Pathways of calcium regulation, electron transport, and mitochondrial protein translation are molecular signatures of susceptibility to recurrent exertional rhabdomyolysis in Thoroughbred racehorses

Kennedy Aldrich; Deborah Velez-Irizarry; Clara Fenger; Melissa Schott; Stephanie J. Valberg

doi:10.1371/journal.pone.0244556

Peer Review History

Original SubmissionSeptember 22, 2020
11 Nov 2020 Decision Letter - Agustín Guerrero-Hernandez, Editor PONE-D-20-29481 Pathways of calcium regulation, electron transport, and mitochondrial protein translation are molecular signatures of susceptibility to recurrent exertional rhabdomyolysis in Thoroughbred racehorses PLOS ONE Dear Dr. Valberg, 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, consider all the variables involved in the experimental protocol to have a better assessment of the conclusions strength. Please submit your revised manuscript by Dec 26 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Agustín Guerrero-Hernandez Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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. In your Methods section, please state the volume of the blood samples collected for use in your study. 3. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing. 4. Thank you for stating the following in the Competing Interests section: "The authors have declared that no competing interests exist.". We note that one or more of the authors are employed by a commercial company: Equine Integrated Medicine, PLC' 1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. 2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. * Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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: I Don't Know ****** 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 ****** 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 ****** 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 search for environmental factors and a genetic predisposition which impact the expression of RER in the horse is of great interest in the practice of sports medicine of this species. The present study has sought the differential expression patterns of specific genes/proteins and GO pathways in RER-susceptible horses. The Authors propose a scenario whereby horses susceptible to RER have enhanced storage of Ca2+ in the sarcoplasmic reticulum. Based on these main results, the authors concluded that it enhances muscle force and energy metabolism within a physiologic range of myoplasmic Ca2+. It is very important but not new finding. However the unique is that the newest and more sophisticated research techniques were used. In addition, this is the first study where RER-susceptible horses were used. In addition, the horses affected by dantrolene were used. The main limitation of the study is low number of horses and different age of them. Thus, the training level of the horses vary. Specific comments L45- side effects of dantrolene should be mentioned. L77 –what was their training regimen? Were their trained according to the same protocol? What was the exertion after which the samples were obtained? Please specify. L78 –“Race-13”, it is not clear what it means. L81- how veterinarian confirmed RER? It was after trotting test or regular training session? Was the LDH measured? L84 – the number of horses in Untreated RER-susceptible and treated group should be mentioned also in those paragraph. L96 – “Samples were obtained in the morning between 1 and 5.5 h after either jogging or galloping exercise on the track” – what do You mean jogging? It is confusing if the horses were exercised or not in this study? Please specify the training session after which the samples were obtained. L243 – whereas there were no statistical differences, there is a huge difference between 2 years old and almost 6 years old race horse during training. The Authors should take it into consideration. Table 2 - Adjusted p value is hard to follow. L499 – what do Authors mean by “tight control”? Expand Your mind. L 535 – The statement is risky. Because during chronic stress it was demonstrated that cortisol release is lower. In addition, it should be taken in to consideration that cortisol plays an essential role in adaptation during exercise. Upregulation of this hormone leads to increase of protein synthesis It can also increase glycogen deposition and storage in the liver. L 595 – the time of sampling should be the same in next study because in 5,5h after exercise the proteins may be degraded. L599 – the timing of samples collection is crucial because of as a result of the vasoconstrictor and vasodilatory response after the exercise. Thus, the blood influx in to the muscles may vary. Reviewer #2: this manuscript is considered new in the field of recurrent exertional rhabdomyolysis in Thoroughbred racehorses and includes a lot of work and its goals are multiple It is clear that the abstract was written in a hurry and it needs to be revised the role of the serum CPK and AST in the diagnosis of exertional rhabdomyolysis should be focused in the text ****** 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.0244556.r001
