Human Molecular Genetics, 2001, Vol. 10, No. 17 1819-1827
© 2001 Oxford University Press
Pathological and genetic analysis of the degenerating muscle (dmu) mouse: a new allele of Scn8a
1Ottawa Health Research Institute, Ottawa, ON K1H 8L6, Canada, and The University of Ottawa Center for Neuromuscular Disease, 2Department of Cellular and Molecular Medicine and 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada and 4Centre de recherche Guy-Bernier de l’Hôpital Maisonneuve-Rosemont, Montreal, QC, Canada
Received May 7, 2001; Revised and Accepted June 19, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Here, we describe a novel spontaneous autosomal recessive mutation in the mouse that is characterized by skeletal and cardiac muscle degeneration. We have named this mutant degenerating muscle (dmu). At birth, mutant mice are indistinguishable from their normal littermates. Thereafter, the disease progresses rapidly and a phenotype is first observed at
11 days after birth; the dmu mice are weak and have great difficulty in moving. The principal cause of the lack of mobility is muscle atrophy and wasting in the hindquarters. Affected mice die at or around the time of weaning of unknown causes. Histopathological observations and ultrastructural analysis revealed muscle degeneration in both skeletal and cardiac muscle, but no abnormalities in sciatic nerves. Using linkage analysis, we have mapped the dmu locus to the distal portion of mouse chromosome 15 in a region syntenic to human chromosome 12q13. Interestingly, scapuloperoneal muscular dystrophy (SPMD) in humans has been linked to this region. SPMD patients with associated cardiomyopathy have also been described in the past. Initial analysis of candidate genes on mouse chromosome 15 reveal that although intact transcripts for Scn8a, the gene encoding the sodium channel 8a subunit, are present in dmu mice, their levels are dramatically reduced. Furthermore, genetic complementation crosses between dmu and med (mutation in Scn8a) mice revealed that they are allelic. Our results suggest that at least a portion of the dmu phenotype is caused by a down-regulation of Scn8a, making dmu a new allele of Scn8a. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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There are a large number of hereditary neuromuscular diseases in humans, and the genetic defects responsible for several of these diseases have been elucidated. Key discoveries include the identification of dystrophin as the protein defective in Duchenne muscular dystrophy (DMD) (1,2), and the demonstration that mutations in some dystrophin-associated proteins (DAPs) are linked to different forms of muscular dystrophies (3–5). Nevertheless, there still remain many human neuromuscular diseases for which the causative gene has yet to be identified. In general, the biological cause of neuromuscular diseases can be intrinsic defects in the motor neurons innervating their target muscles, defects at the neuromuscular junction, or defects in the muscle itself. An invaluable resource to help understand the processes underlying these defects are naturally occurring or laboratory-derived mouse neuromuscular mutants. They allow for a better understanding of the molecular pathogenesis of muscle disease, they help discern the identity of the primary underlying defect, and they provide a model for the evaluation of the efficacy and safety of novel therapeutic strategies (6–8).
Several mouse models have been characterized for the study of human muscle disease and have provided novel insights into disease etiology and gene function. Amongst these, the mdx mouse has mutations in the dystrophin gene and has been used extensively as a model for DMD (9). Another example is the dystrophia muscularis (dy) mouse. This mouse mutant identified by Michelson (10) suffers from muscle degeneration and peripheral nerve dysmyelination (11–13). Several allelic versions of the dy mouse exist and serve as models for the human hereditary disorder congenital muscular disease (CMD). The dy mouse is deficient for the laminin-
2 chain (merosin), a component of the muscle extracellular matrix and a native ligand for -dystroglycan. Mutations in the human merosin gene have been shown to be responsible for CMD (14,15) and the dy mice serve as a model for pathogenic studies.
