Mutation Types and Aging Differently Affect Revertant Fiber Expansion in Dystrophic Mdx and Mdx52 Mice

Duchenne muscular dystrophy (DMD), one of the most common and lethal genetic disorders, and the mdx mouse myopathies are caused by a lack of dystrophin protein. These dystrophic muscles contain sporadic clusters of dystrophin-expressing revertant fibers (RFs), as detected by immunohistochemistry. RFs are known to arise from muscle precursor cells with spontaneous exon skipping (alternative splicing) and clonally expand in size with increasing age through the process of muscle degeneration/regeneration. The expansion of revertant clusters is thought to represent the cumulative history of muscle regeneration and proliferation of such precursor cells. However, the precise mechanisms by which RFs arise and expand are poorly understood. Here, to test the effects of mutation types and aging on RF expansion and muscle regeneration, we examined the number of RFs in mdx mice (containing a nonsense mutation in exon 23) and mdx52 mice (containing deletion mutation of exon 52) with the same C57BL/6 background at 2, 6, 12, and 18months of age. Mdx mice displayed a significantly higher number of RFs compared to mdx52 mice in all age groups, suggesting that revertant fiber expansion largely depends on the type of mutation and/or location in the gene. A significant increase in the expression and clustering levels of RFs was found beginning at 6months of age in mdx mice compared with mdx52 mice. In contrast to the significant expansion of RFs with increasing age, the number of centrally nucleated fibers and embryonic myosin heavy chain-positive fibers (indicative of cumulative and current muscle regeneration, respectively) decreased with age in both mouse strains. These results suggest that mutation types and aging differently affect revertant fiber expansion in mdx and mdx52 mice.


Introduction
Duchenne muscular dystrophy (DMD) is the most common genetic muscular disease and is characterized by progressive muscle degeneration. It occurs with a frequency of about 1 out of 3,500 boys, and is caused by mutations in the dystrophin (DMD) gene [1][2][3]. The mutation leads to progressive myopathy and muscle weakness coupled with cycles of muscle degeneration. Death eventually occurs due to severe respiratory and/or cardiac failure at approximately 20-30 years of age [4,5]. There is currently no effective cure for DMD [6][7][8]. The DMD gene is located on the X-chromosome and is characterized as one of the largest and more complex genes in humans, containing 79 exons and spanning more than 2.4 million base pairs [9,10]. The DMD gene encodes at least 18 protein isoform products of dystrophin with tissue specific alternative promoters [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. The main skeletal muscle isoform is the largest known, consisting of 3,685-amino acids (427 kDa). In skeletal muscles, dystrophin plays a central role in organizing a multi-protein complex at the sarcolemma, and linking cytoskeleton proteins to extracellular matrix proteins [27,28]. The full-length dystrophin protein can be divided into actin-binding NH 2 -(N) terminal, rod, cysteine-rich, and COOH-(C) terminal domains [14,20]. Interestingly, most of the known functions of the protein are assigned to the N-and Cterminals and the cysteine-rich domain [29]. In contrast, the central rod domain, consisting of 24 spectrin repeats with 4 hinges, and spanning about half the length of the protein, appears to be less essential to proper function [30].
Most DMD mutations, which cause disruption of the open reading frame, occur within the central rod domain, thus preventing translation of the crucial C-terminal domain [31][32][33]. In the case of Becker muscular dystrophy (BMD), a milder form of muscular dystrophy also caused by mutations in the DMD gene, most mutations occur in the same rod domain regions, but the mutated mRNA transcripts preserve the open reading-frame and are thus translated into a truncated yet partially functional protein [34]. The mdx mouse, an animal model of human DMD, harbors a natural nonsense point mutation in exon 23, while the mdx52 mouse has a deletion mutation of exon 52 owing to gene-targeting method [35][36][37][38]. Mdx mice were originally discovered with C57BL/10 background, but they were later backcrossed with C57BL/6 background for comparison with other mouse models including mdx52 mice with C57BL/6 background [39]. Both mutants exhibit a lack of dystrophin expression and cycles of muscle degeneration/regeneration [40,41]. Thus, the comparison between mdx and mdx52 mice with the same genetic background should be quite useful in understanding pathogenic mechanisms dependent on different types of nonsense and exon-deletion mutations, which are found in approximately 10-15% and 50-60% of the DMD patients, respectively [42,43].
Interestingly, skeletal muscles in most DMD patients and animal models including mouse and dog models display sporadic dystrophin-positive muscle fibers called ''revertant fibers'' (RFs) in an otherwise dystrophin-negative background [44][45][46][47][48][49][50][51]. Danko et al report distinct frequencies of RFs in different mdx mutants (mdx2cv, 3cv, 4cv, and 5cv mutants) [50]. These dystrophin-positive fibers arise from spontaneous exon skipping (alternative splicing) with a loss of up to 30 exons, leading to the production of in-frame truncated proteins [52]. In mdx mice, these RFs expand with age through cycles of muscle degeneration/regeneration and the activation of muscle precursor cells [40,52]. In human DMD patients, the increase of RFs correlates significantly with their age up to early teens [53]. These revertant events are thought to arise within a subset of muscle precursor cells which proliferate in response to muscle degeneration and participate in regeneration of muscle fibers [40]. These fibers often produce clonal clusters that can expand to up to 100 fibers measuring more than 1 mm in length by 18 months of age [52]. To date, there is no report of either genomic deletion or secondary mutation as the mechanism facilitating the restoration of dystrophin in RFs. Our previous studies using various transgenic mouse models, including microdystrophin transgenic mdx mice and utrophin overexpressing mice, as well as irradiated mdx models, clearly demonstrated that expansion of RF clusters is dependent on muscle regeneration [40,[54][55][56][57]. The expansion of RF clusters reflects the cumulative history of skeletal muscle regeneration and is thought to provide a useful index for functional evaluation of therapies that diminish muscle degeneration [40].
In this study, we employed two newly developed mouse models, mdx mice with C57BL/6 background, and mdx52 mice [37,39]. We compared their RF generation and long-term expansion up to 18 months of age. Interestingly, mdx mice exhibited a higher number of RFs compared to mdx52 mice, indicating the occurrence of revertant events largely depends on the type of mutation present in the DMD gene. To our surprise, although both mouse models showed an increase in RFs through 18 months, the number of centrally nucleated fibers (CNFs) and embryonic myosin heavy chain (eMHC)-positive fibers (as cumulative and current indicators of muscle regeneration, respectively) decreased with age in both the strains except for an increase in CNFs between 2 to 6 months of age in mdx mice. Overall, the dynamics of muscle regeneration associated with age was more markedly altered in mdx than mdx52 mice. These data reported here show that mutation types and aging differently affect RF expansion and muscle regeneration in mdx and mdx52 mice.

Ethics Statement
All animal works were conducted according to relevant national and international guidelines. Animal study was approved by the Ethics Committee for the Treatment of Laboratory Animals of the National Center of Neurology and Psychiatry, and the Animal Care and Use Committee (ACUC) of the University of Alberta. All

