Physiol. Genomics Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 25: 85-95, 2006. First published December 20, 2005; doi:10.1152/physiolgenomics.00226.2005
1094-8341/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Appendix
Right arrow All Versions of this Article:
25/1/85    most recent
00226.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Lunteren, E.
Right arrow Articles by Leahy, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Lunteren, E.
Right arrow Articles by Leahy, P.
Received 8 September 2005; accepted in final form 15 December 2005.
Physiological Genomics 25:85-95 (2006)
American Physiological Society © 2006 American Physiological Society

Gene expression profiling of diaphragm muscle in {alpha}2-laminin (merosin)-deficient dy/dy dystrophic mice

Erik van Lunteren1, Michelle Moyer1 and Patrick Leahy2

1 Pulmonary and Critical Care Division, Department of Medicine, Case Western Reserve University and Cleveland Veterans Affairs Medical Center, Cleveland, Ohio
2 Comprehensive Cancer Center of Case Western Reserve University and University Hospitals Health System, Cleveland, Ohio


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Deficiency of {alpha}2-laminin (merosin) underlies classical congenital muscular dystrophy in humans and dy/dy muscular dystrophy in mice and causes severe muscle dysfunction in both species. To gain greater insight into the biochemical and molecular events that link {alpha}2-laminin deficiency with muscle fiber necrosis, and the associated compensatory responses, gene expression profiles were characterized in diaphragm muscle from 8-wk-old dy/dy mice using oligonucleotide microarrays. Compared with age-matched normal muscle, dystrophic diaphragm was characterized by predominantly augmented gene expression, irrespective of the fold-change threshold. Among the 69 genes with at least plus or minus twofold significantly altered expression, 30 belonged to statistically overrepresented Gene Ontology (GO) biological process groups. These covered four specific themes: development including muscle development, cell motility with an emphasis on muscle contraction, defense/immune response, and cell adhesion. An additional 11 gene transcripts were assigned to more general overrepresented GO biological process groups (e.g., cellular process, organismal physiological process); the remaining 28 did not belong to any overrepresented groups. GO cellular constituent assignment resulted in the highest degree of overrepresentation in extracellular and muscle fiber locations, whereas GO molecular function assignment was most notable for various types of binding. RT-PCR was performed on 38 of 41 genes with at least plus or minus twofold significantly altered expression that were assigned to overrepresented GO biological process groups, with expression changes verified for 36 of 38 genes. These results indicate that several specific groups of genes have altered expression in response to genetic {alpha}2-laminin deficiency, with both similarities and differences compared with data reported for dystrophin-deficient muscular dystrophies.

muscular dystrophy; skeletal muscle; development; contraction; cell adhesion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{alpha}2-LAMININ (MEROSIN) plays an important role in muscle cell attachment by acting as a link between extracellular collagen, on the one hand, and both {alpha}-dystroglycan of the dystrophin-glycoprotein complex and {alpha}7ß1-integrin of the integrin system, on the other (6, 29, 31). Its deficiency underlies human classical congenital muscular dystrophy as well as murine dy/dy muscular dystrophy (12, 15, 31, 48, 49). The human disease presents with hypotonia neonatally or within the first year of life, muscle weakness is profound, most subjects never gain the ability to walk, and various degrees of respiratory failure are commonly problematic (10, 31, 46). The disease in the dy/dy mouse model is manifested by the early onset of marked progressive limb and respiratory muscle weakness, reduced body size, and a life span typically of 2–6 mo (1, 30). Thus both human and mouse {alpha}2-laminin deficiency diseases are phenotypically quite severe.

Human classical congenital muscular dystrophy is quite uncommon. Hence there is a paucity of information available about limb muscles, and even less about respiratory muscles, in this disease. Studies in the mouse dy/dy model have documented the extent of skeletal muscle weakness and the concomitant histological changes. Findings have included reduced force per cross-sectional area (14, 43, 44, 45), slowing of isometric contractile kinetics (14), increased resistance to fatigue (14, 44, 45), reduced passive distensibility (21), changes in fiber subtype distribution to a slower phenotype (14), increased variability in fiber size with evidence of both necrosis and fiber regeneration (7, 14, 30, 42), fragmented basement membranes (48), and increased fibrosis and collagen content (7, 21, 30, 41, 48). However, even in mice there is a very incomplete understanding of the cellular, biochemical, and molecular events underlying these changes that occur as a result of the {alpha}2-laminin deficiency.

Gene expression arrays have been utilized recently for the study of skeletal muscle from dystrophin-deficient human Duchenne muscular dystrophy (DMD) and murine mdx muscular dystrophy (5, 13, 34–36, 38). This approach has not only extended the understanding of previously appreciated cellular processes but also identified formerly unsuspected events that result from dystrophin deficiency, e.g., the highly intense inflammatory process (34). Comparisons of these data with gene expression studies in other genetic muscle diseases (5, 39) are starting to provide insight into disease-specific vs. disease-shared cellular and molecular responses, but as yet a large portion of the spectrum of muscle membrane-associated diseases has not been examined.

The hypothesis of the present study is that {alpha}2-laminin deficiency alters diaphragm muscle gene expression, and does so predominantly in areas involved in muscle injury and attempted repair. Furthermore, the profile of specific genes with changed expression has both similarities and dissimilarities to that reported previously in skeletal muscle from mice and humans with genetic abnormalities of the dystrophin-glycoprotein complex. Studies were performed with a oligonucleotide-based gene expression array system, and genes with significantly altered expression that belonged to overrepresented Gene Ontology biological process groups were verified using quantitative PCR.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Approval for these studies was obtained from the Institutional Animal Care and Use Committee at the Cleveland Veterans Affairs Medical Center. Dystrophic dy/dy mice (129P1/ReJ-Lama2dy) and their normal (+/?) controls were obtained from Jackson Laboratories (Bar Harbor, ME) and studied at an age of 8 wk. Animals were deeply anesthetized, and diaphragm muscle was removed surgically. Muscles from three mice were pooled into a single sample to assure sufficient tissue for analysis without need for double-round amplification protocols and their attendant problems. A total of 12 dystrophic and 12 normal animals provided replicate samples of dystrophic (n = 4) and normal (n = 4) diaphragm, with each sample thus containing pooled muscle from three mice.

