PONE-D-24-30189Oncosis is the Predominant type of cell death in rhabdomyolysis following Exertional heat strokePLOS ONE

Dear Dr. Shan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

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Gokhan Burcin Kubat

Academic Editor

PLOS ONE

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Additional Editor Comments:

As academic editor, I evaluated the reviewers' comments and decided that the manuscript needed major revisions. You can find the reviewers' comments below.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: No

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #4: 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 authors find that EHS rhabdomyolysis is associated with oncosis. However, there are major concerns that reduce the reliability of the study results and enthusiasm for the work. First, the novelty of this work is unclear. In the introduction, the authors suggested that evidence already exists "the mechanism and morphological changes of rhabdo exhibit striking similarities to those observed in oncosis". Second, is this a study of EHS or rhabdo? It is known that only a small portion of EHS patients are associated with rhabdo. It seemed that all the RM rats suffered a heat stroke. Finally, my biggest concern is an insufficient sample size that supports the conclusions. As described, this study included 12 rats, and they were divided into CN and EHS groups (6 per group). Fig 1B indicates that 4 of the 6 rats were lost before 6 hr post-EHS. The animal samples were obtained 6 hrs after EHS. So only 2 rats were included in the EHS group for comparison.

Abstract

ehsRM was defined twice. The conclusions are confusing.

Introduction

Some statements are misleading. For example, there is no evidence for "Oncosis, a form of porimin-dependent non-programmed cell death, is caspase independent..". 

Results

In addition to mean/SD, individual values should be displayed in all the bar graphs

3.1: How do you know "onset"? There are no additional data other than that at  6 hr post-EHS. 

Should this be Table 3? why did CN rats lose weight as well?

3.2: oxidative stress. There are no OS makers in Fig 2

The authors should consider having this manuscript professionally edited.

Reviewer #2: This manuscript investigates the role of oncosis as the primary mechanism of cell death in exertional heat stroke-associated rhabdomyolysis (ehsRM). While the study presents a compelling argument supported by in vitro and in vivo experiments, there are several areas that could be improved or further elaborated. Below are specific comments

1.Mechanistic Link Between Mitochondrial Dysfunction and Oncosis: While the study documents mitochondrial damage and links it to oncosis, the exact molecular mechanisms connecting mitochondrial dysfunction and oncosis could be elaborated further. How mitochondrial swelling directly contributes to oncosis versus other forms of necrosis could be clarified, especially in relation to the release of DAMPs (damage-associated molecular patterns) or mitochondrial permeability transition (MPT).

2.Involvement of Mitochondrial Dynamics: Mitochondrial dynamics (fusion, fission, mitophagy) are critical for maintaining mitochondrial integrity, especially under stress conditions. The manuscript does not address whether there are any disruptions in mitochondrial dynamics that could contribute to the observed mitochondrial swelling and dysfunction. Including markers of fusion (e.g., MFN2, OPA1) and fission (e.g., DRP1) could provide a more comprehensive understanding of how mitochondria fail during ehsRM.

3.Incomplete Exploration of ROS-Mediated Mitochondrial Damage: Although the study demonstrates increased ROS levels and mitochondrial dysfunction, it does not fully explore the interplay between ROS production and mitochondrial damage. Mitochondria are both a source and target of ROS. An investigation into whether mitochondrial ROS production exacerbates the observed dysfunction would be valuable, possibly by using mitochondrial-targeted antioxidants in additional experiments.

4.Mitochondrial Bioenergetics: The study primarily measures ATP levels, but a more detailed analysis of mitochondrial bioenergetics, including the activity of key respiratory chain complexes (e.g., Complex I-IV), would strengthen the conclusions regarding mitochondrial dysfunction.

Recommended Additional Experiments

1.Mitochondrial Dynamics: Assess the expression of proteins involved in mitochondrial fusion (MFN1/2, OPA1) and fission (DRP1, FIS1). This could help elucidate whether altered mitochondrial dynamics contribute to the observed mitochondrial swelling and dysfunction in ehsRM.

2.Mitochondrial Respiration: Perform mitochondrial respirometry to quantitatively assess mitochondrial respiratory function, including oxygen consumption rate (OCR), basal respiration, ATP production, and maximal respiratory capacity in the EHS model.

3.Mitochondrial ROS Scavengers: Introduce mitochondrial-targeted antioxidants to determine whether reducing mitochondrial ROS levels can alleviate mitochondrial damage and oncosis. This could provide a therapeutic angle to prevent or mitigate cell death in ehsRM.

4. Ambiguity in Statistical Presentation: The manuscript does not consistently report statistical significance (e.g., p-values) across experiments. Additionally, error bars on graphs are not always explained (e.g., standard deviation vs. standard error). Ensure that all statistical analyses are clearly reported, with appropriate p-values and error bars labeled. Use more rigorous statistical tests where appropriate.

5. Lack of Functional Outcomes or Behavioral Correlates in Vivo: The manuscript focuses heavily on molecular and structural analysis but lacks a functional or clinical correlate for rhabdomyolysis and EHS severity in the rat model. Behavioral or functional assessments of muscle function (e.g., grip strength, endurance) post-EHS would help translate these findings into clinically relevant outcomes. Include behavioral or physiological assessments in the rat model to assess muscle function after EHS and correlate them with biochemical and histopathological findings.

6. Limited Validation of Oncosis Markers: While the study highlights porimin as a marker for oncosis, the absence of multiple independent markers limits the specificity of the conclusions. Porimin alone may not provide conclusive evidence of oncosis as the dominant mechanism, especially given the presence of apoptotic markers (Bax/Bcl-2). Incorporate additional markers (e.g., HMGB1, or others associated with oncosis) and perform colocalization experiments with mitochondrial damage markers to reinforce the claim of oncosis.

7.Unclear Differentiation between Oncosis and Apoptosis: The manuscript claims that oncosis is the predominant form of cell death, but the presence of apoptotic indicators such as Bax and Bcl-2 raises questions. Although caspase-3 levels did not change, the manuscript does not adequately explain why apoptosis, in addition to oncosis, is not more prominent.Conduct time-course experiments to clearly differentiate the dynamics of oncosis and apoptosis. A detailed analysis of caspase-independent apoptotic pathways could be beneficial to clarify this distinction.

Reviewer #3: 1. Introduction and discussion section should be improved.

The difference between apoptosis and oncosis should be described in detail. Authors should emphasize the importance of this study? Is it really valuable to discriminate oncosis and apoptosis?

2. EHS protocol is stopped when the animals become exhausted. Please give the total duration of protocol for each animal.

3. Authors have provided sterile water. What about chow? Is it also sterile?

4. Authors put cell lines into a hot water bath. How did they gassed the cells? Did authors also consider the solubility change between 37 and 43 °C

5. 30 minutes is a relatively long centrifuge time. Why did authors prefer a long centrifuge time?

6. Authors measured CK. Is it total? Were they able to identify subtypes?

7. Could authors cite the previous study? The study in which they first made the EHS model.

8. In Figure 1: Annexin bars seem relatively different on the other hand your standart deviation is very high. That's why you couldn't observe statistical significance. Do you consider to repeat that data?

9. In Figure 4: There is no mitochondrial membrane potential bar chart. There was only two bar charts for ATP level. Please correct.

10. Figure 5: There is no bar graph for ROS.

11. There is also an increase in Bax BCL levels. How can the authors conclude that there is no apoptosis. And please also give Bax/BCL ratio

12. The survival is very bad in EHS group. How many animals were able to finish the study? Please give “n” for each group.

13. Authors concluded “Therefore, we propose that the mechanism of muscle cell death in skeletal muscle cells undergoing oncosis is primarily due to ATP depletion and mitochondrial damage.” Why this alterations caused oncosis rather than apoptosis? Do authors have any suggestions for mechanism?

Minor

All words in latin should be written in italic.

Reviewer #4: the article presents two models, in vivo and in vitro, to further our understanding of the type of cell death, presumably ischemic and associated with primitive mitochondrial damage, oncosis, in rhabdomyolysis associated with exercise heat stroke. The article is well developed and leads in both models, cellular and animal, to identify mitochondrial damage as the primum movens of the mechanism of oncosis.

The primary damage would result in ATP depletion and mitochondrial damage presumably triggered by free radicals (ROS) acting as the “cause and consequence of mitochondrial damage.”