Animals
Mdx mice with C57BL/6 background, mdx52 mice, and C57BL/6 mice as controls were used in this study [37,39,58]. The genetic background of mdx mice was changed into C57BL/6 as previously described [39]. Male and female mdx and mdx52 mice at 2, 6, 12, and 18 months of age were used in this study. Homozygous mutation of the DMD gene was confirmed in female mice by genotyping. Male C57BL/6 mice at 2 months of age were used as controls. Mice were euthanized by cervical dislocation; then, tibialis anterior (TA) and gastrocnemius (GC) muscles were excised and immediately frozen in liquid nitrogen-cooled iso-pentane. Samples were stored at 280uC until used for immunohistochemistry and histochemistry.
Immunohistochemical analysisAntibodies. The rabbit polyclonal antibody against C-terminal domain (position at 3,661-3,677 amino acids) in human dystrophin was used to detect revertant fibers (Abcam, Bristol, UK). eMHC expressed in newly regenerated muscle fibers was detected with mouse monoclonal anti-rabbit developmental MHC antibody (Leica Biosystems, Newcastle upon Tyne, UK).
Immunohistochemistry. Dystrophin-positive RFs were detected by immunohistochemistry. Transverse frozen sections (7 mm thick) from TA and GC muscles were cut using a Leica CM1900 cryostat (Leica Micro-systems, Wetzlar, Germany). Serial sections were picked up on poly-L-lysine-coated glass microscope slides and air-dried for 30 min. Unfixed sections were then blocked in phosphate-buffered saline (PBS) with 20% goat serum, 0.1% TritonX-100 for one hour at room temperature. Dystrophin was detected with rabbit polyclonal primary antibody against human dystrophin C-terminal (1:400) in the blocking solution by overnight incubation at 4uC. After washing 3 times with PBS, the primary antibody was detected with AlexaFluor TM 488-conjugated goat anti-rabbit IgG secondary antibody (1:2,000) (Molecular Probes, OR, USA) with one-hour room temperature incubation. Nuclear counterstaining was performed with 4',6diamidino-2-phenylindole (DAPI) in a mounting agent (Vectashield; Vector Laboratories, CA, USA). Expression of eMHC and its co-localization with RFs were assessed by triple staining using anti-developmental MHC antibody (1:20), anti-dystrophin anti- Assessment of RFs and eMHC-positive fibers. The muscle RF assessment was performed according to our previous study [40]. RFs and eMHC-positive fibers in entire TA or GC muscle sections were observed using a fluorescence microscope (Nikon Eclipse TE 2000-U; Nikon, Tokyo, Japan) with a 20X objective lens. Muscle fibers were regarded as dystrophin-positive only when more than half the membrane circumference was stained in cross-sections. RFs immediately adjacent to each other were characterized as a single cluster. For closer comparison of RFs and eMHC-positive fibers in mice of different groups, at least 8 serial sections at every 100 mm from the muscle belly were stained with antibodies to assess the following: the number of RFs in a cross-section, the number of revertant clusters, the maximum number of RFs in a cluster, and the number of eMHC-positive fibers. For data per mouse, the highest number among serial sections was averaged between left and right muscles for each of the aforementioned criteria. Large clusters of eMHC-positive fibers (clusters composed of more than one hundred eMHCpositive fibers, which arose due to severe degeneration and were independent of age) were excluded from counting.

Hematoxylin and Eosin staining
Hematoxylin and eosin (HE) staining was performed with Mayer's hematoxylin and eosin solutions as previously described [41]. A DMR microscope (Leica Micro-systems) was used for bright field microscopy with a 20x objective lens. The percentage of CNFs was calculated in 300 to 1,000 fibers in each left and right muscle, and was averaged between two muscles per mouse.

Statistical analysis
All data were reported as mean values 6 standard deviation (S.D.). The statistical differences between the age groups or the strains were assessed by one-way ANOVA with a Tukey-Kramer multiple comparison test. Pearson correlation coefficient was performed between the number of RFs in a section, the number of RF clusters, and the maximum number of RFs in a single cluster. P,0.05 was considered statistically significant.

Results
Distinct patterns of revertant fiber expression and clustering in mdx and mdx52 mice To investigate the effect of different mutation types of the DMD gene on the generation and expansion of RF clusters, we examined RFs in TA and GC muscles of mdx mice with C57BL/6 background and mdx52 mice (also with C57BL/6 background). Mdx mice contain a nonsense mutation in exon 23, and mdx52 mice contain a deletion mutation in exon 52 [36,37]. RFs were observed in all age groups in both mdx and mdx52 mice and centrally-located nuclei were found in most RFs (Figure 1). A rabbit polyclonal antibody against the C-terminal domain of dystrophin was used to detect revertant dystrophin expression because this amino acid-region is reported to be retained in most of the truncated dystrophin or RF proteins in mdx and DMD patients [49,52,59]. Mdx mice clearly showed a higher number of RFs and RF clusters in TA and GC muscles across all age groups in comparison to mdx52 mice (Figures 1, 2A, and B). There were significantly fewer RFs and RF clusters in mdx52 mice when compared to mdx mice. Mdx mice also showed a significantly higher number of RFs per cluster compared with mdx52 mice, except at 2 months of age in GC muscle ( Figure 2C).
We then compared the expression and clustering levels of RFs at 2, 6, 12, and 18 months of age for mdx and mdx52 strains individually. In TA and GC muscles from mdx and mdx52 mice, the number of RF, RF clusters, and the maximum number of RFs per cluster significantly increased compared to 2 months of age (Figures 2A, B and C). The Pearson correlation coefficient showed a strong correlation among these three criteria regarding expression and clustering levels of RFs in each strain (squared correlation coefficient; R 2 = 0.27-0.84, P,0.05). A significant increase in the number of RFs and the maximum number of RFs per cluster at 6 months of age was found only in mdx mice. Mdx mice at 12 months old also showed significant increase in the number of RF clusters compared to 2 months old. In contrast to the dramatic RF expansion with age in mdx mice, mdx52 mice showed only a moderate increase in RFs with age across all measured criteria. A significant increase in RF expression and clustering was found at 18 months in mdx mice compared with mdx52 mice. Levels of RF expression/clustering in mdx52 mice never reached levels seen in mdx mice. This result is not attributable to genetic background, since both mouse strains have a C57BL/6 background. The mdx52 mouse, which has fewer RFs and shows little change in RF expansion with age, may be a good model for testing of new therapies aimed at restoring dystrophin expression, because pre-existing RFs can disturb accurate assessments of the restored dystrophin expression owing to therapeutic treatment.

Dynamics of muscle regeneration with age is different between mdx and mdx52 mice
To examine whether muscle regeneration is physiologically correlated with RF expansion, we assessed CNFs and eMHCpositive fibers, which are cumulative and current indicators of muscle regeneration, respectively, in mdx and mdx52 mice. During muscle regeneration, regenerated skeletal muscle fibers are centrally nucleated as opposed to mature muscle fibers which are peripherally nucleated [60,61]. Also, centrally-located nuclei in mdx mice are reported to persist long-term, indicating that the percentage of CNFs represents the accumulated history of regeneration up to the present [62,63]. Following HE staining, mdx mice showed a significantly lower percentage of CNFs than mdx52 mice at 2 months, followed by an increase in percent CNFs resembling observations in mdx52 mice at 6 months, and a subsequent decrease in CNFs at 12 and 18 months (Figures 3A  and B). At 18 months old in mdx mice, a significant decrease in CNFs was found in both TA and GC muscles when compared to 6 months (the age of the peak CNF percentage). In contrast to the dynamics of CNFs in mdx mice, the percentage of CNFs in mdx52 mice consistently and significantly decreased over time from the peak of 2 months old. This finding indicates that there is a different peak in muscle regeneration between mdx and mdx52 mice, which is before 6 months of age and before 2 months of age, respectively.
To examine the possible correlation between ongoing muscle regeneration and RF expansion, we analyzed the co-localization of eMHC with revertant dystrophin and the number of eMHCpositive fibers, which are found short-term during muscle regeneration. Interestingly, eMHC was not expressed in RFs at any age in mdx and mdx52 mice ( Figure 4A). The number of eMHC-positive fibers was similar between mdx and mdx52 mice at each age ( Figure 4A and B). However, expression dynamics of eMHC with age was different between the strains. In mdx mice, the number of eMHC-positive fibers was the highest at 2 months of age and significantly decreased at 18 months of age in TA and GC muscles, while in mdx52 mice, the number was slightly but not significantly decreased through 18 months.''. These data from CNFs and eMHC-positive fibers indicate that in mdx and mdx52 mice the dystrophic skeletal muscles actively regenerate throughout their life span but the muscle regeneration activity with age is different between the strains.

Discussion
Dystrophin RFs were first described more than 20 years ago by Hoffman et al [44], however, the mechanisms behind both the generation and expansion of RFs remain poorly understood. Lu et al previously reported that revertant events arise in individual muscle satellite cells at around birth [52]. RFs appear at around birth as short segments, approximately 10 mm in length, of sporadic single muscle fibers. One of the most interesting characteristics of the RFs is their substantial expansion with age. In mdx mice, RFs continuously expand at least up to 18 months, resulting in clusters containing more than 100 fibers and traversing 1 mm or more of muscle fiber length. However, RFs never accumulate to a sufficient number to significantly ameliorate dystrophic muscle pathology [40].
Our previous study with irradiated mdx mice aged up to 2 months supported the hypothesis that muscle regeneration is essential for RF expansion in mdx mice [40]. The expansion of RFs has been attributed to the combined effects of cycles of muscle degeneration/regeneration and increased survival of the fibers containing truncated dystrophin [40,64]. However, in this paper, we demonstrated that mutation types and aging differently affect generation and expansion of dystrophin-positive RFs in mdx and mdx52 mice.
Our paradoxical result reported here strongly challenges our previous hypothesis, since rapid expansion of RFs and a decrease in regenerated CNFs and eMHC-positive fibers occur concurrently at older ages (Figures 1-4). This is in sharp contrast to previous experiments with transgenic mdx muscles [40,65]. No expansion of RFs was observed in any transgenic mdx mice expressing mini-or micro-dystrophin in spite of the significant muscle growth before the age of 5 weeks. Instead, the number of RFs decreased with age in these transgenic mdx mice. Our observations here lead us to propose two possibilities to explain RF expansion with age: first, mechanisms other than muscle regeneration, such as secondary DNA mutation, up-regulation of short dystrophin isoforms, or age-related changes affecting splicing machinery may be involved in RF expansion in older muscle fibers. The expansion of RFs independent of muscle regeneration supports the existence of a mechanism in aged muscles that Lu et al described previously; i.e. RF expansion may represent the progressive increase in a territory of factors, each of which determines a specific pattern of exon skipping (alternative splicing), and which spread by diffusion within each fiber and between neighboring fibers [52]. This hypothesis predicts that RF clusters grow within the existing stable (mature) muscle fibers. A second possibility is that the increase of RFs with age may be related to RF stability combined with the expression of partially functional dystrophin proteins; thus, more stable RFs would accumulate with age and prevent degeneration/regeneration cycles in themselves and in closely surrounding fibers. This could lead to a decrease in muscle regeneration with age in both mouse strains. Nevertheless, muscle regeneration appears to be fundamentally required for RF expansion because central nucleation was found in most RFs through 18 months in this study, and our previous study demonstrated that RF expansion does not occur in the absence of regeneration, even when degeneration continues after irradia-tion [40]. Another hypothesis to possibly explain these conflicting observations is that regenerating muscle fibers with central nucleation and/or eMHC in older dystrophic mice might change to mature fibers without them more rapidly than in younger dystrophic mice; thus, a reduced number of CNFs and eMHCpositive fibers might not reflect the frequency of regeneration in older muscles, although there is currently no evidence to support this hypothesis.
As no such phenomenon like RFs has been described in other genetic disorders, the mechanism of their genesis and expansion is still open to speculation, although it seems modification of splicing is involved. Thus, RFs could be an interesting model with which to investigate the mechanisms of spontaneous exon skipping (alternative splicing). Future molecular analysis of RFs will also provide invaluable information toward the development of molecular therapies, such as antisense-mediated exon skipping, which are aimed at inducing the production of revertant-like dystrophinpositive fibers for the treatment of DMD [8,66,67].