Becker muscular dystrophy (BMD), an X-linked muscle disorder, is characterized by progressive muscle wasting and weakness, mostly caused by an in-frame variant in DMD encoding the sarcolemmal protein dystrophin. In BMD muscle tissues, truncated and reduced dystrophin is expressed; therefore, its clinical status is generally milder than that of Duchenne muscular dystrophy (DMD), an allelic disorder with complete loss of dystrophin (Koenig et al., 1989). BMD is clinically heterogeneous, with some affected individuals experiencing a near-normal lifestyle and lifespan, while others lose the ability to walk in their late teens or early 20s (Beggs et al., 1991; Bushby and Gardner-Medwin, 1993; Comi et al., 1994).

A natural history study of patients with BMD revealed that part of the skeletal muscle phenotype may be associated with the genotype of DMD and that there are several patterns of exon deletions associated with severe or milder phenotypes (Nakamura et al., 2023). In-frame exon deletions of BMD accumulate in exons 45–55 (first hotspot) or exons 3–7 (second hotspot) of DMD. Particularly, 80% of all in-frame deletions were included in the first hotspot: exons 45–55. Among these, the deletion of exons 45–47 (d45–47) is the most frequent, approximately 30% of patients with BMD have this in-frame deletion. Deletion of exons 45–48 (d45–48) is the second most frequent; approximately 18% of patients with BMD have this deletion and a milder phenotype than D45–47. In contrast, the deletion of exons 45–49 (d45–49) has a more severe phenotype than D45–47 (Nakamura et al., 2023).

The relationship between the pathomechanisms of disease severity and each exon deletion remains unclear. In the muscle tissue of patients with BMD, truncated dystrophin expression levels are lower than those of healthy controls, and dystrophin immunohistochemistry shows a ‘faint and patchy’ staining pattern (Beggs et al., 1991; Jimi et al., 1992). The correlation between the severity and expression levels of truncated dystrophin is still conflicting, and the truncated dystrophin expression levels in muscle tissues are well correlated with the clinical severity of BMD (Anthony et al., 2011). However, there is another report that truncated dystrophin levels do not appear to be a major determinant of disease severity in BMD (van den Bergen et al., 2014).

Dystrophin is a large filamentous protein with a molecular weight of 427 kDa that protects the sarcolemma from mechanical stress during muscle contraction. Dystrophin connects to cytoskeletal protein families that can assemble into macromolecular structures with a large number of proteins and lipids (Le Rumeur et al., 2010). Recently, a report suggested that the BMD clinical heterogeneity is associated with the changes in dystrophin structure generated by exon deletion using an in silico prediction model (Nicolas et al., 2015). They expect that the d45–48 and d45–51 displayed a structure similar to that of wild-type (WT) dystrophin (hybrid repeat), whereas the d45–47 and d45–49 lead to proteins with an unrelated structure (fractional repeat) among the representative exon deletions in the BMD variant hotspot. Exon deletions associated with fractional repeats are expected to lead to a more severe BMD phenotype than those associated with hybrid repeats; however, the influence of changes in dystrophin structure on clinical BMD severity is not fully understood.

Recently, two animal models of BMD have been developed. One group generated the first BMD rat model carrying a deletion of exons 3–16 of the rat Dmd using CRISPR/Cas9 (Teramoto et al., 2020). This BMD rat model exhibited muscle degeneration, muscle fibrosis, heart fibrosis, and reduced truncated dystrophin levels. Another group generated a BMD mouse model carrying a deletion in exons 45–47 of the mouse Dmd (Heier et al., 2023). The BMD mice show skeletal muscle weakness, heart dysfunction, increased myofiber size variability, increased centronuclear fibers, and reduced truncated dystrophin. However, underlying mechanism of BMD severity remains unclear, and comparative investigations using several BMD animal models carrying exon deletions associated with various severities are necessary.

