10), was linked to that of human versican, a large chondroitin sulfate proteoglycan known to stimulate the proliferation of chondrocytes, and to that of three other chondrocyte- and bone-related transcripts in dystrophic muscles (10, 56).

Using the murine arrays, we identified novel markers not identified on the human array. Most of these markers belonged to the circuits of the proposed pathological flow (10). The central role of calcium homeostasis disruption in dystrophic cells was confirmed by expression variations in several genes: the parvalbumin gene, genes encoding proteins like calcium/calmodulin-dependent protein kinase, and a few protease inhibitors that are controlled by the intracellular calcium concentration. Our murine expression arrays revealed for the first time a concomitant and persistent upregulation of the transcriptionally related components H19 and IGF-II in mdx muscles. Normally, both genes are expressed in many tissues of endodermal and mesodermal origin during embryogenesis (28, 33), but they are downregulated at birth. IGF-II and H19 undergo genomic imprinting in control mouse and human tissues, resulting in parent-specific monoallelic expression. Perturbations in the expression of both genes, including loss of imprinting, were evidenced in several cancers, including rhabdomyosarcoma (9, 55). The persistent increased expression of both genes in mdx muscles (and of IGF-II in human dystrophic hindlimb muscles) underlines the importance of massive muscle cell proliferation and dedifferentiation in each mdx muscle type along the mouse life span. In parallel, it was recently documented that expression of IGF-II ameliorates the mdx phenotype by downregulating programmed cell death, as shown by a reduction of degenerative regions and of the number of centrally located nuclei (42, 43). In addition, a link between IGF-II expression and collagen accumulation was proposed recently, suggesting a role of this protein in the mechanism of fibrosis in dystrophic cells (22, 31). The question whether these factors really do have a beneficial influence in terms of potential therapeutic targets is still open.

Our results on the expression of genes encoding dystrophin-related or -associated proteins in mdx muscles contrasted surprisingly with those obtained in DMD muscles (10). We found that alpha 1-syntrophin and alpha -sarcoglycan genes were not significantly deregulated in mdx hindlimb and diaphragm muscles (ratios ~1.7), whereas both showed a dramatic reduction in human dystrophic muscles. More generally, in murine muscles, we observed that genes encoding utrophin- and dystrophin-associated proteins (like sarcoglycans, dystrobrevins, and nitric oxide synthase) did not display any significant expression changes in mdx muscles. The weak expression levels of the corresponding genes and/or the probe choice could partially explain this discrepancy between human and murine dystrophic muscles.

Conclusion 2: Dystrophin deficiency causes different perturbations in the transcriptomal circuits of mdx diaphragm and hindlimb muscles. The observed expression differences (Fig. 5, subsets II) could explain some of the known histological and functional differences between both mdx muscle types. Thus the opposite expression levels of parvalbumin genes in both mdx muscle types probably reflected differences in the intracellular calcium content. Such differences may in turn modulate the calcium-dependent proteolysis in a muscle type-specific way. Low levels of parvalbumin (<2.5-fold), a protein that links calcium and a relaxing factor in fast muscles (39), together with the concomitant downregulation of genes encoding alpha 1-protease inhibitors type 1, 3, and 5 (<2.2-fold), could provoke an intensed proteolytic activity within the mdx diaphragm cells and a massive degeneration/disorganization with aging. Relatively low levels of calcium, resulting from higher contents of parvalbumins and of protease inhibitors, apparently prevented the mdx hindlimb cells from such a rapid and dramatic degeneration. These observations led us to propose a more precise reexamination of the protective or deleterious role of intracellular calcium levels and their influence on the activity of related proteases in the dystrophic muscle cells. These proteins could be used as either pharmaceutical or genetic targets for future therapy.

