The MRL Mitochondrial Genome Decreases Murine Muscular Dystrophy Severity

Round 1

Reviewer 1 Report

This paper from Jenan Holley-Cuthrell and collaborators demonstrates that super-healing capacity of MRL mice is in part relate to epigenetic. This is an interesting work which open new avenues in the comprehension of MRL super-healing mechanisms by introducing mitochondrial genome to the equation. I have very few criticisms about this work as it is well-conducted, analyzed and discussed.

Major comments:

-My major concern is about pAMPK analysis which is not normalized by AMPK level but to b-actin. This is low rigorous. The authors must quantify pAMK/AMPK to exclude any variation of the protein as always performed in their already published works.

Minor comments:

-The resolution of the figures is very poor and makes reading difficult.

-Table 1 appears after Tables 2 and 3.

-The MRL is not defined when first used.

Author Response

Dear Editors and Reviewers,

 

Thank you very much for the very helpful suggestions and edits for this manuscript. We have amended the manuscript at each reviewer suggestion. In addition, we have proof-read the manuscript again and edited the text to make the manuscript easier to follow.

 

Specific points-

Reviewer 1

• We agree with the reviewer that an AMPK western blot is the industry norm when discussing pAMPK levels, because usually it is the activation of AMPK into pAMPK that researchers are concerned with. In our case we were just looking for the absolute amount of activated protein and the experiments to identify which molecular routes (protein transcription and translation and/or activation) are being used will follow. In addition, we have previously shown that AMPK is not upregulated in skeletal muscles of MRL mice (Berhanu 2014 and Mull 2014). We have rewritten lines 359-362 in the discussion to clarify that AMPK transcription/translation does not appear to be affected by the MRL genomes. Thank you for identifying this rather embarrassing omission.
• We have reworked the figures to enhance the resolution.
• We have defined MRL at its first appearance on page 1 line 39.

Reviewer 2 Report

The manuscript is a correlation study upon the heteroplasmy in MRL mice with the severity in Sarcoglycan deficiency dystrophy. The study was carried out correctly to indicate that fibrotic complications can be alleviated by the heteroplasmic effect of MRL mitochondria as indicated by pAMPK and fibronectin levels. The approach to influence muscle diseases due to mitochondrial genome mutationas is new and straightforward, and fits well into the scope of the special issue. However the therapeutic application of the mutant mitochondria in muscle dystrophies is rather speculative and needs substantial further studes.

Author Response

Dear Editors and Reviewers,

 

Thank you very much for the very helpful suggestions and edits for this manuscript. We have amended the manuscript at each reviewer suggestion. In addition, we have proof-read the manuscript again and edited the text to make the manuscript easier to follow.

 

Specific points-

Reviewer 2

• We have quite extensively reworked the conclusions to be sure we are not over-stepping the results.
• We have now also included a discussion section to include our thoughts on how this work can be translated to the clinic.

Reviewer 3 Report

General comments

As a general remark to the entire manuscript, I would correct the layout of the work so that the tables and graphs are in the place where they are described and do not force the reader to constantly jump into the text, which causes the thread to be lost. The whole work is challenging for the reader to understand.

 

Special comments

100-117: perhaps it would be appropriate to move fragments 100-117, as more generally dealing with mutations in mitochondrial DNA, to position 58, where a specific mutation in mtDNA is already discussed in detail. It seems to be a more causal arrangement of content. I leave it up to the authors to decide.

163-165: as an example, especially in the results section, 1-2 sentences of inclusions are observed, which should be part of the discussion section because they impose on the recipient a scientifically unconfirmed point of view and thus make it challenging to receive content, which in the case of results should be treated only about them, complex data, without directing thinking or suggestions as to what it might have been. I suggest you carefully review the results section in this regard.

218-221: the same applies to the results, combined with inference, which is not bad. However, if the authors of the work agree, I would separate a section for conclusions, which will correlate with the sub-points described in the results.

224-230: this fragment has already been described before, and it seems unnecessary to repeat it. Just write that it was described earlier in a specific section (row range).

254: 2.2. Figures, Tables, and Schemes … have the same number as 2.2. The MRL mitochondrial genome contributes to the muscular dystrophy super-healing phenotype. If these illustrations are intended to end up in this sub-heading in the results, that's fine, but if they're meant to be where they are, continue with the numbering used throughout the manuscript. To be decided by the authors.

 

258-259:Using this randomness, we should be able to generate mitochondrial homoplasmy, mitochondrially pure breeding mouse lines. Additionally, the descriptions under the charts are completely random and are a mix of description, result, conclusion, and discussion. In this case, it has a very negative impact on the reception of the work because we lose the understanding of what hard data is, what a conclusion is, what is a part to be tested at a later stage, and what is only a conjecture, of which there should be as little as possible in scientific work and limited only to the discussion, if at all. Please check and correct the captions for all tables and figures in this manuscript.

Author Response

Dear Editors and Reviewers,

Thank you very much for the very helpful suggestions and edits for this manuscript. We have amended the manuscript at each reviewer suggestion. In addition, we have proof-read the manuscript again and edited the text to make the manuscript easier to follow.

Specific points-

Reviewer 3

• We have added a few points in the methods section to completely describe the procedures.
• We have rewritten the manuscript and placed the figures/tables closer to their descriptions in the text.
• We believe the manuscript is easier to follow in its current form. In particular we have:
• Moved lines 100-117 nearer line 58 so that all of the mtDNA introduction (lines 82-110) is in the same area.
• We have moved multiple lines and ideas from the results to the discussion or conclusion.
• Lines 224-230 have been deleted.

• We have corrected the table order.
• Figure numbers and legends have also been worked on.