Clearly, characterizing new models of neuromuscular disease in mice is of great importance for a better understanding of the molecular mechanism of disease pathogenesis and for the identification of genes implicated in the large number of linked and unlinked human neuromuscular disorders. In the present study, we describe a novel spontaneous autosomal recessive mutation in the mouse that causes an early-onset progressive loss of mobility of the hind limbs and subsequent lethality in the first month of life. The main site of pathology is in skeletal and cardiac muscle, hence we named the mutant degenerating muscle (dmu). We have mapped the dmu locus to the distal arm of mouse chromosome 15, in a region syntenic with human chromosome 12q13. Initial candidate gene analysis demonstrated that the Scn8a locus is down-regulated in dmu mice, thereby explaining part of the phenotype observed in these mice.
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RESULTS |
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The dmu mouse
Recently, a spontaneous mutation occurred in our colony of (C57BL6xC3H) F1 hybrid mice. This mutation is recessive and homozygous mice suffer from muscle degeneration (Fig. 1A). Therefore, we have named the mutant dmu. During the first week of life, homozygous mice are indistinguishable from their normal littermates. By the end of the second week after birth, homozygous dmu mice have an overt phenotype characterized by a progressive loss of mobility in their hind limbs. A normal mouse is able to run and jump using its back legs to spring forward while the dmu mouse is neither able to run nor is it able to jump. Instead, it crawls using its front legs while dragging its hind legs. Although dmu mice weigh the same as their normal littermates at birth, by postnatal day 15 (P15) they weigh
26% less and the weight loss is even more dramatic (43%) at P18 (Fig. 1B). The disorder in dmu mice is fatal within the first month of life. The cause of death of affected mice is not known; however, labored breathing by mice with the most advanced disease state suggests that respiratory or cardiac failure may be responsible. Heterozygotes, on the other hand, are indistinguishable from wild-type mice and breed normally.
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The dmu phenotype is not caused by a neuropathy
We looked at whether the dmu phenotype could be attributed to neurological defects. However, we did not observe any significant abnormalities in sections of brain or spinal cords from dmu mice (data not shown). There was no evidence of neurodegeneration or cellular abnormality. A similar analysis was also performed for sciatic nerves (Fig. 2). In cross-section, the sciatic nerves showed a perineurium surrounding each fascicle of nerve fibers. The diameter of each fascicle in dmu sciatic nerves was comparable to that in wild-type mice. No axonal loss was detected and the neurons appeared normal (Fig. 2A and C).
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Ultrastructural analysis of the sciatic nerves demonstrated that the myelination of axons was normal (Fig. 2B and D). Electron microscopy (EM) of sciatic nerves from dmu mice revealed <1% of the axons with swellings and abnormal myelin structure. These were too infrequent to explain the severe phenotype of dmu mice.
Furthermore, we have assessed whether the expression of nerve-regulated genes is affected in muscle of dmu mice. We would have expected an increase in the level of myogenin mRNA if the muscle was denervated. Using northern blot analysis, we were unable to detect any change in the level of myogenin transcripts between dmu and wild-type muscle at the phenotype stage (data not shown). Together these results indicate that the movement difficulty observed in dmu mice is not caused by a neuropathy.
Skeletal muscle degeneration in dmu mice
Upon dissection, macroscopic observation of the hind limbs revealed a reduction of the muscle mass in dmu mice. In contrast to spinal cord and sciatic nerve, histopathological examination of hind limb skeletal muscle revealed several abnormalities. Hematoxylin & eosin (H&E)-stained cross-sections of hind limb skeletal muscle from P15 dmu mice showed patchy muscle atrophy; with small caliber regenerating fibers intermixed with normal appearing fibers (Fig. 3B). In comparison, the fibers in sections of muscle from wild-type mice had uniform diameters (Fig. 3A). Centrally nucleated fibers, a hallmark of regenerating muscle, are also observed in the dmu sections, albeit only in isolated regions (Fig. 3B). Several cells with large nuclei are detected (Fig. 3B), suggesting macrophage infiltration. Using Evans blue dye to detect damaged muscle fibers, we demonstrated that a few muscle fibers were stained positive at P7, and that this number increased at P16 (data not shown). These results indicate that skeletal muscle degeneration is occurring in dmu mice. The skeletal muscle histology also revealed heterogeneity in the caliber of muscle fibers from dmu mice. To quantitate this aspect, gastrocnemius muscles were dissected from four P15 pups displaying the dmu phenotype as well as from four normal littermates. The cross-sectional surface area of dmu fibers (n = 592) and control fibers (n = 498) was determined. The results demonstrate that on average the muscle fibers from dmu mice had reduced calibers, with a normal distribution centered at 0.10–0.12 arbitrary units (a.u.) while wild-type muscle had a normal distribution centered at 0.20 to 0.24 a.u. (Fig. 3C).
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Mutations in dystrophin and in components of the DAP complex are causative of several muscular dystrophies. Additionally, ablation of dystrophin or members of the DAP complex generally results in a down-regulation of other members of this complex. To determine if the distribution of the DAP complex is affected in dmu skeletal muscle, we carried out an immunofluorescence study using a number of antibodies. P15 dmu and wild-type littermate gastrocnemius muscle were sectioned transversally and immunostained with antisera against the following members of the DAP complex or associated proteins: ß-dystroglycan, dystrophin,
-sarcoglycan, laminin 2-chain (merosin), neuronal nitric oxide synthase, caveolin 3 (Fig. 4) and syntrophin (data not shown). Sections were counterstained with rhodamine-labeled -bungarotoxin to reveal neuromuscular junctions (Fig. 4). In general, the DAP complex and associated protein distribution appeared to be normal in dmu muscle. Furthermore, normal localization of laminin 2 (Fig. 4G and H) and laminin 1, ß1, and 1 chains (not shown) suggests that the basement membrane is deposited properly. Similarly, neuromuscular junctions were readily identified in both wild-type and mutant sections.
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Ultrastructural analysis of skeletal muscle by EM revealed the presence of necrotic muscle fibers in dmu mice (Fig. 5A and B). There is a variation in the size of myofibers and often these are less compact in their organization. We have sometimes observed macrophage infiltration in regions with extensive muscle degeneration. In addition, the number of muscle satellite cells, as determined by EM analysis of several sections, is doubled in dmu mice suggesting that muscle regeneration is occurring (data not shown).
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We have also observed that the sarcomere structure is affected in some regions of skeletal muscle from dmu mice. An irregular pattern of actin and myosin filaments, and a variation in the number of myofilaments has been noted (data not shown).
Cardiac muscle degeneration in dmu mice
Macroscopic examination revealed that hearts from dmu mice were smaller compared to normal littermates at the phenotype stage. This reduction in size was proportional to the overall reduction in body weight of the mutant mice. Histopathological analysis of dmu hearts at P16 showed defects in atria, septum and ventricles (Fig. 6, compare A–C with D–F). Thinning of the epicardium in the atria, and severe degeneration of cardiac muscle in the septum and ventricles were evident (Fig. 6). A morphological variation in the size of the cardiac fibers in the septum is evident (Fig. 6E). Large spaces between the cardiac fibers in the ventricular myocardium were also observed (Fig. 6F). Ultrastructural analysis confirmed the extensive degeneration present in dmu hearts (Fig. 7). Fibroblasts were present in the sections from dmu right atria, and there was an abundance of connective tissue (Fig. 7D). Degenerating cardiac fibers were readily observed in the ventricular myocardium of a right ventricle from a dmu mouse (Fig. 7F).
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Genetic mapping of the mouse dmu locus
dmu is a recessive mutation that arose spontaneously on a mixed background of C57BL6 and C3H, and was maintained by mating of heterozygous animals. The dmu phenotype was observed at a frequency of 28% consistent with the predicted Mendelian ratio for a recessive, fully penetrant trait under monogenic control. This trait was therefore amenable to genetic mapping. Consequently, in the absence of knowledge of the biochemical function of dmu, we decided to map the locus as a first step towards its identification by a candidate gene approach and/or by positional cloning.