Revision 1
2 Dec 2020 Author Response Editors comments 1) We changed the methods section to indicate 7 ml of blood was collected 2) We did upload the data and this was indicated in the paper. I have highlighted the information on where data can be found is in the paper to show where that is located. 3) We would like to amend the funding statement to state. The funding agency provided funds to perform the research study and partial salary for one author (DV) but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript 4) We would like to amend the competing interest statement. One of the authors (CF) is a practicing veterinarian and owner of the business Equine Integrated Medicine PLC. The business did not play a role in the funding or design of the study and Dr. Fenger did not receive any financial remuneration for participating in the study. Dr. Fenger identified horses in her practice that were healthy controls or susceptible to RER and facilitated biopsy samples that were obtained by another author (SV). Dr. Fenger was involved in reviewing the data regarding accuracy of the horse’s clinical phenotypes and approving the manuscript for publication. There are no other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products. Response to reviewers We thank the reviewers for their time and insights into our manuscript. Reviewer #1: The search for environmental factors and a genetic predisposition which impact the expression of RER in the horse is of great interest in the practice of sports medicine of this species. The present study has sought the differential expression patterns of specific genes/proteins and GO pathways in RER-susceptible horses. The Authors propose a scenario whereby horses susceptible to RER have enhanced storage of Ca2+ in the sarcoplasmic reticulum. Based on these main results, the authors concluded that it enhances muscle force and energy metabolism within a physiologic range of myoplasmic Ca2+. It is very important but not new finding. However the unique is that the newest and more sophisticated research techniques were used. In addition, this is the first study where RER-susceptible horses were used. In addition, the horses affected by dantrolene were used. The main limitation of the study is low number of horses and different age of them. Thus, the training level of the horses vary. Specific comments L45- side effects of dantrolene should be mentioned.- This has been added. We are only aware of side effects in horses under anesthesia. Line 57 L77 –what was their training regimen? Were their trained according to the same protocol? What was the exertion after which the samples were obtained? Please specify. The training regime was similar and has been added Line 96. The exercise preceding the biopsy was similar between groups and has been added Line 158 L78 –“Race-13”, it is not clear what it means. This is the brand of horse feed which is further explained now Line 94 L81- how veterinarian confirmed RER? It was after trotting test or regular training session? Was the LDH measured? The AST and CK were measured when a horse had clinical signs of exertional rhabdomyolysis (pain, stiffness, sweating, reluctance to move). This has been added. Line 102. We do not routinely analyze LDH in muscle cases because it is not specific for muscle and because AST provides the needed additional information on the chronicity of rhabdomyolysis. L84 – the number of horses in Untreated RER-susceptible and treated group should be mentioned also in those paragraph. added L96 – “Samples were obtained in the morning between 1 and 5.5 h after either jogging or galloping exercise on the track” – what do You mean jogging? This is the term used at the racetrack for trotting, this has been changed to trotting in the manuscript Line 96, Line 159 It is confusing if the horses were exercised or not in this study? Please specify the training session after which the samples were obtained. Added Line 159 L243 – whereas there were no statistical differences, there is a huge difference between 2 years old and almost 6 years old racehorse during training. The Authors should take it into consideration. We have acknowledged that ideally horses would have been the same age in the discussion line 695. We did not have a significant difference in age between the control and affected groups and all horses, including two-year-olds, were in full race training. Older horses would be expected to have more mitochondrial proteins whereas we found that the RER group which included the one 6 year old had fewer mitochondrial proteins. Table 2 - Adjusted p value is hard to follow. The DEG data had adjusted p-values, the p-values for DEP were not adjusted p-values. The adjusted p-values are q-values, a corrected p-value based on estimations for the proportion of false positives. Significance was determined using a false discovery rate (FDR) ≤ 0.05 to correct for multiple testing in both DEG