Muscular TOR knockdown and endurance exercise ameliorate high salt and age-related skeletal muscle degradation by activating the MTOR-mediated pathway

Shi-jie Wang; Deng-tai Wen; Ying-hui Gao; Jing-feng Wang; Xing-feng Ma

doi:10.1371/journal.pone.0311159

Peer Review History

Original SubmissionMay 30, 2024
23 Jul 2024 Decision Letter - Abhinava Kumar Mishra, Editor PONE-D-24-20830Muscular TOR knockdown and endurance exercise delayed age-related deterioration of skeletal muscle by activating the SIR2/PGC-1α and ATG2/PGC-1α pathway in Drosophila on a high-salt dietPLOS ONE Dear Dr. Wang, 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. ============================== The reviews for the above manuscript have been received. The reviewers' comments are included along with this letter. Three independent reviewers have assessed the manuscript's suitability for publication and solicit additional clarification on certain outstanding issues. While your study offers valuable insights into sarcopenia mechanisms, several critical issues must be addressed. Firstly, the rationale for using flies with TOR knock-down or overexpression requires further elucidation, along with inclusion of data from wild type flies and validation of TOR RNAi efficiency. Additionally, the non-specific expression of MHC-GAL4 outside skeletal muscles warrants clarification with a more specific driver, supported by foundational experiment replication. If you wish to revise the manuscript to address the issues raised by reviewers, editors would be happy to consider the revised version of the manuscript for publication. When revising the manuscript, please consider all the points raised by the reviewers and outline every change made. If you disagree with the reviewers' comments, please provide a suitable rebuttal to their concerns. ============================== Please submit your revised manuscript by Sep 06 2024 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, Abhinava Kumar Mishra, PhD Academic Editor PLOS ONE Journal requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following financial disclosure: [This research was supported by the National Natural Science Foundation of China (No. 32000832), the Natural Science Foundation of Shandong Province (No. ZR2020QC096.]. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: [This work is supported by the Province Natural Science Foundation of Shandong (No. ZR2020QC096) and National Natural Science Foundation of China (NSFC) (No. ] We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [This research was supported by the National Natural Science Foundation of China (No. 32000832), the Natural Science Foundation of Shandong Province (No. ZR2020QC096.]. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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: No ****** 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: No ****** 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: In this article, authors firstly conformed that the muscle TOR gene played an important role in endurance exercise against HSD-induced age-related skeletal muscle degeneration, as it determined the activity of the MTOR/SIR2/PGC-1α and MTOR/ATG2/PGC-1α pathways in skeletal muscle. The author provides a lot of data to support his conclusion. However, there were still some minor issues that need to be fixed to make the article better. 1. The shortened words should be standardized and uniform. For example, the article had TOR and MTOR, these two have the same meaning or different meaning. 2. The format and grammar of the article need to be carefully revised. 3. The author should add some latest references for discussion and analysis. Reviewer #2: The manuscript by Wang et al., studies the mechanism how exercise can ameliorate age-related skeletal muscle deterioration by high salt diet. This study can provide novel insight into how sarcopenia is caused. However there are several issues that need to be addressed: Major issues 1. The reason why the authors used flies where TOR was either knocked-down or overexpressed for the experiments is unclear. The authors need to provide a better explanation. At the same time the authors need to add results of experiments obtained from wild type flies. Also, the authors need to prove that the RNAi for TOR indeed knocked-down the gene. 2. MHC-GAL4 expression in not limited to skeletal muscles. It is expressed in other sites such as cardiac muscles. The authors need to address that in the text. They also need to use a more specific driver (refer to Zappia and Frolov, 2016) and repeat some of their more fundamental experiments. Minor Issues: 1. The manuscript has several grammatical errors. 2. The authors need to provide the full forms of abbreviations while mentioning them for the first time. 3. The authors need to make sure that they consistently use the same nomenclature to refer to the same thing. For isntance they have both used weeks and weekends to denote age of flies. 4. The authors need to make sure that they used proper terminology throughout the paper consistent with previous publications. Also, they need to represent the genotypes of the flies correctly throughout the manuscript. Especially it will be helpful to have complete genotypes in the figures. 5. Information about rearing such as day-night cycle, temperature and humidity have not been mentioned. ****** 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: No ******** [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. https://doi.org/10.1371/journal.pone.0311159.r001
Revision 1
