Human Molecular Genetics, 2001, Vol. 10, No. 24 2745-2750
© 2001 Oxford University Press
Suppression of revertant fibers in mdx mice by expression of a functional dystrophin
1Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109-0618, USA, 2Muscle Cell Biology, Medical Research Council Clinical Science Center, Hammersmith Hospital, London W12 ONN, UK, 3Department of Neurology, University of Washington, Seattle, WA, 98195-7720, USA
Received June 22, 2001; Revised and Accepted September 11, 2001.
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ABSTRACT |
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Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration that results from the absence of dystrophin. Despite null mutations in the dystrophin gene, many DMD patients display a low percentage of dystrophin-positive fibers. These ‘revertant fibers’ are also present in the dystrophin-deficient mdx mouse and are believed to result from alternative splicing or second mutation events that bypass the mutation and restore an open reading frame. However, it is unclear what role dystrophin and the dystrophic pathology might play in revertant fiber formation and accumulation. We have analyzed the role of dystrophin expression and the dystrophic pathology in this process by monitoring revertant fibers in transgenic mdx mice that express truncated dystrophins. We found that newborn transgenic mice displayed approximately the same number of revertant fibers as newborn mdx mice, indicating that expression of a functional dystrophin does not suppress the initiation of revertant fiber formation. Surprisingly, when the transgene encoded a functional dystrophin, revertant fibers were not detected in adult or old mdx mice. In contrast, adult transgenic mice expressing a non-functional dystrophin accumulated increasing numbers of revertant fibers, similar to mdx mice, suggesting that positive selection is required for the persistence of revertant fibers. Finally, we provide evidence that the loss of revertant dystrophin in transgenic mdx muscle fibers overexpressing a functional dystrophin results from displacement of the revertant protein by the transgene-encoded dystrophin.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Duchenne muscular dystrophy (DMD) and the milder Becker muscular dystrophy (BMD) result from mutations within the dystrophin gene (1–3). Dystrophin is a large, 427 kDa multidomain protein that binds multiple integral and peripheral membrane proteins, known as the dystrophin glycoprotein complex (DGC) (4). The DGC links the actin cytoskeleton to the extracellular matrix in muscle, helping to prevent damage to the sarcolemma in contracting and quiescent muscle fibers (5–8). Patients with dystrophin mutations display progressive muscle degeneration, leading to muscle weakness and eventual respiratory and/or cardiac failure (1).
An interesting phenomenon that occurs in 
50% of DMD patients is the presence of revertant fibers that express dystrophin (9–12). Little is known about the molecular mechanisms leading to revertant fibers, but they comprise only 1–7% of all muscle fibers (9,13,14). This phenomenon is not limited to humans, but has also been observed in the dystrophin deficient mdx mouse and the cxmd dog animal models (15–17). Revertant fibers are detectable during all stages of development. Newborn and fetal muscles display isolated, single revertant fibers which grow into clusters that increase in size and number with age (18). There appears to be no correlation between the number of revertant fibers and the severity of the disease, as they are not found in sufficient numbers to prevent dystrophy (9).
Closer analysis of individual revertant fibers has revealed the structure of revertant dystrophins present in mdx muscles. Immunofluorescent analysis using a panel of antibodies specific to the N- and C-terminal regions of dystrophin show that these domains are expressed in revertant fibers (19). However, antibodies recognizing portions of dystrophin encoded by exons near the mdx mutation (a nonsense mutation in exon 23) typically fail to detect the revertant dystrophin. These studies suggest that revertant fibers express dystrophins that arise from alternatively spliced transcripts lacking both the mutant exon and a variable number of adjacent exons (18). Alternatively spliced forms of dystrophin mRNA are detectable by RT–PCR in both dystrophic and wild-type muscle, indicating that such splicing may occur regardless of the muscle phenotype (18–21). It remains unclear what mechanism leads to the stable appearance of revertant dystrophin proteins in only a subset of dystrophic muscle fibers. It is also unclear whether the dystrophic phenotype induces the formation of revertant fibers or simply provides a selective environment to maintain such fibers once they have formed.
We sought to understand the process of dystrophy in the initiation and progression of revertant fibers by asking whether revertant fibers arise in non-dystrophic muscle. This question has been difficult to address since antibodies against dystrophin would not distinguish revertant from wild-type dystrophin in muscles from heterozygous (mdx/+) female mice. However, we have recently described several lines of transgenic mice on the mdx background that express highly functional dystrophins lacking small domains (22–24). These truncated dystrophins lack some epitopes present in full-length and revertant dystrophins, making it possible to distinguish the revertant from the transgene-encoded dystrophins by immunostaining. We found that mice expressing functional dystrophins exhibited normal numbers of revertant fibers in newborn muscle. Surprisingly, almost all revertant fibers disappeared by 2 months of age. We also show that the overexpression of transgene-encoded dystrophin significantly downregulates the endogenous dystrophin levels in wild-type mice, providing a possible explanation for the absence of revertant fibers in adult, transgenic mdx muscles.