To clarify the issue, we generated three types of BMD mice with representative exon deletions in the BMD hotspots: d45–47, d45–48, and d45–49 using CRISPR–Cas9 genome editing and compared the phenotypes and histopathological changes.

In this study, we established three types of BMD mice carrying the d45–48, d45–47, and d45–49. All BMD mice showed muscle weakness and histopathological changes, including muscle degeneration and fibrosis; however, these changes appeared at different times with each exon deletion. In addition, we confirmed the decreased truncated dystrophin levels and the decreased nNOS expression levels in all BMD mice. Furthermore, unlike mdx mice, BMD mice showed site-specific muscle degeneration in particular muscle parts, especially those rich in type IIa fibers.

The phenotypic evaluation confirmed that the d45–49 and mdx mice had larger body weights at 1 and 3 months compared with d45–48, d45–47, and WT mice; however, after 6 months, there was no difference in body weight between the groups. Mdx mice are generally larger in body weight at a young age than WT mice, whereas the difference between mdx and WT mice disappears at old age (Pastoret and Sebille, 1995; Shiba et al., 2015). The body weights of mdx mice in our study were consistent with previous reports, and we demonstrated that the d45–49 mice showed similar changes in body weight as mdx mice.

In this study, muscle weakness in forelimb grip strength in mdx mice was already apparent from 1 month and was consistent toward 12 months compared with WT mice. In contrast, all BMD mice showed muscle weakness in forelimb grip strength compared to WT mice, but the beginning of the appearance differed depending on the type of exon deletion. Muscle weakness was demonstrated in the d45–49 mice at 1 month similar to that of mdx mice, but was apparent in the d45–48 and d45–47 mice after 3 months. All BMD mice reached the same levels of muscle weakness after 6 months whereas milder than those of mdx mice. Furthermore, analysis of the hanging wire test revealed decreased power to pull up the trunk in BMD mice, especially in the d45–49 mice. Generally, mdx mice show muscle weakness in grip strength (Takeshita et al., 2017) and hanging wire tests (Klein et al., 2012), and muscle weakness is already seen at 1 month (McDonald et al., 2015; Aartsma-Rus and van Putten, 2014). Furthermore, mdx mice are difficult to pull themselves up from their hanging position (Hübner et al., 1996), which was consistent with our results. The BMD rats did not show significant muscle weakness, and the BMD mice carrying the d45–47 showed muscle weakness at 10–15 weeks compared to WT mice, whereas it was milder than that in mdx mice (Teramoto et al., 2020; Heier et al., 2023). On the other hand, we were able to demonstrate differences in severity due to exon deletions in BMD mice, consistent with the severity of human BMD carrying d45–48, d45–47, and d45–49.

The histopathological changes in our mdx mice correspond to previous reports (Pastoret and Sebille, 1995; Pessina et al., 2014; Giovarelli et al., 2022). BMD rats showed muscle degeneration accompanied by necrotic fibers and inflammatory cell infiltration at 1 month and became conspicuous after 2 months, and muscle fibrosis was shown after 2 months (Teramoto et al., 2020). In addition, BMD mice carrying d45–47 showed muscle degeneration with necrotic fibers and immune cell infiltration, and an increase in fibrotic areas (Heier et al., 2023). In this study, all BMD mice exhibited muscle degeneration and fibrosis correspond to previous reports, furthermore, we revealed the timing of its appearance varied depending on the type of exon deletion.

All BMD mice showed faint and patchy dystrophin staining patterns and decreased truncated dystrophin expression levels. These findings are typical in the muscles of patients with BMD (Beggs et al., 1991; Jimi et al., 1992). Furthermore, sarcolemmal nNOS reduction was observed in all BMD mice to levels similar to those in mdx mice, consistent with the lack of an nNOS-binding site from exon deletions. Unlike normal, sarcolemmal nNOS expression is decreased and altered in localization not only in patients with DMD and mdx mice (Brenman et al., 1995), but also in patients with BMD (Torelli et al., 2004; Chao et al., 1996). The sarcolemmal expression of aSG was remaining in BMD mice; however, the expression level was slightly decreased. In patients with BMD, proteins expression of DGC including aSG are usually detected in the sarcolemma (Chao et al., 1996; Anthony et al., 2014). In our BMD mice, utrophin expression was slightly increased compared with WT mice, but this change was smaller than that in mdx mice. Utrophin is overexpressed in the muscles of human DMD (Anthony et al., 2014; Arechavala-Gomeza et al., 2010) and mdx mice (Pons et al., 1994; Banks et al., 2014), but not in the muscles of patients wih BMD (Torelli et al., 2004; Arechavala-Gomeza et al., 2010; Pons et al., 1994; Janghra et al., 2016). All of our BMD mice showed reduced nNOS expression and residual aSG expression on the sarcolemma and no overexpression of utrophin.

Our BMD mice showed differences in the beginning of muscle weakness, muscle degeneration, and fibrosis accompanied by exon deletions, but the mechanisms underlying these differences from exon deletions remain unclear. We confirmed decreased levels of truncated dystrophin in all BMD mice; however, the expression levels were almost the same despite differences in phenotypic severity. Therefore, the severity of BMD in mice might be influenced by the qualitative shift in truncated dystrophin proteins along with exon deletions, and not by quantitative changes. Indeed, using an in silico prediction model, it has been reported that in-frame exon deletions possibly induce a structural shift in dystrophin, and the collapse of the dystrophin structure might influence BMD severity (Nicolas et al., 2015).

We demonstrated site-specific muscle degeneration in BMD mice, unlike mdx mice, which, showed diffuse and non-specific muscle degeneration. Even in mdx mice, multiple and heterogeneously distributed MRI hyperintensities are seen in a short period of approximately 13–19 weeks (Pratt et al., 2013), although BMD mice showed selective and site-specific muscle degeneration, especially in the deep muscle or the inner part of the same muscle. In human BMD, specific muscles are known to change the intensity of muscle MRI images (Tasca et al., 2012). Serum CK levels were two- to fourfold higher in all BMD mice than those of WT mice after 3 months and similar to that in a previous study on a BMD rat model (Teramoto et al., 2020). However, the difference in serum CK levels between BMD and WT mice was less than that between mdx and WT mice. These results may be due to the small area of muscle degeneration in BMD mice because of its uneven distribution in the deep part of each muscle compared with that in mdx mice, which showed diffuse muscle degeneration. It has been known that muscles located near the body surface are rich in white muscle fibers, while the deep muscles are rich in medium and red muscle fibers (Ogata, 1958). In our BMD mice, type IIa fibers were decreased compared to WT mice, especially in the inner muscle region, where type IIa fibers are abundant. And we found that type IIa fiber reduction started after 3 months in BMD mice, whereas WT mice showed type IIa fiber increment after 3 months. In contrast, type I, IIx, and IIb fibers were unchanged in BMD mice compared with WT mice (Figure 5C).

Type IIa fibers are also known as fast-oxidative fibers (Talbot and Maves, 2016) and are known to have fatigue resistance compared with type IIb and IIx fibers, known as having the least and the second least fatigue-resistant fibers (Larsson et al., 1991) type IIa fiber reduction is thought to contribute to muscle fatigability (Percival et al., 2010). Recently, it was reported that mice lacking the RNA-binding protein Musashi-2, which is predominantly expressed in slow-type muscle fibers, showed a reduction in type IIa fibers along with reduced muscle contraction force (Furuichi et al., 2023). These findings suggest that decrease in type IIa fibers contributes to the muscle weakness and fatigability in BMD mice. In addition, only type I, IIa, and IIx fibers are present in human skeletal muscle, and type IIb fibers appear to be absent (Talbot and Maves, 2016; Schiaffino and Reggiani, 2011). Thus, the reduction of type IIa fibers might have a greater effect on the skeletal muscle function of human BMD than that of BMD mice. We examined the association between type IIa fiber reduction and muscle atrophy, but there was no remarkable CSA reduction or changes in muscle atrophy inducing factors. The vulnerability of type IIa fibers to CTX-induced muscle damage has been reported (Dalle et al., 2020), and we also observed that the recovery of type IIa and IIx fibers was delayed compared to that of type IIb fibers after CTX injection. This vulnerability and delayed recovery of type IIa fibers may partly explain the type IIa fiber reduction in BMD mice, but the recovery of type IIx fibers was slower than that of type IIa fibers after CTX injection. Therefore, the type IIa fiber-specific decay in BMD mice might not be explained by this vulnerability and delayed recovery during muscle degeneration and regeneration. A decrease in type IIa fibers was also observed in microgravity-induced muscle atrophy in WT mice, where a decrease in sarcolemmal nNOS occurred (Sandonà et al., 2012). It has been reported that the expression level of nNOS is higher in type IIa fibers than in type I and IIb fibers (Planitzer et al., 2001) and that nNOS-deficient mice have reduced type IIa fibers compared to WT mice (Percival et al., 2010) and reduced capillary density only in the inner part of the muscle (Baum et al., 2023).

By examining BMD mice, we found for the first time morphological capillary changes from a ‘ring-pattern’ to a ‘dot-pattern’ with decreased nNOS expression in the sarcolemma and fewer capillaries in circumferential contact with type IIa fibers. Capillary changes to a ‘dot-pattern’ were also seen around type IIx fibers in BMD mice, but in WT mice, capillaries around type IIx fibers were poor compared with that of type IIa fibers, and showed an ‘incomplete ring-pattern’. In addition, capillaries around type IIb and I fibers showed a ‘dot-pattern’ even in WT mice. These results suggest that type IIa fibers may require numerous capillaries and maintained blood flow, compared with other muscle fibers, and this high requirement for blood flow might be associated with the type IIa fiber-specific decay in BMD mice. Between the main muscle capillaries running parallel to the muscle fibers, there are transversely interconnected branches and capillary loops (Haas and Nwadozi, 2015), and the capillary changes in BMD mice may be associated with the deterioration of these interconnected branches and capillary loops (Figure 6D). We examined transversely interconnected branches and capillary loops, using longitudinal muscle sections. We confirmed that there were fewer interconnected capillaries in BMD and mdx mice than in WT mice (Figure 6E). Vascular dysfunction has been implicated in muscle damage in canine model of DMD (Kodippili et al., 2021) and in human DMD (Thomas et al., 1998), and our analysis of BMD mice also suggests that the reduction of type IIa fibers may be influenced by vascular dysfunction with reduced sarcolemmal nNOS as well as fragility and delayed recovery.

All data generated or analyzed during this study are included in the manuscript and supporting files; data files for primers used in RT-PCR have been provided as supplementary file 1.

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Author details

1. Daigo Miyazaki

   1. Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, Japan
   2. Intractable Disease Care Center, Shinshu University Hospital, Matsumoto, Japan

   Present address

   Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, Japan

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Writing – original draft

   For correspondence

   miyajiro@shinshu-u.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0032-0077

2. Mitsuto Sato

   1. Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, Japan
   2. Department of Brain Disease Research, Shinshu University School of Medicine, Matsumoto, Japan

   Contribution

   Data curation, Visualization

   Competing interests

   No competing interests declared

3. Naoko Shiba

   Department of Regenerative Science and Medicine, Shinshu University, Matsumoto, Japan

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

4. Takahiro Yoshizawa

   Research Center for Advanced Science and Technology, Shinshu University, Matsumoto, Japan

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

5. Akinori Nakamura

   1. Department of Clinical Research, NHO Matsumoto Medical Center, Matsumoto, Japan
   2. Third Department of Medicine, Shinshu University School of Medicine, Matsumoto, Japan

   Contribution

   Conceptualization, Data curation, Project administration, Writing - review and editing

   For correspondence

   anakamu@shinshu-u.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8801-6688