The Role of Mitochondria in Mediation of Skeletal Muscle Repair
Round 1
Reviewer 1 Report
Stephen E. Always et al., showed an effort to highlight the role of mitochondria in the mediation of skeletal muscle repair. The manuscript is written well and is interesting, though many publications are available for muscle injury or biogenesis. By considering the importance of the mitochondrial role, authors could improve the manuscript with additional information along with required figures for better understanding. Concerns are highlighted below for author consideration.
1. Line 43: Skin is the largest organ in the body! Please edit it.
2. Line 76-77: This is a review of skeletal muscle injury; authors can highlight toxic injury models of skeletal muscle with more examples.
2. Please discuss more on the following:
a) Mitochondria-derived signaling pathways controlling inflammation, Reactive oxygen/nitrogen species for protecting muscle injury vs. muscle-mass recovery.
b) Highlight all the relevant markers of the above.
c) Illustrate pathways with relevant molecules. It helps researchers to understand at best.
3. Line 200: Typo error. Please check other areas carefully while reviewing.
4. Please provide details with the figure for mitochondria respiratory function (overview of mitochondrial function), which helps to understand ATP synthesis's (an example) role in muscle differentiation or regeneration.
5. Please discuss-mitophagy markers more.
6. Please provide more details (Make a table if necessary) on alterations in mitochondrial mRNA, miRNA, and proteins for injury vs. recovery.
7. Line 324: Not sure why the author discussed only on TNFa and why not other cytokines. Please highlight markers of M1 vs. M2 macrophages in the article.
8. Please discuss more on pharmacological or biological strategies for attenuating mitochondrial aging or degradation vs. augmentation.
Overall, this article shall improve by providing more details with relevant mechanistic figures which favor researchers or readers.
Author Response
Response to Reviewer #1
Reviewer’s comments: Stephen E. Alway et al., showed an effort to highlight the role of mitochondria in the mediation of skeletal muscle repair. The manuscript is written well and is interesting, though many publications are available for muscle injury or biogenesis. By considering the importance of the mitochondrial role, authors could improve the manuscript with additional information along with required figures for better understanding. Concerns are highlighted below for author consideration.
Authors’s response: Thank you for your thoughtful comments. Our original intention was to be more focused on the areas that we are currently investigating in the area of mitochodnria and muscle repair and particularly in muscle stem cells. Many of the comments from the Reviewer have been considered and we have revised our manuscript accordingly. As a result, the review is now much broader and spends more time in mitochondria that regulate non-regenerative areas such as inflammation as a negative regulator of muscle regeneration. However, we do agree that this additional information does provide a more rounded review and therefore we have proceeded accordingly. We have added the revision to the review material with the MS Word tracking functions so that the changes to the manuscript can be easily identified. We have also uploaded a “clean” copy of the manuscript for review. We hope that this revision has now captured the essence of the Reviewer’s suggestions for improving our manuscript, and we are grateful for the comments and the opportunity to do so.
Reviewer’s comments: 1. Line 43: Skin is the largest organ in the body! Please edit it.
Authors’s response: Thank you. Yes we should have said one of the largest organs.
Reviewer’s comments: 2. Line 76-77: This is a review of skeletal muscle injury; authors can highlight toxic injury models of skeletal muscle with more examples.
Authors’s response: We have added new references for each of the examples of muscle injury and have added other examples of injury types that have been studied and reported.
Reviewer’s comments: 2. Please discuss more on the following:
Reviewer’s comments: a) Mitochondria-derived signaling pathways controlling inflammation, Reactive oxygen/nitrogen species for protecting muscle injury vs. muscle-mass recovery.
Authors’s response: (a1) We have added considerable information in the revised section 3.1 and have added Table 1 to describe important proteins and pathways related to the role of mitochondria in inflammation. Although some of this is not solely for injury/regeneration which was the original intention of this paper, (although much of the inflammation is still relevant to the injury side, and ROS also can be injury inducing as we had already included or injury protective which we have now included in this revision) we have added the broader discussion to the paper as suggested by the Reviewer.
Authors’s response: (a2) We recognize that ROS/RNS have dual roles where excess can induce damage, but some is needed for proper adaptions. We have now added a section to describe the reactive oxygen/nitrogen species for protecting muscle injury vs. muscle-mass recovery.
Reviewer’s comments: b) Highlight all the relevant markers of the above.