Muscle was frozen in liquid nitrogen and stored at –80°C. Subsequently, total RNA was extracted using Trizol (Gibco BRL, Rockville, MD). The RNA pellets were resuspended at 1 µg RNA/µl diethyl pyrocarbonate (DEPC)-treated water. Samples were subjected to the cleanup protocol from a Qiagen (Valencia, CA) RNeasy Total RNA minikit. Total RNA was prepared for use on Affymetrix (Santa Clara, CA) mouse microarrays, according to the manufacturer's protocol. Briefly, 8 µg of RNA were 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. After purification and fragmentation, 15 µg of biotin-labeled cRNA were used in a 300-µl hybridization cocktail containing spiked transcript controls. Two hundred microliters of cocktail were loaded onto Affymetrix MOE 430A microarrays and hybridized for 16 h at 45°C with agitation. Standard posthybridization washes and double-stain protocols used an Affymetrix GeneChip Fluidics Station 400. Arrays were scanned using a Hewlett Packard Gene Array scanner and analyzed with Affymetrix MAS 5.0 software. The data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE3252.

Statistical analysis was done with Bayesian analysis of variance for microarrays (BAM), using BAMarray 1.0 software (http://www.bamarray.com/) (19). BAM relies on a special type of inferential regularization (i.e., borrowing strength across the data), allowing it to balance the number of false detections against false nondetections. Genes whose expression was significantly changed by BAM were then further selected based on consistent and appropriate present and absent calls in all four samples of each group per Affymetrix MAS 5.0 software (marginal calls were accepted only if that gene was called present for all 3 other samples in that group). For each group, signals were averaged, and then fold changes were calculated based on group average data. Except as indicated otherwise, analysis focused on genes whose expression changed at least plus or minus twofold in dystrophic compared with control muscle. Hierarchical clustering was performed with Spotfire Decision Site software (version 8.1.1; Spotfire, Somerville, MA). To assign biological meaning to changes, the subsets of genes that met the above criteria were analyzed with the Gene Ontology (GO) classification system, using DAVID 2.0 software (http://apps1.niaid.nih.gov/david/) (8). Overrepresentation of genes with altered expression within specific GO categories was determined with the one-tailed Fishers exact probability modified by the addition of a jackknifing procedure, which penalizes the significance of categories with very few (e.g., 1 or 2) genes and favors more robust categories with larger numbers of genes (17).

Confirmation of changes in gene expression was performed with real-time PCR. Testing was done with two normal muscle samples and three dystrophic muscle samples, using the same tissue that had been used for gene expression arrays, and was performed for 48 genes. Real-time PCR studies were performed with an Applied Biosystems ABI 7900HT unit with automation attachment (Foster City, CA). This unit is capable of collecting spectral data at multiple points during a PCR run. To execute the first step and make archive cDNA, 3 µg of total RNA were reverse transcribed in a 100-µl reaction using Applied Biosystems enzymes and reagents in accordance with the manufacturer's protocols. Three candidate genes were used for endogenous control determination: GAPDH and ß-actin (general housekeeping genes) and TATA-binding protein (TBP; a general transcription factor associated with the RNA polymerase II transcription apparatus). RNA samples were accurately quantitated using a Nanodrop Technologies ND-1000 spectrophotometer (Wilmington, DE). Equal amounts of total RNA were reverse transcribed and then used in PCR amplifications. GAPDH had very little variation in expression across the sample set and therefore was chosen as the endogenous control. Because many of the target genes of interest were signaling molecules and likely to be expressed at low levels, we opted for a low dilution factor so as to create an environment more conducive to obtaining reliable results. The cDNA reaction from above was diluted by a factor of 10. For the PCR step, 9 µl of this diluted cDNA were used for each of three replicate 15-µl reactions carried out in a 384-well plate. The genes tested and their respective Applied Biosystems mouse assay catalog numbers are listed in Supplemental Appendix S1 (available at the Physiological Genomics web site).1 Standard PCR conditions were used for the Applied Biosystems assays: 50°C for 2 min, followed by 95°C for 10 min, followed by 40 cycles of 95°C for 15 s alternating with 60°C for 1 min each. PCR confirmation of gene expression array data required that the direction of the change in expression had to be the same with quantitative PCR as with gene expression arrays and, in addition, that gene expression measured with PCR had to be consistently higher (for increases) or lower (for decreases) in all three dystrophic samples compared with both normal muscle samples for all six pairwise comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
There were 66 known genes with increased and 3 known genes with decreased expression in dystrophic compared with normal diaphragm, using the cutoff of at least a plus or minus twofold change in addition to consistent present calls by Affymetrix software and statistical significance by BAM. The substantially larger number of increases compared with decreases was also noted when other fold-change cutoffs were chosen: e.g., 104 increases vs. 5 decreases for a fold change of at least ±1.8, and 218 increases vs. 27 decreases for a fold change of at least ±1.5. Results of hierarchical clustering of the 69 genes with at least plus or minus twofold changed expression in dystrophic compared with control diaphragm muscle are presented in Fig. 1. Some variability in gene expression was noted within each group, but this was modest compared with the differences between groups.


Figure 1
View larger version (43K):
[in this window]
[in a new window]
  		Fig. 1. Hierarchical clustering of 69 genes with more than ±2-fold changed expression in dystrophic compared with control diaphragm muscle. Fold-change values are presented for 4 control samples (left) and 4 dy/dy dystrophic samples (right). Red indicates increased expression compared with average values in control muscle, whereas green indicates reduced expression.

 
GO biological processes.
GO biological function assignment was possible for 41 of the 69 genes with at least plus or minus twofold changed expression in dystrophic compared with normal diaphragm. The categories with overrepresentation were most notable for belonging to the following four themes: development including muscle development, cell motility with an emphasis on muscle contraction, defense/immune response, and cell adhesion. These four themes together covered 30 genes, with several genes assigned to more than one category. In addition, there were a number of generally less specific overrepresented GO terms (e.g., cellular process, cellular physiological process, organismal physiological process) that covered an additional 11 genes. The above genes with at least plus or minus twofold expression are listed in Table 1. The remaining 28 genes with at least plus or minus twofold expression did not belong to any overrepresented GO biological process groups and are listed in Table 2.


View this table:
[in this window]
[in a new window]
  		Table 1. Genes with at least ±2-fold altered expression that belonged to overrepresented GO biological process groups

 

View this table:
[in this window]
[in a new window]
  		Table 2. Genes with at least ±2-fold altered expression that did not belong to overrepresented GO biological process groups

 
The cell motility theme was composed of the terms cell motility (7 genes, P = 0.000012), muscle contraction (7 genes, P = 0.000000057), and striated muscle contraction (4 genes, P = 0.000017). This group includes three contractile proteins (Myh3, Myh8, and Myl4) as well as two molecules that bind to contractile proteins (Mybph and Tnnt2).

The development theme included the general term development (17 genes, P = 0.000034), the specific term muscle development (8 genes, P = 0.000000041), and other terms such as morphogenesis (15 genes, P = 0.000014), organogenesis (14 genes, P = 0.000012), cell differentiation (7 genes, P = 0.0012), and regulation of development (5 genes, P = 0.0012). Several of these genes are shared with the cell motility group (e.g., Myh3, Myl4, Myh8, Tnnt2, Mybph). Ankrd2 is involved in muscle hypertrophy, whereas Myog and Igf2 are involved in cellular proliferation and differentiation.