9 Aug 2024 Author Response Reviewer #1: In this article, authors firstly conformed that the muscle TOR gene played an important role in endurance exercise against HSD-induced age-related skeletal muscle degeneration, as it determined the activity of the MTOR/SIR2/PGC-1α and MTOR/ATG2/PGC-1α pathways in skeletal muscle. The author provides a lot of data to support his conclusion. However, there were still some minor issues that need to be fixed to make the article better. 1. The shortened words should be standardized and uniform. For example, the article had TOR and MTOR, these two have the same meaning or different meaning. The full names of TOR and MTOR have been added to distinguish them 2. The format and grammar of the article need to be carefully revised. The format has been modified according to the requirements of the journal, and the syntax has been Revised. Please see the blue font in the body of the Revised Manuscript with Track Changes 3. The author should add some latest references for discussion and analysis. It has been modified. For details, see the section highlighted in blue in Revised Manuscript with Track Changes. Reviewer #2: The manuscript by Wang et al., studies the mechanism how exercise can ameliorate age-related skeletal muscle deterioration by high salt diet. This study can provide novel insight into how sarcopenia is caused. However there are several issues that need to be addressed: Major issues 1. The reason why the authors used flies where TOR was either knocked-down or overexpressed for the experiments is unclear. The authors need to provide a better explanation. At the same time the authors need to add results of experiments obtained from wild type flies. Also, the authors need to prove that the RNAi for TOR indeed knocked-down the gene TOR knockdown/overexpression flies were used to verify the role of TOR in this study. For example, from the differences in motor performance, antioxidant capacity and gene expression levels between the TOR overexpression group and the TOR normal expression group, we can see the role of TOR genes in these aspects..The construction of TOR overexpression/knockdown system was demonstrated by comparing the expression level of MTOR gene in flies with normal TOR expression group at three week-old without intervention.The results showed that there were significant differences in the expression level of MTOR gene between overexpression group/knockdown group and normal expression group(See text 3.1 for details). 2. MHC-GAL4 expression in not limited to skeletal muscles. It is expressed in other sites such as cardiac muscles. The authors need to address that in the text. They also need to use a more specific driver (refer to Zappia and Frolov, 2016) and repeat some of their more fundamental experiments. The experimental methods and procedures related to skeletal muscle have been completed (related to overexpression/knockdown MTOR gene expression levels in Drosophila melanogaster over three weeks).We do not deny that MHC-Gal4 is expressed in other muscles. However, we only examined the skeletal muscle of fruit flies, and we plan to study the specific changes of TOR gene in other muscles in the future. Minor Issues: 1. The manuscript has several grammatical errors. The format has been modified according to the requirements of the journal, and the syntax has been Revised. Please see the blue font in the body of the Revised Manuscript with Track Changes 2. The authors need to provide the full forms of abbreviations while mentioning them for the first time. It has been modified. For details, see the section highlighted in blue in Revised Manuscript with Track Changes. 3. The authors need to make sure that they consistently use the same nomenclature to refer to the same thing. For isntance they have both used weeks and weekends to denote age of flies. It has been modified. For details, see the section highlighted in blue in Revised Manuscript with Track 4. The authors need to make sure that they used proper terminology throughout the paper consistent with previous publications. Also, they need to represent the genotypes of the flies correctly throughout the manuscript. Especially it will be helpful to have complete genotypes in the figures. It has been modified. For details, see the section highlighted in blue in Revised Manuscript with Track 5. Information about rearing such as day-night cycle, temperature and humidity have not been mentioned. For details, see Revised Manuscript with Track Changes 2.1. The information the editor says about the journal format, funding, supporting documents, etc. has been corrected. Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0311159.r002
16 Sep 2024 Decision Letter - Abhinava Kumar Mishra, Editor Muscular TOR knockdown and endurance exercise ameliorate high salt and age-related skeletal muscle degradation by activating the MTOR-mediated pathway PONE-D-24-20830R1 Dear Dr. Wang, 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, Abhinava Kumar Mishra, PhD 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: All comments have been addressed Reviewer #2: All comments have been addressed ******** 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: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #1: Yes Reviewer #2: No ****** 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 #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 #1: This study confirms that the muscle TOR gene plays an important role in endurance exercise against HSD-induced age-related skeletal muscle degeneration, as it determines the activity of the mammalian target of rapamycin(MTOR)/SIR2/PGC-1α and MTOR/ATG2/PGC-1α pathways in skeletal muscle. The data supports the conclusions, and the statistical analysis is performed appropriately and rigorously, and the manuscript is presented in an intelligible fashion and written in standard English after the paper has been revised. Reviewer #2: The authors have satisfactorily addressed all my concerns and have also made the necessary changes. The authors need to share the data sets and provide a summary table for the stats. ****** 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 #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0311159.r003
Formally Accepted
24 Sep 2024 Acceptance Letter - Abhinava Kumar Mishra, Editor PONE-D-24-20830R1 PLOS ONE Dear Dr. Wang, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Abhinava Kumar Mishra Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0311159.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .