Becker muscular dystrophy mice showed site-specific decay of type IIa fibers with capillary change in skeletal muscle
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eLife Assessment
The authors present three valuable transgenic models carrying three representative exon deletions of the dystrophin gene. The findings are supported by rigorous biochemical assays and state-of-the-art microscopy methods, although the evidence, while overall solid, is only partially developed, and some points could be improved.
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Abstract
Becker muscular dystrophy (BMD), an X—linked muscular dystrophy, is mostly caused by an in—frame deletion of DMD. BMD severity varies from asymptomatic to severe, associated with the genotype of DMD. However, the underlying mechanisms remain unclear. We established BMD mice carrying three representative exon deletions: ex45—48 del., ex45—47 del., and ex45—49 del. (d45—48, d45—47 and d45—49), with high frequencies and different severities in the human BMD hotspot. All three BMD mice showed muscle weakness, muscle degeneration, and fibrosis, but these changes appeared at different times for each exon deletion, consistent with the severities obtained by the natural history study of BMD. BMD mice showed site—specific muscle changes, unlike mdx mice, which showed diffuse muscle changes, and we demonstrated selective type IIa fiber reduction in BMD mice. Furthermore, BMD mice showed sarcolemmal neuronal nitric oxide synthetase (nNOS) reduction and morphological capillary changes around type IIa fibers. These results suggest that capillary changes caused by nNOS reduction may be associated with the mechanism of skeletal muscle degeneration and type IIa fiber reduction in BMD mice. BMD mice may be useful in elucidating the pathomechanisms and developing therapeutic strategies for human BMD.
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eLife Assessment
The authors present three valuable transgenic models carrying three representative exon deletions of the dystrophin gene. The findings are supported by rigorous biochemical assays and state-of-the-art microscopy methods, although the evidence, while overall solid, is only partially developed, and some points could be improved.
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Reviewer #1 (Public review):
Summary:
In this article the authors described mouse models presenting with backer muscular dystrophy, they created three transgenic models carrying three representative exon deletions: ex45-48 del., ex45-47 19 del., and ex45-49 del.. This article is well written but needs improvement in some points.
Strengths:
This article is well written. The evidence supporting the authors' claims is robust, though further implementation is necessary. The experiments conducted align with the current state-of-the-art methodologies.
Weaknesses:
This article does not analyze atrophy in the various mouse models. Implementing this point would improve the impact of the work
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Reviewer #2 (Public review):
Miyazaki et al. established three distinct BMD mouse models by deleting different exon regions of the dystrophin gene, observed in human BMD. The authors demonstrated that these models exhibit pathophysiological changes, including variations in body weight, muscle force, muscle degeneration, and levels of fibrosis, alongside underlying molecular alterations such as changes in dystrophin and nNOS levels. Notably, these molecular and pathological changes progress at different rates depending on the specific exon deletions in the dystrophin gene. Additionally, the authors conducted extensive fiber typing, revealing a site-specific decline in type IIa fibers in BMD mice, which they suggest may be due to muscle degeneration and reduced capillary formation around these fibers.
Strengths:
The manuscript introduces …
Reviewer #2 (Public review):
Miyazaki et al. established three distinct BMD mouse models by deleting different exon regions of the dystrophin gene, observed in human BMD. The authors demonstrated that these models exhibit pathophysiological changes, including variations in body weight, muscle force, muscle degeneration, and levels of fibrosis, alongside underlying molecular alterations such as changes in dystrophin and nNOS levels. Notably, these molecular and pathological changes progress at different rates depending on the specific exon deletions in the dystrophin gene. Additionally, the authors conducted extensive fiber typing, revealing a site-specific decline in type IIa fibers in BMD mice, which they suggest may be due to muscle degeneration and reduced capillary formation around these fibers.
Strengths:
The manuscript introduces three novel BMD mouse models with different dystrophin exon deletions, each demonstrating varying rates of disease progression similar to the human BMD phenotype. The authors also conducted extensive fiber typing across different muscles and regions within the muscles, effectively highlighting a site-specific decline in type IIa muscle fibers in BMD mice.
Weaknesses:
The authors have inadequate experiments to support their hypothesis that the decay of type IIa muscle fibers is likely due to muscle degeneration and reduced capillary formation. Further investigation into capillary density and histopathological changes across different muscle fibers is needed, which could clarify the mechanisms behind these observations.
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Author response:
Reviewer #1 (Public review):
Summary:
In this article the authors described mouse models presenting with backer muscular dystrophy, they created three transgenic models carrying three representative exon deletions: ex45-48 del., ex45-47 19 del., and ex45-49 del. This article is well written but needs improvement in some points.
Strengths:
This article is well written. The evidence supporting the authors' claims is robust, though further implementation is necessary. The experiments conducted align with the current state-of-the-art methodologies.
Weaknesses:
This article does not analyze atrophy in the various mouse models. Implementing this point would improve the impact of the work
We thank the reviewer for their constructive suggestions and comments on this work. Muscle hypertrophy is shown with growth in …
Author response:
Reviewer #1 (Public review):
Summary:
In this article the authors described mouse models presenting with backer muscular dystrophy, they created three transgenic models carrying three representative exon deletions: ex45-48 del., ex45-47 19 del., and ex45-49 del. This article is well written but needs improvement in some points.
Strengths:
This article is well written. The evidence supporting the authors' claims is robust, though further implementation is necessary. The experiments conducted align with the current state-of-the-art methodologies.
Weaknesses:
This article does not analyze atrophy in the various mouse models. Implementing this point would improve the impact of the work
We thank the reviewer for their constructive suggestions and comments on this work. Muscle hypertrophy is shown with growth in dystrophin-deficient skeletal muscle in mdx mice; thus, we did not pay attention to the factors associated with muscle atrophy in BMD mice. As the reviewer suggested, the examination of the association between type IIa fiber reduction and muscle atrophy is important, and the result is considered to be helpful in resolving the cause of type IIa fiber reduction in BMD mice.
Thus, we are planning to:
(1) Evaluate the cross-sectional areas (CSA) of muscles and compare them with the changes in the proportion of type IIa fibers.
(2) Evaluate the expression levels of Murf1 and Atrogin1 as markers of muscle atrophy using RT-PCR.
Reviewer #2 (Public review):
Summary
Miyazaki et al. established three distinct BMD mouse models by deleting different exon regions of the dystrophin gene, observed in human BMD. The authors demonstrated that these models exhibit pathophysiological changes, including variations in body weight, muscle force, muscle degeneration, and levels of fibrosis, alongside underlying molecular alterations such as changes in dystrophin and nNOS levels. Notably, these molecular and pathological changes progress at different rates depending on the specific exon deletions in the dystrophin gene. Additionally, the authors conducted extensive fiber typing, revealing a site-specific decline in type IIa fibers in BMD mice, which they suggest may be due to muscle degeneration and reduced capillary formation around these fibers.
Strengths:
The manuscript introduces three novel BMD mouse models with different dystrophin exon deletions, each demonstrating varying rates of disease progression similar to the human BMD phenotype. The authors also conducted extensive fiber typing across different muscles and regions within the muscles, effectively highlighting a site-specific decline in type IIa muscle fibers in BMD mice.
Weaknesses:
The authors have inadequate experiments to support their hypothesis that the decay of type IIa muscle fibers is likely due to muscle degeneration and reduced capillary formation. Further investigation into capillary density and histopathological changes across different muscle fibers is needed, which could clarify the mechanisms behind these observations.
We thank the reviewer for these positive comments and the very important suggestion about type IIa fiber reduction and capillary change around muscle fibers in BMD mice. From the results of the cardiotoxin-induced muscle degeneration and regeneration model, type IIa and IIx fibers showed delayed recovery compared with that of type-IIb fibers. However, this delayed recovery of type IIa and IIx could not explain the cause of the selective muscle fiber reduction limited to type IIa fibers in BMD mice. Therefore, we considered vascular dysfunction as the reason for the selective type IIa fiber reduction, and we found morphological capillary changes from a “ring pattern” to a “dot pattern” around type IIa fibers in BMD mice. However, the association between selective type IIa fiber reduction and the capillary change around muscle fibers in BMD mice remains unclear due to the lack of information about capillaries around type IIx and IIb fibers. The reviewer pointed out this insufficient evaluation of capillaries around other muscle fibers (except for type IIa fibers), and this suggestion is very helpful for explaining the association between selective type IIa fiber reduction and vascular dysfunction in BMD mice.
Thus, we are planning to:
(1) Evaluate the changes in capillary formation around other muscle fibers, except for type IIa fibers (e.g., type IIx and IIb fibers).
(2) Evaluate the endothelial area around other muscle fibers, except for type IIa fibers.
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