The defense response theme included the general terms defense response (9 genes, P = 0.040) and immune response (8 genes, P = 0.034). There were a large number of specific terms identified, seven of which dealt with antigen presentation and processing (each with 3–5 genes, range P = 0.000000078 to P = 0.0016) and five of which dealt with lymphocyte and T cell activation and differentiation (each with 2–4 genes, range P = 0.0064 to P = 0.036).

The cell adhesion theme consisted of only a single GO term, cell adhesion (9 genes, P = 0.0014). One of the genes in this group was Lama2, whose expression was decreased as expected based on the known genetic defect in dy/dy mice. All eight of the other genes in this group had increased expression, among which Mybph was already mentioned above. Most of the others (Postn, Spp1, Lgals3, Thbs4, Ncam1, Col1a2) have well-established roles in extracellular adhesion or the structure of the extracellular matrix.

The genes with at least plus or minus twofold significantly changed expression that belonged to other overrepresented GO biological function groups (n = 11) or did not belong to any groups (n = 28) generally cover a range of different functions. However, a few of these may be of particular relevance to skeletal muscle function, including two acetylcholine receptor subunits, Chrng and Chrna1, as well as Ankrd1 (which is related to Ankrd2, a gene that was assigned to the developmental theme, as noted above).

GO cellular constituent.
GO cellular constituent assessment was performed on the group of genes with at least plus or minus twofold altered expression in dystrophic compared with normal diaphragm muscle, and assignment was possible for 59 of the 69 genes. The categories with overrepresentation were most notable for the following themes: extracellular, muscle fiber, and cytoplasm. The extracellular and cell surface theme included the term extracellular (29 genes, P = 0.00000036), extracellular space (28 genes, P = 0.000000082), extracellular matrix (11 genes, P = 0.00000086), and external side of plasma membrane as well as cell surface (the same 5 genes, P = 0.000080 and P = 0.00051, respectively) (Table 3). The muscle fiber theme included the terms sarcomere, myofibril and muscle fiber (the same 6 genes, P = 0.0000010, P = 0.0000011, and P = 0.0000016, respectively), striated muscle thick filament and myosin (the same 4 genes, P = 0.000016 and P = 0.0012, respectively), and actin cytoskeleton (5 genes, P = 0.0093). The cytoplasm term had a more modest level of significance (24 genes, P = 0.0070).


View this table:
[in this window]
[in a new window]
  		Table 3. Genes assigned to overexpressed GO cellular constituent terms related to extracellular and cell surface locations

 
GO molecular function.
GO molecular function assignment was possible for 58 of 69 genes with at least plus or minus twofold changed expression in dystrophic muscle. The molecular function categories with overrepresentation revolved predominantly around the theme of binding. These included the general category of binding (41 genes, P = 0.014) as well as subcategories of protein binding (18 genes, P = 0.00031), calcium ion binding (7 genes, P = 0.020), cytoskeletal protein binding and actin binding (the same 4 genes for both, P = 0.012 and P = 0.0043, respectively), heparin binding and glycosaminoglycan binding (the same 3 genes for both, P = 0.013 and P = 0.022, respectively), and immunoglobulin binding (2 genes, P = 0.033).

Genes with lesser degrees of altered expression.
There are several previous studies of mdx mouse muscular dystrophy that have used lower fold-change values as a cutoff for inclusion, most commonly ±1.5-fold change (35, 36). The GO annotation analysis was redone using a threshold of at least ±1.5-fold change in gene expression to determine whether incorporation of a larger number of genes in the functional classification analysis would alter any of the functional groups identified. The major biological processes themes of cell motility, development, defense/immune responses, and cellular adhesion were confirmed in this larger group, as were the general conclusions derived from the cellular constituent and molecular function analyses.

A number of new specific groups with statistical significance emerged from this larger group of genes, of which two are of particular interest from a muscle biology perspective. Both groups had limited representation in the smaller group of genes with at least ±2-fold changes, and both were readily apparent from visual inspection of the list of genes with at least ±1.5-fold changes. These were a group of genes related to collagen regulation (cellular constituent GO term collagen, 6 genes, P = 0.00021) and another group related to the complement system (biological process GO term complement activation, 5 genes, P = 0.0020) (Table 4).


View this table:
[in this window]
[in a new window]
  		Table 4. Selected groups of genes with at least ±1.5-fold altered expression in dystrophic compared with normal diaphragm

 
Confirmation of gene expression with real-time PCR.
Real-time PCR was done for the vast majority of genes with at least plus or minus twofold altered expression in dystrophic muscle, consistent present calls by Affymetrix software, significant changes by BAM, and assignment to overrepresented GO biological process groups. Among these 41 genes, which are listed in Table 1, expression of 3 genes was not tested due to lack of availability of reagent; expression of 36 genes was verified by quantitative PCR; expression of 1 gene was tested, but it was present in very low abundance, by PCR making the results unreliable; and altered expression of 1 gene could not be ascertained with certainty, because of high variability of expression in normal muscle, by PCR. One of the genes with altered expression but lack of assignment to overrepresented GO biological process groups listed in Table 2 was tested with PCR, and increased expression was verified. Altered expression of all nine genes with increased expression in the 1.5- to 2-fold range listed in Table 4 was tested. Among this group, seven were confirmed, and two could not be confirmed largely because of variability in expression either among normal samples or among dystrophic samples. Results of quantitative PCR testing are listed in each of the tables. Thus, among genes with at least plus or minus twofold changed expression by gene expression arrays that underwent PCR testing, verification occurred in 37 of 39 cases (95%) (Tables 1 and 2). Within this group, among genes with at least ±2.3-fold changed expression by gene array, the verification rate was 100% (24 of 24) but dropped to 87% in the fold-change range of 2.0–2.3 (13 of 15). Among genes with 1.5- to 2-fold changes in expression by gene expression arrays that underwent PCR testing, verification occurred in 7 of 9 cases (78%) (Table 4).