and DEP. The p-values for proteomics were determined through permutation, the cutoff equivalent to an FDR ≤ 0.05 is a p-value ≤ 0.01 (for this analysis). Line 220. Multiple test correction was performed with false discovery rate less than 0.05. q-values, an adjusted or corrected p-value that is based on estimations of the proportion of false positives, were used in tables to show significance. L499 – what do Authors mean by “tight control”? Expand Your mind. We reworded this to make the sentence clearer. Line 581 Our results suggest that regulation of myoplasmic Ca2+homeostasis is perturbed in horses susceptible to- but not currently experiencing RER- when housed in a race-training environment. Marked alterations in the expression of mitochondrial proteins in RER susceptible horses could be related to the role of mitochondria in buffering myoplasmic Ca2+ which could have a detrimental down-stream effect on the mitochondrial respiratory chain and apoptosis [39]. L 535 – The statement is risky. Because during chronic stress it was demonstrated that cortisol release is lower. In addition, it should be taken in to consideration that cortisol plays an essential role in adaptation during exercise. Upregulation of this hormone leads to increase of protein synthesis It can also increase glycogen deposition and storage in the liver. We have changed this to indicate that “Conditions of chronic stress with associated prolonged beta-adrenergic stimulation could produce post-translational modification of RYR1 in young fillies that trigger enhanced Ca2+ release in RER-susceptible horses.” Line 623 We did not mean to imply that cortisol was detrimental but meant that cortisol levels were an indicator in RER horses that they could be undergoing chronic stress. The beta-adrenergic stimulation of CALM2 and its effect on hyperphosphorylation of RYR1 could be a reason why stress impacts the expression of RER in a fashion similar to what has been demonstrated with chronic heart failure. L 595 – the time of sampling should be the same in next study because in 5,5h after exercise the proteins may be degraded. Yes this would definitely be ideal to have been able to make this all the same. L599 – the timing of samples collection is crucial because of as a result of the vasoconstrictor and vasodilatory response after the exercise. Thus, the blood influx in to the muscles may vary. This is an excellent point. Fortunately, there was not a significant difference in the timing of the sample relative to exercise across the three groups. We have add post exercise blood flow as a fact Line 689. Reviewer #2: this manuscript is considered new in the field of recurrent exertional rhabdomyolysis in Thoroughbred racehorses and includes a lot of work and its goals are multiple It is clear that the abstract was written in a hurry and it needs to be revised We have revised the abstract. The role of the serum CPK and AST in the diagnosis of exertional rhabdomyolysis should be focused in the text We have added additional information on the way the horses were diagnosed with RER Line 102. Horses were diagnosed with RER based on both clinical signs and on the presence of abnormal elevations in CK and AST at the time clinical signs were present. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0244556.r002
14 Dec 2020 Decision Letter - Agustín Guerrero-Hernandez, Editor Pathways of calcium regulation, electron transport, and mitochondrial protein translation are molecular signatures of susceptibility to recurrent exertional rhabdomyolysis in Thoroughbred racehorses PONE-D-20-29481R1 Dear Dr. Valberg, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Agustín Guerrero-Hernandez 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: I Don't Know ****** 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 Response) ****** 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: Dear Authors, Now I have no doubts about an article. It is important and interesting topic according to muscle failure in the race horses. Reviewer #2: This manuscript reported that recurrent exertional rhabdomyolysis in Thoroughbred racehorses is associated with alterations in proteins, genes and pathways impacting myoplasmic Ca2+ regulation, the mitochondrion and protein degradation with opposing effects on mitochondrial transcriptional/translational responses and mitochondrial protein content. Great care has been observed to improve the work in the revised manuscript. ****** 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.0244556.r003
Formally Accepted
20 Jan 2021 Acceptance Letter - Agustín Guerrero-Hernandez, Editor PONE-D-20-29481R1 Pathways of calcium regulation, electron transport, and mitochondrial protein translation are molecular signatures of susceptibility to recurrent exertional rhabdomyolysis in Thoroughbred racehorses Dear Dr. Valberg: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Agustín Guerrero-Hernandez Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244556.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 .