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RESULTS |
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Revertant fibers in adult transgenic mice
We initially sought to determine whether expression of dystrophin from transgenes might affect the number of revertant fibers found in mdx muscle. For this purpose, single muscle sections from a variety of muscle groups were immunostained with dystrophin antisera. The mice analyzed were adult C57Bl/10, mdx, and transgenic mice expressing either the
71–78, H2–R19 or Dp71 forms of dystrophin (Fig. 1A). One-year-old 71–78 mice displayed no revertant fibers in quadriceps, extensor digitorum longus (EDL), tibialis anterior (TA), diaphragm or soleus muscle (Fig. 1B). Similarly, no revertant fibers were detected in the TA or EDL from 2-year-old H2–R19 mice. In contrast, mice expressing the Dp71 isoform of dystrophin displayed large numbers of revertant fibers, similar to the number in mdx mice (Fig. 1). The number of revertant fibers observed in each muscle group is tabulated in Table 1. Since we observed so few revertant fibers in the 71–78 mice, we chose to examine a larger number of sections from several different muscles of young (2 months) and old (2 years) mice. Analysis of 100 sections from each muscle of the 71–78 and H2–R19 mice identified very few revertant fibers, all of which were found in a single muscle from each strain (Table 2). Two isolated revertant fibers were detected in the TA of one 2-year-old 71–78 mouse. The H2–R19 mice displayed a single cluster of five revertant fibers in the soleus muscle from a 2-year-old mouse.
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Revertant fibers in newborn transgenic mice
Since the 2-month-, 1-year- and 2-year-old
71–78 and H2–R19 mice displayed a striking paucity of revertant fibers, we decided to also examine neonatal muscles. Quadriceps muscles from 5-day-old 71–78 mice displayed a similar number of revertant fibers as did those from mdx mice (Fig. 2; Table 2). These revertant fibers were approximately the same length (30 µm) and were present as single isolated fibers (not clustered) in both strains. Immunostaining with antisera against the N-terminus of dystrophin confirmed that the 71–78 protein was expressed at this age (Fig. 2).
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Absence of revertant fibers is not likely due to mRNA downregulation
To determine whether the reduced number of revertant fibers in adult transgenic mice was due to a downregulation of the endogenous dystrophin transcript, we performed RT–PCR. Amplification of the dystrophin mRNA segment encoded by exons 74–76 generates a product from the endogenous, but not from the
71–78 transcript. Semi-quantitative RT–PCR was performed in C57Bl/10, mdx and 71–78 mice by amplifying this exon 74–76 fragment in parallel with a fragment from the hypoxanthine phosphoribosyltransferase (HPRT) gene transcript. When compared with HPRT controls, the dystrophin mRNA levels were approximately equal in muscle RNA isolated from 71–78 and mdx mice (Fig. 3). Dystrophin mRNA levels were higher in C57Bl/10 muscle than in mdx muscles as reported previously (25).
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Endogenous dystrophin levels are downregulated by overexpression of exogenous dystrophin
We next asked whether the transgene-encoded dystrophin could affect the accumulation of endogenous wild-type C57Bl/10 or revertant (mdx) dystrophin. For these studies, we initially examined two different transgenic mouse lines that express the
71–78 transcript at different levels on a wild-type (C57Bl/10) background (Fig. 4). Line 1 expresses less dystrophin 71–78 protein than Line 2, as detected with the N-terminal dystrophin antibody. Muscles with the highest expression of dystrophin 71–78 protein accumulated the least amount of endogenous dystrophin, as detected with the C-terminal dystrophin antibody. A similar downregulation of the endogenous protein was also seen in a transgenic line that expresses high levels of a functional, truncated dystrophin (R2–21+H3). Furthermore, we have observed a similar downregulation of endogenous dystrophin in at least three other transgenic mouse lines (unpublished data). This striking downregulation of endogenous dystrophin on a wild-type background suggests that low levels of revertant dystrophin in mdx mice might be similarly suppressed by expression of stable and functional transgenic dystrophins.
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DISCUSSION |
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The precise mechanism by which revertant fibers express dystrophin is unknown, but appears to involve exon skipping around mutations such that an open reading frame is restored (18–21). It is unclear what role dystrophin and the dystrophic pathology play in revertant fiber formation. We had the opportunity to study revertant fibers on a non-dystrophic background, and found evidence that a lack of functional dystrophin is required for the maintenance, but not the formation of revertant fibers.
Revertant fibers have previously been identified in newborn mdx mice before any signs of dystrophy were apparent (9,18). This observation suggested that dystrophic pathology is not required for the initial events leading to the formation of revertant fibers. We showed that overexpression of a highly functional dystrophin transgene does not prevent revertant fibers from forming in newborn mice. Therefore, the events leading to the initial formation of revertant fibers in mdx mice appear to be independent of a dystrophic phenotype and the presence of dystrophin.
Revertant fibers increase in size, number and length with age in both humans and the mdx mouse (18). Surprisingly, we found that in adult 71–78 mice, revertant fibers were almost entirely absent. This lack of revertant fibers indicates the importance of the dystrophic phenotype in growth and expansion of revertant fibers. Mice expressing the Dp71 isoform of dystrophin have the same number of, if not more, revertant fibers than do mdx mice. Since Dp71 is not able to prevent dystrophy in mdx mice (26), this result suggests that simple expression of an exogenous dystrophin protein is not sufficient to prevent the accumulation of revertant fibers. Instead, the exogenous dystrophin must be functional to suppress an increase in the number of revertant fibers. We have previously noted that Dp71 mice appear more dystrophic than mdx mice (26), suggesting that the severity of dystrophy may correlate with the number of revertant fibers present. We found it intriguing that the TA muscle was the only muscle group from the 71–78 transgenic mouse in which any revertant fibers were detected (Table 2). This muscle displays a partially mosaic expression of the transgene and significantly higher levels of centrally nucleated myofibers, indicating an increased rate of regeneration (22). These data suggest that ongoing regeneration may be required to maintain revertant fibers in adult mice, and support the idea that positive selection is necessary for persistence and expansion of revertant fibers. When normal dystrophin is absent, as in mdx mice, the revertant dystrophin may persist, mechanically protecting the revertant muscle fiber from injury.
Expression of a functional dystrophin not only prevented an increase in the number and size of revertant clusters, but also led to a dramatic decrease in detectable revertant dystrophin in adult mice. Semi-quantitative analysis of endogenous dystrophin mRNA and protein levels in transgenic/mdx and transgenic/wild-type mice suggested that functional transgenic dystrophin can actively downregulate endogenous dystrophin protein, but not mRNA. Although the mechanism of downregulation is unclear, there are several possibilities. First, the transgenic dystrophin may saturate the sarcolemmal membrane, displacing or out-competing the endogenous normal and revertant dystrophin. This competition may be less pronounced in newborn mice since neonatal muscles are undergoing a rapid phase of growth and myocyte fusion that is producing a large surface area of new sarcolemma that has not yet become saturated with dystrophin. There is also the possibility that transgenic 71–78 and H2–R19 protein is more functional or stable than smaller, internally truncated, revertant dystrophins. Therefore, these revertant proteins might be unable to compete with the highly functional transgenic proteins. Indeed, the precise structure of different mini- and micro-dystrophins has an enormous impact on the relative functional capacity of the truncated proteins (23,27; unpublished data).
Our data suggest that multiple events influence revertant fiber formation. The initial appearance of revertant fibers results from events early in development that are independent of dystrophin or the dystrophic pathology. The apparent clonal origin of revertant fiber clusters (18) suggests that the initiating events occur before muscle precursor cells differentiate into myocytes. The dystrophin gene is not transcribed in muscle precursor cells, so it is not surprising that dystrophin expression has no influence on initial revertant fiber appearance. In contrast, maintenance and expansion of revertant fibers occurs only in the absence of a functional dystrophin. We suggest that revertant fibers are not maintained in adult transgenic mice due to a competitive advantage of the transgenic versus the revertant dystrophin. Dp71 lacks both the rod and actin-binding domains, and may not effectively compete with nearly full-length ‘revertant’ dystrophin. However, since the H2–R19 protein lacks the rod domain but is able to suppress reversion, effective competition between exogenous and endogenous dystrophins appears to require the N-terminal actin-binding domain. Alternatively, the failure to detect expanded clusters of revertant fibers in old transgenic/mdx mice might be due to a lack of ongoing necrosis and regeneration in these protected muscles, which also eliminates any selective advantage for revertant fibers.
The natural occurrence of revertant fibers in DMD patients and animal models for the disease has potential implications for therapy (16). Although individual revertant fibers appear to be protected from dystrophic degeneration, they are not present in sufficient numbers to be clinically significant. Developing a better understanding of the events underlying the initiation of revertant fiber formation could facilitate the development of therapeutic strategies aimed at increasing endogenous dystrophin expression in the muscles of DMD patients (28–30).
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MATERIALS AND METHODS |
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Transgenic mice and immunofluorescence
Mice expressing dystrophin deleted for exons 71–78 (
71–78) or for hinge 2 to spectrin-like repeat 19 (H2–R19), or for spectrin-like repeats 2–21 [but including hinge 3 (R2–21+H3)] were generated as described by Crawford et al. (22). Unless explicitly stated otherwise, all transgenic strains were carried on the mdx dystrophin deficient background. To analyze revertant fibers, muscles were isolated and frozen in liquid nitrogen cooled OCT (Tissue Tek). Serial sections (50–100), 10 µm thick, were analyzed for each mouse strain. Tissue sections from the 71–78 mice were stained with a rabbit antibody specific for the C-terminal domain of dystrophin (26). This antibody does not recognize the 71–78 dystrophin protein (Figs 1 and 2). Dp71 mice express only the Dp71 isoform of dystrophin, encoded by exons 63–79, and are dystrophic (26). To identify revertant fibers in Dp71 mice, a rabbit antibody specific for the dystrophin N-terminal domain was used (31). After incubation with antiserum for 2 h, sections were washed and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies. Images were collected on a Nikon E1000 microscope using fixed exposure times. Muscles from H2–R19 mice were incubated with a monoclonal antibody specific for the rod domain. Muscle fibers that were dystrophin positive were scored as revertant. Every fiber within each muscle cross section was scored for the presence of revertant dystrophin, and similar numbers of fibers were counted from each test group. Revertant fiber counts were performed as a blind study and the mean average number of revertant fibers per serial section was determined. Individual revertant fibers were followed through multiple sections to determine their approximate length.
RT–PCR
To estimate the relative ratios of endogenous dystrophin from wild-type, mdx and 71–78 (on the mdx background) transcripts, RT–PCR was performed as described previously (32). Briefly, 1 µg of total RNA from 1-year-old mice was used in a reverse transcription reaction, using either an exon 77-specific reverse primer (5'-GGGAAGGAGTTGTTGAGTTGCTC-3') for dystrophin or an oligo(dT) primer for HPRT. PCRs used either HPRT primers (5'-GCTGGTGAAAAGGACCTCT-3' and 5'-CACAGGACTAGAACACCTGC-3') or dystrophin primers specific for exons 74–76 (5'-GAGAATCCTAGCAGATCTTGAGG-3' and 5'-GAAGTTTGACTGCCAACCAC-3'). Note that these latter primers do not amplify the 71–78 transcript. Products were separated after 40 cycles of PCR by electrophoresis on 1% agarose gels and the approximate levels of the dystrophin products were determined relative to the HPRT products by densitometry using Molecular Analyst software (Bio-Rad).
Western analysis
Protein was extracted from mouse quadriceps as described previously (33). Briefly, tissue was ground to a fine powder in a liquid nitrogen cooled mortar and pestle. Frozen tissue was boiled and vortexed in homogenate solution (1% SDS, 5 mM EGTA) plus protease inhibitor Complete (Boehringer). Protein concentrations were determined by Bradford assay and confirmed by Coomassie blue staining of SDS–PAGE separated samples. Forty micrograms of protein was separated by SDS–PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in TBST (10 mM Tris–Cl pH 8.0, 150 mM NaCl, 0.05% Tween 20) and incubated with primary Dys1 or Dys2 monoclonal antibodies (Novacastra) for 3 h at room temperature. After three washes in TBST, the membranes were incubated for an additional 2 h in secondary goat anti-mouse antibody conjugated to horseradish peroxidase (HRP; Jackson Labs) and washed again three times in TBST. Immunoreactive proteins were visualized using an ECL kit (Amersham).
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ACKNOWLEDGEMENTS |
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We thank Dennis Hartigan-O’Connor, Scott Harper and Michael Hauser for helpful discussions and Robert Crawford for excellent technical assistance. This study was supported by NIH grants AR 40864 and AR 44533, and a grant from the Muscular Dystrophy Association (USA) to J.S.C.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Department of Neurology, University of Washington School of Medicine, K243 HSB, Box 357720, Seattle, WA 98195-7720, USA. Tel: +1 206 221 5363; Fax: +1 206 616 8272; Email: jsc5@u.washington.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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