A comparison of fold changes with gene expression arrays vs. quantitative PCR indicated a significant correlation between changes in gene expression with the two methods (r = 0.92, P < 0.001) (Fig. 2). At fold changes ranging from 1.5 to ~4, values determined by the two methods were generally similar to each other (albeit with some scatter). In contrast, at higher fold changes, values were consistently larger for PCR than for gene expression arrays.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Relationship between fold change in gene expression in dystrophic compared with normal muscle determined by gene expression arrays vs. that determined by quantitative PCR for 44 genes for which PCR confirmed gene expression data. A logarithmic scale was chosen because of high variability among genes in extent of altered expression. Data are expressed as absolute values to include the 2 genes with reduced expression in dystrophic compared with normal muscle.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Changes in gene expression.
We found in the dy/dy diaphragm that the genes with altered expression belonged to overrepresented GO groups in four relatively specific themes: cell motility (but mainly muscle contraction), development (with the greatest fold increases seen with the muscle development subgroup), defense/immune responses, and cellular adhesion. In addition, GO cellular constituent analysis demonstrated in particular altered gene expression relating to extracellular and muscle fiber locations. These themes are quite robust in that they were identified when analyzing the restricted set of genes with at least 2-fold changed expression as well as a larger group of genes with at least 1.5-fold changed expression. Analysis of the larger group also yielded two additional specific GO groups, those of collagen synthesis and complement activation.

Several of these findings can be related directly to the known features of muscle in {alpha}-laminin deficiency. First, dy/dy muscle has evidence of muscle fiber regeneration (7, 14). Furthermore, diseased muscle has altered contractile properties, with slower isometric twitch kinetics in some studies (14) and higher resistance to fatigue (14, 44, 45). The present study is consistent with this by demonstrating changes in myosin gene expression patterns, with a shift toward embryonic and perinatal forms, as well as increased expression of other genes that participate in cell differentiation and development. Second, {alpha}-laminin is located on the extracellular surface of the muscle. It is known to play important roles in adhesion between the cellular membrane and surrounding collagen, and dy/dy muscle has disrupted basement membrane (48). However, there is less functional evidence of sarcolemmal disruption than is observed in dystrophin-deficient mdx muscle (41). Gene expression patterns from the present study indicate especially prominent alterations among genes encoding for molecules with extracellular location as well as in genes involved in cellular adhesion. Upregulated expression of these gene groups may very well be a compensatory mechanism for the critical loss of {alpha}-laminin and therefore might present opportunities for compensatory therapeutic interventions. Third, dy/dy muscle has increased connective tissue, fibrosis and collagen content, with reduced passive distensibility (21, 30, 41). Increased expression of a number of different procollagen genes was noted in the present study, suggesting a widespread activation of collagen synthesis. On the other hand, one finding that was not expected based on descriptions of muscle histology in dy/dy mice was the upregulation of genes related to defense/immune response and complement activation, in that inflammation is generally not a prominent feature reported in histological studies.

Regarding specific genes with altered expression, among the genes in the cellular motility group, the two for myosin heavy chain components are both developmental forms (Myh3 and Myh8), and the one gene for myosin light chain component (Myl4) is normally expressed in adult cardiac atrial cells as well as fetal skeletal muscle and cardiac ventricular tissue. Mybph binds to myosin and is believed to be involved in the interaction of thick filaments in the region of the A band. Tnnt2 encodes for the tropomyosin-binding subunit of troponin, which confers calcium sensitivity to striated muscle actomyosin ATPase activity. Spp1 is involved in muscle regeneration after injury (11). All six of these genes also belonged to the development theme. Other notable genes in the development group include Ankrd2, which is normally expressed in both skeletal and cardiac muscle, has increased expression in response to skeletal muscle stretch (23), interacts with transcription factors (25), and is believed to be involved in intracellular signaling relating muscle stretch or stress with subsequent hypertrophy. Myog is an important regulator of muscle-specific gene expression, is involved in differentiation of fibroblasts into myoblasts, and plays a role in regulating enzyme activity, favoring oxidative relative to glycolytic metabolism (18). The latter effect could explain the increased resistance to fatigue found in dy/dy mouse diaphragm and sternohyoid muscle (44, 45). Igf2 is normally expressed in low levels during myoblast proliferation but in much higher levels during myoblast differentiation. Thus over one-half of the genes comprising the development theme have known functions in skeletal muscle.

Among genes of the defense response theme, Fcgr2b, H2-Eb1, H2-Aa, Ii, and H2-Ab1 are involved with antigen presentation and processing, whereas Fcgr2b, Spp, H2-Aa, and Ii deal with lymphocyte and T cell activation and differentiation. Other genes in the defense response group included Cxcl14, a C-X-C chemokine, and Lsp1, a binding protein found in lymphocytes, macrophages, and neutrophils. There also was upregulation of the complement cascade system, which included one gene with >2-fold increased expression, C1qb, and four genes with 1.5- to 2-fold increased expression, C3ar1, C1qa, C1qg, and C4. The complement system is a key component of innate immunity but also has important modulatory roles in adaptive immunity (3, 32). Among its functions is disposing products of inflammatory injury, such as immune complexes, to allow subsequent healing.

The final group of genes assigned to specific GO biological functions were those related to cellular adhesion and collagen formation. Many of these genes are not central components of laminin's normal interactions with the dystroglycan-glycoprotein complex or the integrin system. Postn binds to heparin, induces cell attachment and spreading, and acts as a cellular adhesion molecule by binding to cell surface integrins. Postn is involved in muscle regeneration after injury (11). Furthermore, in zebrafish, Postn is critical for adhesion of muscle fibers to the myoseptum and for muscle fiber differentiation (26). Spp1 is also involved in muscle regeneration after injury (11, 16). Of note, Spp knockout mice have impaired wound healing, with greater disorganization of matrix and an alteration of collagen fibrillogenesis (27). Thbs4 is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions and can bind to fibrinogen, fibrinectin, laminin, and type V collagen. Lgals3 is involved in a number of processes, among them wound healing (2). Ncam1 is best known for its role in neuron-neuron as well as neuron-muscle adhesion but also participates in skeletal muscle development and regeneration (4, 20). Mybph binds to myosin and is involved in the interaction of thick filaments in the region of the A band. It thus normally has intracellular adhesive roles, in contrast to most of the others listed above, which are involved in extracellular adhesion. Finally, there were a number of procollagen genes with increased expression, one with a >2-fold increase (Col1a2) and five with a 1.5- to 2-fold increase (Col6a3, Col6a1, Col5a1, Col6a2, and Col8a1). It is not surprising to see upregulation of this group in view of the increased collagen content and fibrosis of laminin-deficient dystrophic muscle (7, 21, 30, 41, 48).

Among genes with significantly changed expression that did not belong to the above groups, two closely related genes stand out: Chrng and Chrna1. These encode for two components of skeletal muscle nicotinic acetylcholine receptors and hence are key to neuromuscular transmission (28). The {gamma}-subunit is found in immature neuromuscular junctions and is replaced by an {epsilon}-subunit in adult tissue. The {alpha}-subunit comprises part of both fetal and adult acetylcholine receptors. Another gene of particular interest is Ankrd1, which is related to Ankrd2, with the former found preferentially in cardiac muscle and the latter preferentially in skeletal muscle. Ankrd1 expression is increased (along with Ankrd2 expression) in the mouse model of muscular dystrophy with myositis, a disease in which the primary abnormality is in the titin filament system (47).