Authors’s response: (b) We have discussed relevant signaling proteins and markers for the pathways for inflammation and ROS. This was added with a new section 3.2.2.1 - 3.2.2.7.
Reviewer’s comments: c) Illustrate pathways with relevant molecules. It helps researchers to understand at best.
Authors’s response: (c) We have made a new figure (Figure 3) that highlights relevant signaling proteins and molecules in the pathways for ROS signaling.
Reviewer’s comments: 3. Line 200: Typo error. Please check other areas carefully while reviewing.
Authors’s response: We have read through the manuscript and identified several typos and have corrected those.
Reviewer’s comments: 4. Please provide details with the figure for mitochondria respiratory function (overview of mitochondrial function), which helps to understand ATP synthesis's (an example) role in muscle differentiation or regeneration.
Authors’s response: We generated a new figure 2 which shows an overview of oxidative phosphorylation and ATP production that is required for myoblast differentiation and protein assembly in muscle regeneration.
Reviewer’s comments: 5. Please discuss-mitophagy markers more.
Authors’s response: We have added additional information regarding mitophagy and the important proteins of mitophagy in the revised section 3. We have also referenced several good papers that provide a review of the topic.
Reviewer’s comments: 6. Please provide more details (Make a table if necessary) on alterations in mitochondrial mRNA, miRNA, and proteins for injury vs. recovery.
Authors’s response: We have added additional information in a new Table 2 to describe some examples of mRNA and protein changes related to mitochondrial in injured and regenerating muscle after an injury.
Reviewer’s comments: 7. Line 324: Not sure why the author discussed only on TNFa and why not other cytokines. Please highlight markers of M1 vs. M2 macrophages in the article.
Authors’s response: We have extensively increased the scope of cytokines from TNF on IL-1b, IFNb1 and IL-6, IL-10 etc. to provide a better description of M1 and M2 activation by cytokines.
Reviewer’s comments: 8. Please discuss more on pharmacological or biological strategies for attenuating mitochondrial aging or degradation vs. augmentation.
Authors’s response: Thank you for your comments. Although it is beyond the scope of this paper to extensively discuss pharmacological and/or biological strategies that have been used in aging and mitochondria we have added to 3.4.4.5 some areas that have been shown to have treatment effects on mitochondrial and satellite cells.
Reviewer’s comments: Overall, this article shall improve by providing more details with relevant mechanistic figures which favor researchers or readers.
Authors’s response: Thank you for your comments. We have extensively revised the manuscript and we hope that the new information and figures will provide the context for the manuscript that will help readers understand this exciting area of research.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
First, I would like to express my gratitude for the opportunity to review this excellent work. The authors highlighted in detail the role of mitochondria in skeletal muscle repair, and also presented modern techniques for managing this process. I have a few small suggestions that I hope can improve the presentation of the work.
Comments:
1. Along with induced muscle injuries, it is noted that mitochondrial dysfunction also plays an important role in the progression of congenital muscular dystrophies (Duchenne muscular dystrophy is probably one of the most studied in this case). In this case, it is known that an improvement in mitochondrial biogenesis (10.1016/j.phrs.2021.105751) or an improvement in the calcium-buffering capacity of organelles (10.3390/ijms22189780) leads to mitigation of the pathology and normalization of tissue regenerative capacity. I suggest, if possible, that the authors also discuss these mechanisms.
2. Lines 22-23. Repair and remodeling during muscle repair is energy demanding and mitochondria provide a primary source for energy production during regeneration.
I think this sentence sounds a little unfortunate, it's better to rewrite it.
3. Line 60. Perhaps it would be better to talk about ion homeostasis, and not just about calcium. Mitochondria also regulate potassium ion homeostasis, which is also important in the case of muscles, especially the heart.
Author Response
Response to Reviewer 2
Reviewer’s comments: First, I would like to express my gratitude for the opportunity to review this excellent work. The authors highlighted in detail the role of mitochondria in skeletal muscle repair, and also presented modern techniques for managing this process. I have a few small suggestions that I hope can improve the presentation of the work.
Authors’s response: Thank you for your comments and encouragement. We have made some extensive revisions by adding new material that has been requested by other reviewers. The paper is now broader, but it does provide a greater level of perspective for the involvement of mitochondria in muscle repair.
Reviewer’s comments: 1. Along with induced muscle injuries, it is noted that mitochondrial dysfunction also plays an important role in the progression of congenital muscular dystrophies (Duchenne muscular dystrophy is probably one of the most studied in this case). In this case, it is known that an improvement in mitochondrial biogenesis (10.1016/j.phrs.2021.105751) or an improvement in the calcium-buffering capacity of organelles (10.3390/ijms22189780) leads to mitigation of the pathology and normalization of tissue regenerative capacity. I suggest, if possible, that the authors also discuss these mechanisms.
Authors’s response: The Reviewer is quite correct concerning other myopathies in which mitochondrial dysfunction plays a large role. We have added a new section 3.3 to the manuscript that highlights this area of mitochondria dysfunction in myopathy and particularly DMD as suggested by the Reviewer.
Reviewer’s comments: 2. Lines 22-23. Repair and remodeling during muscle repair is energy demanding and mitochondria provide a primary source for energy production during regeneration.
I think this sentence sounds a little unfortunate, it's better to rewrite it.
Authors’s response: This sentence was changed to read “Muscle repair is energy demanding and mitochondria provide the primary source for energy production during regeneration.”
Reviewer’s comments: 3. Line 60. Perhaps it would be better to talk about ion homeostasis, and not just about calcium. Mitochondria also regulate potassium ion homeostasis, which is also important in the case of muscles, especially the heart.
Authors’s response: This is a good point. Although calcium regulation was probably most relevant to mitochondrial function (and overloading mitochondria with calcium induces. mitochondrial dysfunction and opening of the mitochondrial permeability pore) other ions are indeed regulated in injury and DMD. We have changed calcium to ion in several places, but we do describe calcium dysregulation as well as mentioning potassium dysregulation in DMD.
Author Response File: Author Response.pdf
Reviewer 3 Report
It was the thought of this reviewer that the paper was just a compilation of the already known for long time, which may be helpful for teaching purposes but not to the advance of science. However, as I went through the manuscript there were some interesting points that changed this vision. I will, nevertheless, propose several changes that will (in my opinion) move the scientific sound of the manuscript forward.
One of the main concerns is regarding the physiological or pathological injury, I feel that these terms are interchangeable throughout the manuscript. This need to be considered when discussed the mechanisms as the role of mitochondria could differ between these kinds of injuries.
In addition, the role of mitochondria-nucleus axis in the context of physiological injury can be disused (DOI: 10.1126/science.abe5620).
Regarding the immunometabolism section (mitochondria-macrophage connection), a new line of thought shows that cardiomyocytes eject dysfunctional mitochondria (via exophers) which are captured and eliminated by macrophages, see ( DOI: 10.3390/life11010061) for a discussion of this mechanism in the context of skeletal muscle. Discussion of this mechanism in the context of repair can move the topic forward.
It would be interesting to present the MSCs in the Introduction.
Minor
L46. I don’t think that there is so much difference between disuse and bed rest.
L146. Are you suggesting the formation of a new muscle fiber?
Line 179. PGC-1 is not only responsible of mitochondrial adaptations, but also other oxidative-related adaptations such as angiogenesis.
Line 226. What is healthy mitochondria? It can be easily confounded mitochondrial hyperactivity with an improve in mitochondrial function. For instance, overweight and obese subjects can show an increased mitochondrial respiration within SKM which can be down-regulated through calorie restriction ( DOI: 10.1016/j.metabol.2022.155336). It is important to define it. Revise through the manuscript.
Line 230 (see the comment of mitochondria-macrophage connection).
Author Response
Response to Reviewer 3
REVIEWER’S COMMENT. It was the thought of this reviewer that the paper was just a compilation of the already known for long time, which may be helpful for teaching purposes but not to the advance of science. However, as I went through the manuscript there were some interesting points that changed this vision. I will, nevertheless, propose several changes that will (in my opinion) move the scientific sound of the manuscript forward.
RESPONSE: Thank you for your comments and suggestions for improving our manuscript. We have inserted more information on other topics as requested by the other Reviewers and this has broadened the paper somewhat. We have carefully reviewed and considered your suggestions and have implemented these changes in the revision. We have included both a copy of the manuscript with the word tracking functions so all of the extensive changes can be seen, and we have uploaded a "clean" copy of the manuscript without the tracking changes.
REVIEWER’S COMMENT One of the main concerns is regarding the physiological or pathological injury, I feel that these terms are interchangeable throughout the manuscript. This need to be considered when discussed the mechanisms as the role of mitochondria could differ between these kinds of injuries.
RESPONSE: Thank you for your comment. Reviewer 2 asked us to include Duchene Muscular Dystrophy as a pathophysiological response and many of the mechanisms are in fact very similar to the damage that occurs expreiementally by for example cardiotoxin injury. The paper is primarily focused on non-physiological (e.g, not much is devoted to exercidse induced damage) whereas the maginitude of the dysfunction and injury is very high in pathological or experimental injury. We have gone back through the manuscripot to attempt to better clarify the injury status where needed as pathological or physiological as it could certainly affect mitochondrial involvement if for no other reason than the magnitude of the changes because of the types of injury that were used.
REVIEWER’S COMMENT In addition, the role of mitochondria-nucleus axis in the context of physiological injury can be disused (DOI: 10.1126/science.abe5620).
RESPONSE: Thank you. Although the idea that muscle repair can occur without satellite cell activation has not been widely explored it is extremely interesting, and the fact that mitochodnria have a role in more modest physiological injury at least through calcium buffereing is very interesting. This has been added to the paper as a nuclei to mitochodnria link in repair.
REVIEWER’S COMMENT Regarding the immunometabolism section (mitochondria-macrophage connection), a new line of thought shows that cardiomyocytes eject dysfunctional mitochondria (via exophers) which are captured and eliminated by macrophages, see ( DOI: 10.3390/life11010061) for a discussion of this mechanism in the context of skeletal muscle. Discussion of this mechanism in the context of repair can move the topic forward.
RESPONSE: Thank you. We have included a discussion of mitochodnria-macrophage interactions in the context of muscle repair as suggested by the Reviewer. We have also added a section on extracellular vesicles and the potential for mitochondrial transport in EV and macrophages for affecting muscle regeneration.
REVIEWER’S COMMENT It would be interesting to present the MSCs in the Introduction.
RESPONSE: We have introduced MSCs in the introduction as suggested.
Minor
REVIEWER’S COMMENT L46. I don’t think that there is so much difference between disuse and bed RESPONSE: Disuse can come in many forms such as casting, or in rodents tail suspension (hindlimb suspension). Yes, bed rest would be considered a type of disuse atrophy.
REVIEWER’S COMMENT L146. Are you suggesting the formation of a new muscle fiber?
RESPONSE: We know from multiple animal studies and some supportive data in human studies that loading and particular stretch loading is capable of forming new fibers. When a muscle undergoes removal/degeneration as a result of cardiotoxin injury or some other severe pathological injury, yes, satellite cells can divide, fuse into myoblasts and form new fibers or attach to damaged fibers (really filling a gap between two ends of a fiber).
REVIEWER’S COMMENT Line 179. PGC-1 is not only responsible of mitochondrial adaptations, but also other oxidative-related adaptations such as angiogenesis.
RESPONSE: Yes the Reviewer is correct. Although PGC1a is often described as a master reguiator or mitochondrial biogenesis, and mitochondria is the focus of this paper, PGC1a also has other roles. This was mentioned here.
REVIEWER’S COMMENT Line 226. What is healthy mitochondria? It can be easily confounded mitochondrial hyperactivity with an improve in mitochondrial function. For instance, overweight and obese subjects can show an increased mitochondrial respiration within SKM which can be down-regulated through calorie restriction ( DOI: 10.1016/j.metabol.2022.155336). It is important to define it. Revise through the manuscript.
RESPONSE: Thank you. To us a normally respiring mitochondria, with appropriate mitochondrial dynamics (fission/fusion functions), not overloaded with calcium nor have an excessively open mitochodnria permeability pore would be healthy. We have added the following :
Healthy mitochondria for this review are defined as mitochondria that are respiring normally (not “hyperactive” metabolically), do not have an excessive Ca2+ load, have normal mitochodnrial dynamics (fission/fusion), normal metabolism and do not have an excessively open mitochondria permeability pore which allows the contents of the mitochondria to leak into the cyotplasm.
REVIEWER’S COMMENT Line 230 (see the comment of mitochondria-macrophage connection).
RESPONSE: We have revised the sections on mitochondria and macrophages.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
The authors have made a substantial effort to improve the manuscript. The paper can be published as such. Thanks for the contribution to the field.