Mitochondrial damage is definitely related to ROS accumulation, which in turn is triggered by inflammatory damage especially of a chronic nature.

Markers of inflammation were evaluated in the presented experimental mouse model? From the most common, such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and plasma viscosity (PV) in blood, to the more cell-specific, such as pro-inflammatory and anti-inflammatory cytokines?

ROS damage is definitely quantitative; there is a correlation between extent of damage and amount of ROS. Have ROS levels been quantified? Is it possible to show an image of the DCFH-DA fluorescent probe?

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6. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #2: Yes:  Meghnad G Joshi

Reviewer #3: No

Reviewer #4: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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PONE-D-24-30189R1Oncosis is the Predominant type of cell death in rhabdomyolysis following Exertional heat strokePLOS ONE

Dear Dr. Shan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 01 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Gokhan Burcin Kubat

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments :

Editor' Comments: There are some minor revisions to the manuscript.

Reviewer's Comments:

Clarity and Readability: The abstract is rich in data but dense, making it difficult for readers to quickly grasp the key findings and implications. Simplify and organize the content for better flow.

Consistency: There are inconsistencies in spacing (e.g., missing spaces after commas) and formatting (e.g., inconsistent use of abbreviations). Ensure uniformity throughout.

Grammar and Syntax: Certain phrases could be reworded for grammatical correctness and conciseness.

Structure: The abstract lacks a clear division between the background, objectives, methods, results, and conclusions. These sections should be clearly identifiable for better readability.

Introduction: The manuscript effectively introduces exertional heat stroke (EHS) and rhabdomyolysis (RM), but the research hypothesis could be stated more clearly.

Materials and Methods: Detailed descriptions of the experimental design, including EHS modeling and assays, are provided. However, statistical methods could be elaborated for transparency.

Results: Results are well-organized and supported by figures and tables. However, the results section could benefit from a more narrative flow linking findings to the study objectives.

Discussion: The discussion highlights the significance of oncosis but should better integrate findings with existing literature to emphasize novelty.

Conclusion: The conclusion is concise but would benefit from a clearer statement of implications and future directions.

[Note: HTML markup is below. Please do not edit.]

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 #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: 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 #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: 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 #2: No

Reviewer #3: Yes

Reviewer #4: 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 #2: General Comments:

Clarity and Readability: The abstract is rich in data but dense, making it difficult for readers to quickly grasp the key findings and implications. Simplify and organize the content for better flow.

Consistency: There are inconsistencies in spacing (e.g., missing spaces after commas) and formatting (e.g., inconsistent use of abbreviations). Ensure uniformity throughout.

Grammar and Syntax: Certain phrases could be reworded for grammatical correctness and conciseness.

Structure: The abstract lacks a clear division between the background, objectives, methods, results, and conclusions. These sections should be clearly identifiable for better readability.

Introduction: The manuscript effectively introduces exertional heat stroke (EHS) and rhabdomyolysis (RM), but the research hypothesis could be stated more clearly.

Materials and Methods: Detailed descriptions of the experimental design, including EHS modeling and assays, are provided. However, statistical methods could be elaborated for transparency.

Results: Results are well-organized and supported by figures and tables. However, the results section could benefit from a more narrative flow linking findings to the study objectives.

Discussion: The discussion highlights the significance of oncosis but should better integrate findings with existing literature to emphasize novelty.

Conclusion: The conclusion is concise but would benefit from a clearer statement of implications and future directions.

Reviewer #3: Thank you for your editing. I think manuscript is ready for publication after this revision. I have no further concerns.

Reviewer #4: (No Response)

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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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #2: No

Reviewer #3: No

Reviewer #4: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Oncosis is the Predominant type of cell death in rhabdomyolysis following Exertional heat stroke

PONE-D-24-30189R2

Dear Dr. Shan,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gokhan Burcin Kubat

Academic Editor

PLOS ONE

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 #2: 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 #2: Yes

**********

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

Reviewer #2: Yes

**********

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 #2: Yes

**********

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 #2: Yes

**********

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 #2: All comments addressed and manuscript has improved in the present form and suitable for publication.

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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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #2: No

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