Besides differences in calcium homeostasis, a global comparison of the differential patterns observed in mdx diaphragm and hindlimb muscles (Fig. 5, subsets IIa and IIb, respectively) indicated that most differential genes were upregulated in diaphragm muscle but downregulated in hindlimb muscles. According to the corresponding encoded functions, the strongest represented differential gene sets were involved either in structure/organization of the muscle fibers or in their energy machinery. Markers transiently expressed during control muscle development were found to be continuously upregulated in adult mdx diaphragm muscle, e.g., myosin light chain II (>2.1-fold), troponin I (>2.1-fold), helicase II/Gu (>2.7-fold), and atrial/fetal myosin light chain I and III (>3.1-fold; see Refs. 50 and 54). The regeneration processes, associated with the upregulation of such genes, may function to compensate for the lifelong structural disorganization of mdx diaphragm fibers, resulting from a continuous and intense intracellular proteolytic activity. During the same period, a few other genes, also implied in the assembly of sarcomeric and cytoskeletal structures during cellular differentiation and the maintenance of sarcomeric integrity (53), e.g., titin (<1.7-fold) and myosin heavy chain IIa and IIb (respectively <1.8-fold and <3-fold), were downregulated in mdx hindlimb muscles. Such contrasted expression patterns do not explain why, in adult mdx mice, a sarcomeric organization remains present in hindlimb fibers, whereas it is finally replaced by connective tissue in diaphragm muscle (as human dystrophic hindlimb fibers) fibers (46, 47).

The other main differences observed between the expression profiles of both mdx muscles concerned numerous genes involved in energy metabolism and mitochondrial functions [e.g., glucose transporter GLUT4, pyruvate kinase, and NADH dehydrogenase (Fig. 3, Table 3, and Refs. 3 and 57)]. Downregulation of these genes, exclusively observed in mdx hindlimb muscles, indicated an impaired ATP synthesis and an alteration of metabolite supply for muscle activity. These findings served as a confirmation of observations by others that showed that the hindlimb (peripheral) muscles in DMD patients (2, 11, 49) and mdx mice (19, 20, 27) are affected by a generalized dysfunction of their energy machinery. The concomitant observation that the same gene set did not display differential expression in mdx diaphragm muscle indicated that the progressive and dramatic destruction of fibers from this muscle type possibly occurs without large perturbations of the metabolic machinery. Taken together, our findings suggested that the metabolic crisis is a process that occurs secondary to other decisive inputs, like membrane leakage and the related deregulation of intracellular calcium concentration, which appear to be acting on all dystrophic cells.

Finally, the present results show that neither mdx diaphragm muscle nor mdx hindlimb muscles are animal muscle models accounting entirely for the complex pathophysiological cascades occurring in human DMD muscles. This finding is especially important with regard to the numerous pharmaceutical and genetic therapy experiments in which murine peripheral muscles were used for drug/gene injection. Our observation of different involvement of the functional circuits in the long-term evolution of both mdx muscle types should now be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.


    ACKNOWLEDGEMENTS

We thank Agnes Hivonnait, Patrick Guyot, and Jacques Noireaud for assistance with animal care and facilities. We are grateful to Denis Escande, Jean-Jacques Schott, and Jean Rossier for helpful discussions and reading of the manuscript and to Philippe Marc for initial assistance in interpreting microarray image data.


    FOOTNOTES

M. Steenman was supported by a grant from the Institut National de la Santé et de la Recherche Médicale. This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National à la Recherche Scientifique, l'Association Française contre les Myopathies, and la Fondation de la Recherche Médicale.

Address for reprint requests and other correspondence: J. J. Léger, Inserm U533, Faculté de Médecine, 1 rue Gaston Veil, 44093 Nantes, France (E-mail: Jean.Leger{at}nantes.inserm.fr).

1 Supplemental material to this article is available online at http://ajpcell.physiology.org/cgi/content/full/283/3/C773/DC1.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 24, 2002;10.1152/ajpcell.00112.2002

Received 12 March 2002; accepted in final form 17 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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