Muscle injury and repair is felt to be an ongoing process in dy/dy mice. A good description of the normal temporal sequence of recovery from acute muscle injury comes from a study of cardiotoxin-induced muscle damage, which found an orderly progression of gene groups with altered expression during 14 days after injury (11). Changes in expression for transcriptional genes peaked at 6 h and then gradually tapered for the remainder of time. Alterations in expression levels of transcripts of genes involved in signal transduction and cell cycle were maximal between 6 h and 2 days, while those for inflammatory/immune response genes were maximal between 12 h and 2 days. For extracellular matrix/cell adhesion gene transcripts, maxima occurred between 5 and 14 days. The early response gene groups (transcription, signal transduction, cell cycle) seen in the cardiotoxin acute injury model were not identified as belonging to overrepresented GO groups among the genes with altered expression in dy/dy diaphragm per statistical testing in the present study. However, there were some examples of such genes with increased expression in dystrophic muscle for all three early gene groups per GO assignment: six genes for the GO term transcription (Ankrd1, Asb15, Hes6, Myog, Runx1, Sox11), seven genes for the term signal transduction (Asb15, Dcamkl1, Fcgr2b, Grb14, Lsp1, Ms4a7, Mt2), and two genes for the term cell cycle (Igf2, Pycard). Thus genes from these early response groups are certainly upregulated, but not in a large enough proportion for these groups to have been identified as preferentially altered by statistical testing.

In the present study, there were considerably more genes whose expression was upregulated than downregulated by {alpha}2-laminin deficiency. A similar pattern of greater over- than underexpression has been noted in skeletal muscle from dystrophin-deficient mdx mice (34) and humans with DMD (13) and has been attributed to a broad increase in protein turnover associated with degeneration and regeneration.

Comparison with dystrophin-deficient muscular dystrophy.
Several studies have reported skeletal muscle gene expression in the mdx mouse. Porter et al. (34) studied leg muscle using Affymetrix microarrays (albeit an older chip array than that used in the present study) and found that the most prominent change was an upregulation of numerous components of the chronic inflammatory response system. This included complement system activation, invasive cell type-specific markers, leukocyte adhesion and diapedesis, and cytokine and chemokine signaling. Other identified themes included proteolysis, muscle regeneration, and extracellular matrix. In a subsequent study from the same group comparing diaphragm with limb muscle (36), generally similar overall themes of altered gene expression were identified in the two muscle groups. However, there were divergences between diaphragm and limb muscle within themes of which specific genes were altered, especially within the inflammatory and muscle-specific gene categories. Rouger et al. (38) studied both diaphragm and limb muscle using cDNA microarrays that tested expression of a much more focused group of 1,082 genes. They found some genes with altered expression in both muscle groups, others with altered expression in diaphragm only or limb muscle only, and yet others with altered but different expression in the two muscle groups. Major categories of genes with changed expression included cell surface and extracellular matrix, muscle structure and developmental genes, and intracellular signaling and cell-cell communication. Haslett et al. (13) examined gene expression in quadriceps muscle from humans with DMD and identified several themes including muscle structure, muscle development, immune response, and extracellular matrix and cytoskeleton. However, the intensity of the inflammatory profile was much less than that described in mdx limb muscle (34).

The present study identified several overall themes in the dy/dy model that are similar to those described in dystrophin deficiency: muscle structure, muscle development, immune response, and extracellular matrix and adhesion. This is not surprising in view of both disease entities being manifested by muscle inflammation, degeneration, and regeneration. A more important issue is the extent to which muscles with dystrophin deficiency and {alpha}2-laminin deficiency have conserved vs. discrepant alterations in expression of individual genes within these themes, in that it will lead to a better understanding of which changes represent a standard muscle repertoire in response to abnormalities involving the dystrophin-glycoprotein complex and its attachments and which changes are specific to the exact underlying genetic defect. Several genes with altered expression in dy/dy mice have been identified previously as altered in murine and/or human dystrophin deficiency, including Myh3, Myh8, Myog, Mybph, Igf2, Spp1, Lsp1, Lgals3, Col1a2, Chrna1, Ankrd 1, Ankrd2, and Tnnt2 (5, 13, 34–36, 38). In other cases, related but not identical gene changes occur, e.g., Myl4 in dy/dy mice but Myl1 or Myl5 in mdx mice (38) and human DMD (5, 13), and both Chrng and Chrna1 in dy/dy mice but Chrnb1, Chrng, and Chrna1 in mdx mice (36) and only Chrna1 in human DMD (5). Studies in mdx mice have performed over a range of animal ages, and alterations in gene expression vary over time, with some being limited to narrow age ranges and others being present over a larger time frame. Disease progression is considerably faster and more severe in dy/dy than mdx mice. Studies correlating changes in muscle contractile performance with changes in gene expression over time are needed to better compare these two models and to determine whether gene expression differs between models as a function not only of age but also of extent of muscle involvement.

On the basis of this comparison among disease models, certain biological themes emerge. First, dystrophin deficiency leads to a much broader activation of genes involved with inflammation than does {alpha}2-laminin deficiency. Whether inflammation contributes to muscle damage or merely is a mechanism for dealing with necrosed myocytes is not well understood. If the former is the case, anti-inflammatory interventions might be more useful therapeutically to manage dystrophin- than laminin-deficient muscular dystrophy. Second, increased expression of procollagen genes is common among several of the muscular dystrophies. Assuming that the fibrosis interferes with muscle regeneration, strategies to reduce procollagen gene expression or translation might be equally effective across a number of different types of muscular dystrophy. Third, increased expression is more pronounced for fetal and neonatal than for adult contractile protein isoforms across diseases, suggesting that fiber regeneration occurs but fiber maturation is incomplete. Improving fiber maturation might present another therapeutic opportunity applicable to a range of muscular dystrophies. Finally, the presence of both differences and similarities in the specific repertoire of increased cell adhesion-related gene expression among diseases could lead to both disease-specific and disease-shared therapeutic opportunities.

Verification of gene expression by quantitative PCR.
The present study verified microarray results using quantitative PCR on a high proportion of genes identified as having increased expression that belonged to overrepresented GO biological process groups, with 100% verification in genes with at least ±2.3-fold changed expression, 87% verification in the fold change range of ±2.0–2.3, and 78% verification in the fold change range of ±1.5–2. Although we only performed PCR on one gene among those with at least plus or minus twofold changed expression that did not belong to overrepresented GO biological function groups (i.e., those listed in Table 2), the high (95%) rate of PCR verification on the genes listed in Table 1 suggests that the majority of the genes listed in Table 2 would have been validated had they also undergone PCR testing.

Previous studies of muscle gene expression in dystrophin-deficient mdx mice and humans with DMD using the Affymetrix microarray system have independently verified a small percentage of genes with altered expression. Chen et al. (5) used immunohistochemistry to verify 9 of 144 genes with altered expression, Haslett et al. (13) used quantitative PCR to verify 12 of 105 genes with altered expression, Porter et al. (34) tested 5 of 242 differentially expressed genes using immunoblot and/or PCR, and Porter et al. (36) tested 8 of ~700 differentially expressed genes using quantitative PCR (albeit at 5 different ages). In the present study, of the 78 genes identified with altered expression (Tables 1–4), 48 were tested with quantitative PCR, and 44 of these were confirmed, a much higher rate of confirmation testing than previous studies of the dystrophin-deficient muscular dystrophies.

Methodological issues.
One limitation of the present study is that it was performed at a single age, namely 8 wk. This age was chosen because the animals already manifest severe clinical weakness but are not yet at the limits of their markedly shortened life span, thereby minimizing any survivor effects (30, 49). In addition, sufficient data are available about diaphragm and limb muscles at this age to document aberrant structure and function. Of note is that the adverse effects of {alpha}2-laminin deficiency are progressive over time, so that data from this single age may not reflect events either early on or during the terminal stage of the disease. Further studies are needed at additional ages to determine the time course of changes in expression of the genes identified in the present study, and to determine whether pathways in addition to the ones described in the present study also have alterations in gene expression but at different points in time.

A second issue is that diaphragm but not limb muscle was studied. Morbidity and disability in the neuromuscular diseases result from both respiratory and limb muscle dysfunction, whereas mortality is determined predominantly by the extent of respiratory muscle impairment. In diseases where there is disconcordance between respiratory and limb muscle involvement, a better understanding of the diaphragm is especially important in designing therapeutic strategies that can prolong life in these disorders, hence the choice of diaphragm over limb muscle for the present study. The mdx mouse has much more severe diaphragm than limb muscle involvement (9, 40). This contrasts with human DMD, human classical congenital muscular dystrophy, and dy/dy mouse muscular dystrophy in which the degree of impairment among limb and respiratory muscles is more homogeneous. Whether limb and diaphragm muscles have different molecular signatures in laminin-deficient muscular dystrophies is not known, but one might anticipate smaller differences than those seen in the dystrophin-deficient mdx mouse. Further studies are needed to clarify this matter.

A third methodological issue is the use of pooled samples. The primary reason for this approach was to ensure sufficient amount of tissue per sample without having to perform double rounds of RNA amplification, as undetected nonlinearities in this kind of amplification can alter measured expression levels. Sample pooling has been done in a number of previous studies of dystrophin-deficient mdx mice (34–36, 38) and has been advocated as a method of minimizing the effects of interanimal variability (38). A recent gene expression array toxicology study (22) has found that pooling may lead to increased numbers of false positive and negative results and, furthermore, does not allow the identification of individual outliers that contribute to pooled samples. The extent to which this is an issue depends on the degree of heterogeneity among animals, which in fact was not inconsiderable in the toxicology study of Jolly et al. (22). An advantage of studying a disease produced by a single gene mutation (as in the present study) is a greater degree of similarity among animals or subjects than in studies of more complex diseases produced as a result of the interaction of multiple genes, especially those complex diseases in which there are also prominent environmental influences. Furthermore, the data of Kendziorski et al. (24) suggest that the 12-on-4 design (i.e., 12 samples placed into 4 pools) used in the present study has an acceptable level of performance compared with designs in which smaller numbers of samples are analyzed without pooling (specifically, 3-on-3 and 6-on-6 designs).

Finally, the present study used the GO assignment to attribute function to genes with altered expression. Many genes have been implicated in multiple cellular functions, so that assignment of a single gene to multiple GO categories is not uncommon. The GO approach was chosen for the present study because it is a widely accessible and independently curated system. This has advantages of using a single nomenclature that can be applied consistently among studies by different laboratories and avoiding any preconceived biases on the part of individual investigators in the assignment of functional attribution. In addition, it allows statistical testing of overrepresentation within categories, so that gene categories are not merely identified based on their overall large size in the transcriptome as a whole. A potential weakness with this system is that, in some cases, genes are assigned to categories based on relatively minor involvement (e.g., Spp1 assigned to muscle contraction), or, alternatively, genes are not assigned to categories in which it could be argued they belong (e.g., Chrng, which is a fetal subunit of the acetylcholine receptor in muscle, not assigned to development). The former issue is potentially problematic in terms of themes being falsely identified. However, the addition of the jackknifing procedure to the Fishers exact probability test used for identifying overrepresented GO groups addresses this problem by recalculating the statistic after removal of a single data point, ensuring that the statistical result remains robust even in the event of a gene being wrongly assigned to a category. The latter issue may lead to the underidentification of some GO categories but should not negate the validity of themes that were identified.

In conclusion, the primary defect in dy/dy dystrophic muscle is deficiency of {alpha}2-laminin, a molecule that plays an important role in adhesion of the cell to the extracellular matrix and hence in the structural integrity of the muscle as a whole. This primary defect results in a variety of downstream events such as fiber necrosis, inflammation, and regeneration, evidence for which is seen histologically as well as in patterns of gene expression. The cycle of muscle injury and incomplete repair is in many respects similar to that noted in dystrophin-deficient muscle, although with a less prominent degree of inflammation in laminin than dystrophin deficiency. Not clear from previous studies of {alpha}2-laminin-deficient mouse or human muscle is the extent to which compensatory mechanisms that address the primary genetic and biochemical defect (the impairment of the extracellular adhesive system) are initiated. One event known to occur is an upregulation of {alpha}4-laminin; however, {alpha}4-laminin fails to bind {alpha}-dystroglycan with significant affinity, so that compensation for the loss of {alpha}2-laminin is incomplete (33, 37). The present study indicates that there is also a robust upregulation of alternative cell adhesion genes and, in particular, of genes that are not central components of laminin's normal interactions with the dystroglycan-glycoprotein complex or the integrin system. This includes Postn (periostin), Spp1 (secreted phosphoprotein 1; also known as osteopontin), Thbs4 (thrombospondin 4), Lgals3 (lectin, galactose binding, soluble 3; also known as galectin-3), and Ncam1 (neural cell adhesion molecule 1), all of which had increased expression of at least threefold in magnitude. Activation of these alternative cell adhesive systems could potentially ameliorate the adverse effects of {alpha}2-laminin deficiency on muscle structural and contractile integrity. Further studies are needed to determine the extent to which these alternative cell adhesion genes undergo upregulated expression in limb muscles, how increased expression varies as a function of age, the degree to which similar compensatory mechanisms are activated in mice with other variants of {alpha}2-laminin deficiency (e.g., dy2J/dy2J mice, which have near-normal levels of dysfunctional truncated {alpha}2-laminin), and the extent to which increased expression of these genes correlates with preservation of muscle mechanical properties.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-70697, by a Merit Review Grant from the Department of Veterans Affairs Medical Research Service, and by the Gene Expression Array Core Facility of the Comprehensive Cancer Center of Case Western Reserve University and University Hospitals of Cleveland (P30 CA43703).


