PONE-D-23-21038Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophyPLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: No

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Hassani et. al. investigates the beneficial impact of muscle activity in mouse models of muscular dystrophy. Specifically, the authors show that synergistic ablation induces muscle hypertrophy, maximal force production, and resistance to eccentric damage. While demonstrated previously, this has been expanded to be present even with lower levels of hypertrophy, in a more severely affected mouse model and to not be dependent on calcineurin signaling. The manuscript is generally well written. The influence of exercise and muscle loading in DMD is of importance to the field with questions that this paper can help to address. However, there are substantial concerns that dampen enthusiasm listed below.

The decrease fragility could simply be more sarcomeres in series leading to less sarcomeric strain or strain rate with the eccentric contraction. The description of how lengths are recorded is incomplete. It appears Lo was set to where maximum force was achieved, but how was Lo then measured in situ? Where calipers used and was any tendon length included? This is important given that the lengthening contractions are set to 10% of Lo and Lo is expected to change with overload.

Although it did not turn out to induce major effects the treatment with CsA is thought to act through inhibiting slow fiber programs through Calcineurin. However, only one myosin heavy chain molecule was examined. It would be informative to look at fiber types more directly to see if that was related at all to the susceptibility to damage as postulated.

The experimental design section is helpful to show what was done. However, the rationale for some decisions could be more fully explained. Particularly, it may be helpful to include wildtype animals so the degree of recovery could be stated for certain parameters. Also, it isn’t clear why a bilateral design is not used to compare the contralateral limb to that of the OVL.

There are a number of associations that are proposed between parameters studied, such as that between AKT and muscle growth. Given that many of the parameters are investigated within the same muscle it would be helpful to directly test for significant relationships between parameters such as hypertrophy and force loss or pAKT levels and muscle mass.

Minor comments:

Methods: It is stated that a velocity of 5.5 mm/s was used that is equivalent to a 0.85 fiber length/s velocity. This would assume a constant fiber length of ~6.5 mm. How is this determined, especially in light of potentially changing Lo?

Methods: The methods state that lengthening contractions were induced in the TA, however that is not consistent with the rest of the manuscript.

Fig 2C: Weight is misspelled on the axis.

Fig 2D: The rationale for separating out the different phases of force development is not clear. It may be easier to just show one value (perhaps 0-50%).

Fig 2D: The rate of force development is slowed with CsA, but this would seem counter to expectations of CaN promoting slower fibers (thus blocking it would promote faster fibers). A comment in the discussion could be warranted.

Fig 3C: It isn’t clear if error bars are present in this figure.

Fig 5: The lane labels are not very clear.

Discussion: The statement that improvement in fragility cannot be explained by an increase in less fragile fibers could e overly strong. While a significant change in MHC2a was not observed there is a shift in the mean and with low sample sizes here this appears under powered.

Discussion: Research is cited that gene therapy does not increase muscle force in the D2 model. However, there is data showing substantial increases in muscle force with microdystrophin gene therapy (Cernisova et. al. Int J Mol Sci 2023).

Reviewer #2: The study describes the benefits of resistance training in two mouse DMD models, offering preclinical data that might be relevant to human disease. Results are solid and well presented, yet the study is purely descriptive and the manuscript needs revision before it might be acceptable for publication.

Main concerns with this study are that it offers little mechanistic insight and no direct link to applicability to human medicine. The title should be improved to better phrase the study.

The underlying mechanisms of benefit have not been investigated in depth. Administering CsA leads the authors to conclude that calcineurin signalling is not involved, and it is speculated that Akt/mTor might be involved. However, no specific data is offered that might corroborate this statement, and it therefore needs to be downplayed. The study appears to me somewhat immature., and authors need to clearly acknowledge limitations and future routes forward. Lines 344-370 need to be reduced in fit with the shortcomings of the study.

The manuscript would benefit from comparison with other studies given in a comprehensive table. Also, relevance to human medicine should be better explored. What could be a recommended (improved) training strategy based upon the study’s findings? What is the added value for the clinic? Are there any significant differences between mdx and D2-mdx that aid in comparison of severity? Becker MD is not mentioned anywhere, give this some thought and add to the discussion. In view of human DMD being severe, how can resistance training be implement in the window of ambulation? Authors should more thoroughly discuss the translational value of the study.

There are several method concerns. Describe how the CsA dose and administration was determined. Was this based on literature or on own preliminary tests? Give concentrations of antibodies used for western blots in µg/ml IgG and not as a dilution factor. Why was gamma-actin chosen as a loading control, while most use alpha- or beta-? Also, the use of HSP60 as a mitochondrial marker needs to be explained (as well as its preference to VDAC). Legends to fig 5 and 6 do not mention HSP60, nor if the graphs of protein levels are ratio’s to HSP60. Explain rationale of normalization to mitochondrial content. Full western blots need to be provided as a supplement. Figure legends need to specify precise numbers of mice per group, and not ranges.

Minor remarks:

Line 280 benefic

Line 293 cytoplamic

Reviewer #3: I think this research paper is very detailed and thorough. It is technically sound, the data supports the conclusions and the statistical analysis is very rigorous. It just needs some small revisions.

I have one main area of confusion. For the first sub -header of the discussion section, I was very confused by the wording. "OVL improved fragility in both mdx and D2-mdx mice, treated or not with CsA" makes it sound like both the mdx and the D2-mdx mice were treated with CsA. I had to go back to the methods section to double-check that the D2-mdx mice were never treated with CsA.

In addition, I was wondering why you all did not include a CsA group in the D2-mdx mice?

Lastly, I believe almost every time the cytoplasmic gamma-actin are mentioned in the paper, cytoplasmic is spelled incorrectly. I saw "cytoplamic" multiple times throughout the paper.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy

PONE-D-23-21038R1

Dear Dr. Ferry,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Aldrin V. Gomes, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The revised manuscript by Hassani et. al. makes notable improvements in the investigation of muscle overload in dystrophic muscles. The added relationships between hypertrophy and eccentric damage and pAKT support the conclusions. Importantly the limitations of the study have been more thoroughly addressed by the authors which prevent more strong conclusions from being drawn. The topic of overloading dystrophic muscle is of interest and the result of decreased susceptibility to damage is clear although the mechanistic insight is limited.

Reviewer #3: The author addressed all comments and concerns made to the manuscript. The manuscript is sound, the data supports the conclusions, and the statistical analysis has been performed appropriately and rigorously.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

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