To map dmu, we produced a (dmu/+xDBA/2) F2 population where animals presenting the dmu phenotype were scored at P18. Only mutant animals (n = 55), whose genotypes can be inferred to be homozygous, were used for the genetic analysis. A first scan was done with 23 SSLP markers polymorphic between C57BL6, C3H and DBA/2, and distributed across the mouse genome. Genotype data were analyzed using the interval-mapping program Map Manager QTb11. This analysis provided significant evidence for a single locus determining the dmu phenotype in the vicinity of D15Mit105 (P = 7.3 x 10–7) and prompted us to saturate this region of chromosome 15 with additional markers. The significance of linkage increased for markers distal on the chromosome and was accompanied by an excess of C57BL6 alleles, indicating that the dmu mutation arose on this background. These data were used to produce a genetic map of distal chromosome 15 by minimizing the occurrence of double-crossovers. Locus order and calculated locus distances are shown in Figure 8. dmu was localized to the most telomeric region of chromosome 15. The closest marker to dmu is D15Mit35, presenting three crossovers that define the proximal boundary of the dmu genetic interval. Interestingly, several genes associated with mutant neuromuscular phenotypes in the mouse have been localized to this region and stand as potential candidates for dmu, including Scn8a, a sodium channel subunit gene that is mutated in motor end-plate disease (med) (16). The syntenic region in humans to this region of mouse chromosome 15 is chromosome 12q13 (Fig. 8). A scan of the OMIM database revealed that scapuloperoneal muscular dystrophy (SPMD), in which focal muscle atrophy has been reported, has been linked to 12q13.3-q15 (17).
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Scn8a transcript levels are reduced in dmu mice
To assess whether candidate genes at the dmu locus are affected, we carried out northern blot analysis of total RNA isolated from tissues of mutant and wild-type mice. Interestingly, when an Scn8a cDNA probe was used, a dramatic reduction in the level of transcripts was detected in brain RNA from dmu homozygotes when compared with that from wild-type mice (Fig. 9A). Although the levels were reduced, the size of the Scn8a transcripts appeared to be unaltered. Two transcripts of
10 and 12 kb are detected in both mutant and wild-type samples (Fig. 9A). In contrast, when a cDNA probe from another closely linked gene, Cacnb3, was used, we did not observe any difference in the level of transcripts or in their size when comparing wild-type to dmu mice (Fig. 9B).
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dmu is a new allele of Scn8a
Analysis of the candidate genes and the clinical course of the disease in the dmu mouse suggested that Scn8a depletion was responsible for at least part of the phenotype. To determine whether dmu is a new allele of Scn8a, a genetic complementation experiment was carried out. Mice heterozygous for the med allele (Scn8amed) were obtained from the Jackson Laboratory. These mice have previously been shown to have an insertion of a small LINE element into exon 2 of the Scn8a gene, thereby causing exon skipping and production of a very short truncated protein (18). This allele is therefore predicted to be a null allele. Heterozygous Scn8amed mice were crossed with heterozygous dmu mice. From four resulting litters, 9 of 25 offspring expressed a neurological phenotype. Like the dmu homozygous mice, the dmu/Scn8amed mice displayed an abnormal phenotype in the second week after birth. These mice had severe muscle atrophy, they suffered from a progressive loss of mobility in their hind limbs, and they did not survive past the weaning stage. These results indicate that dmu and Scn8a are allelic. However, it should be noted that the phenotype of the compound heterozygotes was not entirely identical to that of dmu homozygotes. For example, there was a delay of a couple of days in the appearance of the phenotype in the compound heterozygotes. In addition, the dmu/Scn8amed mice were more prone to positioning themselves on their sides and displayed a wriggling movement. This is more akin to the original description of the med mice when they were called ‘seal’ (19). Nevertheless, these results implicate Scn8a in the dmu disorder.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse models to study neuromuscular disorders can be very useful in our understanding of the various steps in the pathogenesis of these diseases. In addition, these mice often serve to facilitate the identification of genes implicated in muscle disease in humans. Here, we have described a novel locus in the mouse that when mutated gives rise to skeletal and cardiac muscle degeneration. We have named this locus dmu for degenerating muscle. The dmu mutation is recessive and homozygous mice are indistinguishable from their normal littermates at birth. However, by P11, dmu mice are smaller and display a severe phenotype characterized by a progressive loss of mobility of their hind limbs. The mice appear to be paralyzed in their hindquarters but do respond to sensory stimuli such as pinching of the toes. Our analysis demonstrated the absence of significant neurological pathology and correspondingly we did not find evidence for skeletal muscle denervation. The loss of weight of dmu mice can largely be attributed to muscle atrophy and wasting. Consistent with this, histopathological and ultrastructural analysis of the dmu mouse revealed severe skeletal muscle defects typical of muscular dystrophy including myofiber atrophy and areas of degeneration/regeneration. The observed muscle pathology in dmu mice does not appear to be caused by defects in the DAP complex or from basement membrane defects. Myocardium degeneration is also a prominent feature of dmu pathology and may be the cause of the early lethality perhaps in combination with a respiratory problem due to degenerating diaphragm.
An intriguing observation from the ultrastructural analysis of the skeletal and cardiac muscle defect was the abnormal organization of the sarcomere structure. In some regions, an atypical number and arrangement of actin filaments surrounding each myosin filament characterized this structural change. The normal repetitive hexagonal pattern was perturbed such that 8 or 10 actin filaments often surrounded one myosin filament. The change in molecular architecture and the resulting irregular patterns of the myofilament matrix may influence the overall muscle contractility. A mechanism by which this might arise could be a mis-registration between the actin binding sites and the myosin cross-bridges. Whether this is a primary or secondary defect in dmu mice remains to be determined.
In our initial mapping of the dmu locus, we narrowed down the genetic interval to lie between the distal portion of mouse chromosome 15 and the telomere. A number of genes have been mapped to this region including the Hoxc gene cluster, several keratin genes, Wnt1, Wnt10b, Cntn1, Itgb7, Cacnb3 and Scn8a. Of these, Cntn1, Cacnb3 and Scn8a are interesting candidates for the dmu locus. Cntn1 codes for the protein contactin, a cell adhesion molecule of the immunoglobulin gene superfamily. This protein has been shown to regulate axonal growth and fasciculation in vitro (20–22). Gene targeting inactivation of contactin in mice caused cerebellum defects and resulted in a severe ataxic phenotype (23). The second gene, Cacnb3, codes for the voltage-dependent ß-3 subunit of the type L calcium channel (24). Although mutations in this gene have not yet been identified, it is interesting to note that mutations in the calcium channel -1 and ß-4 subunits have been identified in the mouse mutant tottering (25) and in its allelic mutants leaner and rolling and in lethargic (26), respectively. Preliminary experiments have not revealed a change in the level of Cacnb3 transcripts in dmu mice (Fig. 9B).
Of the candidate genes residing at the distal end of mouse chromosome 15, Scn8a is the most attractive. This gene codes for the voltage-gated sodium channel subunit 8a. The mouse neurologic mutant med is caused by mutations in Scn8a (16,18,27). The med mouse is characterized by early-onset progressive paralysis of the hind limbs, severe muscle atrophy, degeneration of Purkinje cells and juvenile lethality (16,28). Variation in muscle fiber diameter and appearance of centrally nucleated fibers has also been described in med mice (29). In general, the phenotype of the med mouse is quite similar to that of the dmu mouse described here. We therefore performed genetic complementation crosses between dmu and med mice and demonstrated that they are allelic. In support of this conclusion, we have shown that in dmu mice, transcript levels for the Scn8a gene are dramatically reduced (Fig. 9A). The phenotype of the dmu/Scn8amed compound heterozygotes was not completely identical to that of dmu homozygotes, perhaps due to differences in mutation and/or genetic background. Our results are consistent with dmu being an allele of Scn8a, with a possibility that other genes may also be implicated in the dmu disorder. Since Scn8a is expressed in brain and spinal cord, but not in cardiac or skeletal muscle (16), the observed defects in muscle of dmu mice are likely a result of secondary effects. Further experiments are necessary to determine if a mutation is present within the Scn8a gene and to delineate the exact contribution of Scn8a deficiency to the overall disease in dmu mice.
The dmu locus on distal mouse chromosome 15 is in a region syntenic to human chromosome 12q13 (Fig. 8). Of interest is the linkage of the human syndrome SPMD to this region of chromosome 12 (17). Scapuloperoneal syndromes are a heterogeneous collection of neuromuscular disorders characterized by weakness in the distribution of the shoulder girdle and peroneal muscles (30,31). These syndromes have been divided into scapuloperoneal spinal muscular atrophy (SPSMA), a neurogenic form of the disease, and SPMD, the myogenic form of the disease. SPSMA has been linked to human chromosome 12q24 at a locus distinct from SPMD (17,32). The dmu mouse may represent an animal model of SPMD since patients suffering from the disease have muscle fibers with focal atrophy (17). Particularly striking is the evidence that some patients suffering from SPMD also present with cardiomyopathy (33,34), a feature of the dmu mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice
The dmu mutation arose spontaneously within our breeding colony and was maintained on a C57BL6xC3H F1 hybrid background. Since the mutation is recessive, the allele was maintained by pairing presumptive heterozygotes and only parents giving rise to offspring with the dmu phenotype were retained. Heterozygous Scn8amed mice were obtained from The Jackson Laboratory (Bar Harbor, ME). This allele was originally described by Searle (19) and subsequently shown to have a splicing mutation in the Scn8a gene (18).
Intercross analysis
Tail biopsies were collected and genomic DNA was purified according to a standard protocol (35). Primer pairs were obtained from Research Genetics (Huntsville, AL). F2 genotypes were obtained by PCR amplification of tandemly repeated sequences (microsatellites) that were polymorphic between C57BL6, C3H and DBA/2J, essentially as described previously (35). Linkage analysis was performed using Map Manager QTb11 (http://micro.med.buffalo.edu/mmm/). An arbitrary P-value of 0.0001 was used to indicate support of linkage in accordance with accepted standards (36).
Evans blue staining of skeletal muscle in vivo
To detect damaged muscle fibers, vital staining using Evans blue dye was performed. Two litters from heterozygous dmu/+ parents were examined at P7 and P16. Each pup was weighed, identified and injected intraperitoneally with 0.1 ml of Evans blue (10 mg/ml in PBS)/10 g of body weight. All pups were killed 20 h later, hind limb skeletal muscle was fixed in 4% paraformaldehyde in PBS, embedded in paraffin, sectioned at 4 µm and examined under a fluorescent microscope (Zeiss Axioplan).
Histological analysis
Brain, spinal cord, sciatic nerve, heart, skeletal muscle and diaphragm were collected from wild-type and homozygous dmu mice at P15 or P16. Tissues were fixed in 4% paraformaldehyde in PBS pH 7.4 for 24 h, embedded in paraffin, sectioned at 4 µm thickness, stained with H&E and examined by light microscopy using a Zeiss Axioplan microscope. For fiber caliber measurements, gastrocnemius muscles were dissected out and embedded in JB4 glycol metacrylate resin, sectioned at 2 µm thickness, stained with H&E, and photographed using a Zeiss Axioplan microscope equipped with a digital camera. Images were imported into the NIH Image analysis program and a blinded measurement of the cross-sectional area of each muscle fiber was determined.
Immunofluorescence
Skeletal muscles (tibialis, semitendinosus, and gastrocnemius) from P15 homozygous dmu and wild-type mice were dissected in PBS, embedded in OCT compound (Sakura) and frozen in isopentane, the isopentane having been cooled in liquid nitrogen beforehand. Cryostat sections of 14 µm thickness were stored at –80°C before using. Sections were thawed in a moist chamber at room temperature for 15 min. After two washes in blocking buffer (0.5% BSA, 0.15% glycine in PBS) for 15 min, sections were incubated in blocking buffer supplemented with 5% normal horse serum at room temperature for 15 min. The relevant primary antibodies diluted in blocking buffer were incubated with sections for 1 h at room temperature. After three washes with blocking buffer for 15 min, the sections were incubated with the secondary antibody diluted in blocking buffer for 1 h at room temperature. Sections were subsequently washed three times in PBS for 15 min, mounted (DAKO fluorescent mounting medium) and visualized by fluorescent microscopy (Zeiss Axioplan). Tetramethylrhodamine-conjugated -bungarotoxin (Molecular Probes, 1:200 dilution) was used to visualize the acetylcholine receptors in skeletal muscle.
Primary antibodies. Monoclonal anti-dystrophin antibody, which is derived from mandra 1 (Sigma), was used at a 1:200 dilution. The -sarcoglycan antiserum was kindly provided by Dr E.McNally (University of Chicago, Chicago, IL; 37). The anti-laminin 2 (merosin) antibody was kindly provided by Dr P.Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ). Syntrophin, monoclonal antibody 2101 A used at 1:100 dilution was kindly provided by Dr S.Froehner (University of North Carolina, Chapel Hill, NC). Antiserum to ß-dystroglycan was kindly provided by Dr S.Carbonetto (McGill University, Montreal, QC).
Secondary antibody. Anti-mouse IgG-FITC conjugated (Sigma) was used at 1:200 dilution.
Electron microscopy
Homozygous dmu mice and wild-type mice at P18 were anesthetized by i.p. injection of avertin. The chest was shaved and the thoracic cage was opened. The mice were transcardially perfused with 10 ml of PBS followed by 20 ml of Karnovsky’s fixative (4% paraformaldehyde, 2% glutaraldehyde and 0.1 M cacodylate in PBS pH 7.4). Tissues were dissected out and for the heart, the four cavities (left and right ventricles, and left and right atria) as well as the septum were isolated. The central part of each sample was used for EM. After incubation for 4–6 h in the same fixative, samples were post-fixed with osmium tetroxide for 1 h. The samples were subsequently dehydrated and embedded in EMbed-812 (from Electron Microscopy Sciences). Sections (60 nm) were collected onto grids, pre-treated with 2% aqueous uranyl acetate, stained with lead citrate and observed by EM. For light microscopy, sections of 0.5 µm were stained with 1% toluidine blue.
RNA analysis
Total RNA was prepared from brain and liver using the Trizol protocol (Life Technologies, Grand island, NY). Approximately 20 µg of total RNA was resolved by electrophoresis through 1% agarose in the presence of 2.2 M formaldehyde and transferred to a Hybond membrane (Amersham Pharmacia Biotech). Filters were hybridized to radioactively labeled probes as described previously (38). RT–PCR products corresponding to nucleotides 4563–5146 of the Scn8a cDNA and nucleotides 1558–2049 of the Cacnb3 cDNA were used as probes in the northern analysis. The membranes were autoradiographed at –80°C for 19 h.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs C.DiDonato, C.Boudreau-Larivière and D.Picketts for critical reading of the manuscript, and the rest of the Kothary laboratory for helpful discussions. We thank Martine Mathieu for excellent technical assistance, and Ian Robb (Children’s Hospital of Eastern Ontario) and Louise Pelletier (University of Ottawa) for carrying out the EM. Drs Sal Carbonetto, Paul Holland, Elizabeth McNally, Stanley Froehner and Peter Yurchenco kindly provided various antibodies used in this study. This work was supported by grants from the Canadian Institutes of Health Research (CIHR, grant no. MT-12622) and the Muscular Dystrophy Association (USA) to R.K. P.D.C. is supported by a CIHR Postdoctoral Fellowship.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada. Tel: +1 613 737 8707; Fax: +1 613 737 8803; Email: rkothary@ohri.ca
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REFERENCES |
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