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: E. van Lunteren, Pulmonary 111J(W), Cleveland VA Medical Center, 10701 East Boulevard, Cleveland, OH 44106 (e-mail: exv4{at}cwru.edu).

1 The Supplemental Material for this article (Supplemental Appendix S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00226.2005/DC1. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Allamand V and Campbell KP. Animal models for muscular dystrophy: valuable tools for the development of therapies. Hum Mol Genet 9: 2459–2467, 2000.[Abstract/Free Full Text]
  2. Cao Z, Said N, Amin S, Wu HK, Bruce A, Garate M, Hsu DK, Kuwabara I, Liu FT, and Panjwani N. Galectins-3 and -7, but not galectin-1, play a role in re-epithelialization of wounds. J Biol Chem 277: 42299–42305, 2002.[Abstract/Free Full Text]
  3. Carroll MC. The complement system in regulation of adaptive immunity. Nat Immun 5: 981–986, 2004.[CrossRef][ISI][Medline]
  4. Cashman NR, Covault J, Wollman RL, and Sanes JR. Neural cell adhesion molecule in normal, denervated and myopathic human muscle. Ann Neurol 21: 481–489, 1987.[CrossRef][ISI][Medline]
  5. Chen YW, Zhao P, Borup R, and Hoffman EP. Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology. J Cell Biol 151: 1321–1336, 2000.[Abstract/Free Full Text]
  6. Colognato H and Yurchenco PD. Form and function: the laminin family of heterotrimers. Dev Dyn 218: 213–234, 2000.[CrossRef][ISI][Medline]
  7. Connolly AM, Keeling RM, Mehta S, Pestronk A, and Sanes JR. Three mouse models of muscular dystrophy: the natural history of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin {alpha}2-deficient mice. Neuromuscul Disord 11: 703–712, 2001.[CrossRef][ISI][Medline]
  8. Dennis G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, and Lempicki RA. DAVID: data base for annotation, visualization, and integrated discovery. Genome Biol 4: R60, 2003.[CrossRef]
  9. Dupont-Versteegden EE and McCarter RJ. Differential expression of muscular dystrophy in diaphragm versus hindlimb muscles of MDX mice. Muscle Nerve 15: 1105–1110, 1992.[CrossRef][ISI][Medline]
  10. Emery AEH. The muscular dystrophies. Lancet 359: 687–695, 2002.[CrossRef][ISI][Medline]
  11. Goetsch SC, Hawke TJ, Gallardo TD, Richardson JA, and Garry DJ. Transcriptional profiling and regulation of the extracellular matrix during muscle regeneration. Physiol Genomics 14: 261–271, 2003.[Abstract/Free Full Text]
  12. Guo LT, Zhang XU, Kuang W, Xu H, Liu LA, Vilquin JT, Miyagoe-Suzuki Y, Takeda S, Ruegg MA, Wewer UM, and Engvall E. Laminin {alpha}2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice. Neuromuscul Disord 13: 207–215, 2003.[CrossRef][ISI][Medline]
  13. Haslett JN, Sanoudou D, Kho AT, Bennett RR, Greenberg SA, Kohane IS, Beggs AH, and Kunkel LM. Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle. Proc Natl Acad Sci USA 99: 15000–15005, 2002.[Abstract/Free Full Text]
  14. Hayes A and Williams DA. Examining potential drug therapies for muscular dystrophy utilising the dy/dy mouse: I. Clenbuterol. J Neurol Sci 157: 122–128, 1998.[CrossRef][ISI][Medline]
  15. Hebling-Leclerc A, Zhang X, Topaloglu H, Cruaud C, Tesson F, Weissenbach J, Tome FMS, Schwartz K, Fardeau M, Tryggvason K, and Guicheney P. Mutations in the laminin {alpha}2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy. Nat Genet 11: 216–218, 1993.
  16. Hirata A, Masuda S, Tamura T, Kai K, Ojima K, Fukase A, Motoyoshi K, Kamakura K, Miyagoe-suzuki Y, and Takeda S. Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection. A role for osteopontin. Am J Pathol 163: 203–215, 2003.[Abstract/Free Full Text]
  17. Hosack DA, Dennis G, Sherman BT, Lane HC, and Lempicki RA. Identifying biological themes within lists of genes with EASE. Genome Biol 4: R70, 2003.[CrossRef][Medline]
  18. Hughes SM, Chi MM, Lowry OH, and Gundersen K. Myogenin induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice. J Cell Biol 145: 633–642, 1999.[Abstract/Free Full Text]
  19. Ishwaran H and Rao JS. Detecting differentially expressed genes in microarrays using Bayesian model selection. J Am Stat Assoc 98: 438–455, 2003.[CrossRef]
  20. Jamali AA, Afshar P, Abrams RA, and Leiber RL. Differential expression of neural cell adhesion molecule (NCAM) after tenotomy in rabbit skeletal muscle. J Orthop Res 20: 364–369, 2002.[Medline]
  21. Jannapureddy SR, Patel ND, Hwang W, and Boriek AM. Merosin deficiency leads to alterations in passive and active skeletal muscle mechanics. J Appl Physiol 94: 2524–2533, 2003.[Abstract/Free Full Text]
  22. Jolly RA, Goldstein KM, Wei T, Gao H, Chen P, Huang S, Colet JM, Ryan TP, Thomas CE, and Estrem ST. Pooling samples within microarray studies: a comparative analysis of rat liver transcription response to prototypical toxicants. Physiol Genomics 22: 346–355, 2005.[Abstract/Free Full Text]
  23. Kemp TJ, Sadusky TJ, Saltisi F, Carey N, Moss J, Yang SY, Sassoon DA, Goldspink G, and Coulton GR. Identification of Ankrd2, a novel skeletal muscle gene coding for a stretch-responsive ankyrin-repeat protein. Genomics 66: 229–241, 2000.[CrossRef][ISI][Medline]
  24. Kendziorski C, Irizarry RA, Chen KS, Haag JD, and Gould MN. On the utility of pooling biological samples in microarray experiments. Proc Natl Acad Sci USA 102: 4252–4257, 2005.[Abstract/Free Full Text]
  25. Kojic S, Medeot E, Guccione E, Krmac H, Zara I, Martinelli V, Valle G, and Faulkner G. The Ankrd2 protein, a link between the sarcomere and the nucleus in skeletal muscle. J Mol Biol 339: 313–325, 2004.[CrossRef][ISI][Medline]
  26. Kudo H, Amizuka N, Araki K, Inohaya K, and Kudo A. Zebrafish periostin is required for the adhesion of muscle fiber bundles to the myoseptum and for the differentiation of muscle fibers. Dev Biol 267: 473–487, 2004.[CrossRef][ISI][Medline]
  27. Liaw L, Birk DE, Ballas CB, Whitsitt JS, Davidson JM, and Hogan BLM. Altered wound healing in mice lacking a functional osteopontin gene (spp1). J Clin Invest 101: 1468–1478, 1998.[ISI][Medline]
  28. Lindstrom JM. Acetylcholine receptors and myasthenia. Muscle Nerve 23: 453–477, 2000.[CrossRef][ISI][Medline]
  29. Martin PT. Dystroglycan glycosylation and its role in matrix binding in skeletal muscle. Glycobiology 13: 55R–66R, 2003.[Abstract/Free Full Text]
  30. Michelson AM, Russell ES, and Harman PJ. Dystorphia muscularis: a hereditary primary myopathy in the house mouse. Proc Natl Acad Sci USA 41: 1079–1084, 1955.[Free Full Text]
  31. Miyagoe-Suzuki Y, Nakagawa M, and Takeda S. Merosin and congenital muscular dystrophy. Microsc Res Tech 48: 181–191, 2000.[CrossRef][ISI][Medline]
  32. Morgan BP, Marchbank KJ, Longhi MP, Harris CL, and Gallimore AM. Complement: central to innate immunity and bridging to adaptive responses. Immunol Lett 97: 171–179, 2005.[CrossRef][Medline]
  33. Patton BL, Miner JH, Chiu AY, and Sanes JR. Distribution and function of laminins in the neuromuscular system of developing, adult and mutant mice. J Cell Biol 139: 1507–1521, 1997.[Abstract/Free Full Text]
  34. Porter JD, Khanna S, Kaminski HJ, Rao JS, Merriam AP, Richmonds CR, Leahy P, Li J, Guo W, and Andrade FH. A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice. Hum Mol Genet 11: 263–272, 2002.[Abstract/Free Full Text]
  35. Porter JD, Merriam AP, Khanna S, Andrade FH, Richmonds CR, Leahy P, Cheng G, Karathanasis P, Zhou X, Kusner LL, Adams ME, Willem M, Mayer U, and Kaminski HJ. Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice. FASEB J 17: 893–895, 2003.[Abstract/Free Full Text]
  36. Porter JD, Merriam AP, Leahy P, Gong B, Feuerman J, Cheng G, and Khanna S. 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 13: 257–269, 2004.[Abstract/Free Full Text]
  37. Ringelmann B, Roder C, Hallmann R, Maley M, Davies M, Grounds M, and Sorokin L. Expression of laminin alpha1, alpha2, alpha4 and alpha5 chains, fibronectin, and tenascin-C in skeletal muscle of dystrophic 129ReJ dy/dy mice. Exp Cell Res 246: 165–182, 1999.[CrossRef][ISI][Medline]
  38. Rouger K, Le Cunff M, Steenman M, Potier MC, Gibelin N, Dechesne CA, and Leger JJ. Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice. Am J Physiol Cell Physiol 283: C773–C784, 2002.[Abstract/Free Full Text]
  39. Sanoudou D, Haslett JN, Kho AT, Guo S, Gazda HT, Greenberg SA, Lidov HG, Kohane IS, Kunkel LM, and Beggs AH. Expression profiling reveals altered satellite cell numbers and glycolytic enzyme transcription in nemaline myopathy muscle. Proc Natl Acad Sci USA 100: 4666–4671, 2003.[Abstract/Free Full Text]
  40. Stedman HH, Sweeney HL, Shrager JB, Maguire HC, Panettieri RA, Petrof B, Narusawa M, Leferovich JM, Sladky JT, and Kelly AM. The MDX mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 342: 536–538, 1991.
  41. Straub V, Rafael JA, Chamberlain JS, and Campbell KP. Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J Cell Biol 139: 375–385, 1997.[Abstract/Free Full Text]
  42. Tsuji S and Matsushita H. Successful treatment of murine muscular dystrophy with the protease inhibitor bestatin. J Neurol Sci 72: 183–194, 1986.[CrossRef][ISI][Medline]
  43. van Lunteren E and Moyer M. Improvement of dy/dy dystrophic diaphragm by K+ channel blocker 3,4-diaminopyridine. Muscle Nerve 26: 71–78, 2002.[CrossRef][Medline]
  44. van Lunteren E and Moyer M. Sternohyoid muscle fatigue properties of dy/dy dystrophic mice, an animal model of merosin-deficient congenital muscular dystrophy. Pediatr Res 54: 547–553, 2003.[Medline]
  45. van Lunteren E and Moyer M. Reduced fatigue in diaphragm muscle of merosin-deficient dy/dy dystrophic mice. Respiration 70: 636–642, 2003.[Medline]
  46. Voit T. Congenital muscular dystrophies: 1997 update. Brain Dev 20: 65–74, 1998.[CrossRef][ISI][Medline]
  47. Witt CC, Ono Y, Puschmann E, McNabb M, Wu Y, Gotthardt M, Witt SH, Haak M, Labeit D, Gregorio CC, Sorimachi H, Granzier H, and Labeit S. Induction and myofibrillar targeting of CARP, and suppression of the Nkx2.5 pathway in the MDM mouse with impaired titin-based signaling. J Mol Biol 336: 145–154, 2004.[CrossRef][ISI][Medline]
  48. Xu H, Christmas P, Wu XR, Wewer UM, and Engvall E. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc Natl Acad Sci USA 91: 5572–2276, 1994.[Abstract/Free Full Text]
  49. Xu H, Wu XR, Wewer UM, and Engvall E. Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene. Nat Genet 8: 297–302, 1994.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. Hager, M. G. Bigotti, R. Meszaros, V. Carmignac, J. Holmberg, V. Allamand, M. Akerlund, S. Kalamajski, A. Brancaccio, U. Mayer, et al.
Cib2 Binds Integrin {alpha}7B{beta}1D and Is Reduced in Laminin {alpha}2 Chain-deficient Muscular Dystrophy
J. Biol. Chem., September 5, 2008; 283(36): 24760 - 24769.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
E. van Lunteren and M. Moyer
Oxidoreductase, morphogenesis, extracellular matrix, and calcium ion-binding gene expression in streptozotocin-induced diabetic rat heart
Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E759 - E768.
[Abstract] [Full Text] [PDF]

This Article
Right arrow