PONE-D-21-09743

Longitudinal Impact on Rat Cardiac Tissue Transcriptomic Profiles due to Acute Intratracheal Inhalation Exposures to Isoflurane

PLOS ONE

Dear Dr. Park,

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.

Two expert reviewers have studied your manuscript. Although both find the topic of interest they have made several comments that you will need to address in a revised manuscript. Particularly, reviewer #1 has identified flaws in your experimental design that can only be repaired by including additional groups. Hence, experiments should be redone in SHR rats receiving free, spontaneous inhalation with ISO compared to air only. In addition, control WKY rats receiving free, spontaneous inhalation with ISO compared to air only, should also be included. However, I find it acceptable if these additional groups are not followed for 240 and 360 days. The earlier time points (D1 and D30) will suffice.

If you will need more time than 6 months 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Jaap A. Joles, DVM, PhD

Academic Editor

PLOS ONE

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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: Partly

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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: 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: 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: Aim of the study described in the manuscript by Park et al., is to elucidate the effect of the inhalation anesthetic isoflurane (ISO) on gene expression patterns of cardiac tissue. While ISO may not lead to long-lasting side-effects, short- and long-term effects on gene regulation remain unexplored.

Park et al., investigated the role of ISO on SHR rats by 2 consecutive days of 2hour ISO (2%) intra-trachael inhalation (IHIH) versus air only inhalation. Hearts were explanted on D1, D30, D240 and D360 post-exposure. Their main finding was that ISO exposure leads to changes in cardiac gene expression, some of which last up until D360. The primary pathways involved included general immune response e.g. wound healing and cytokine-mediated pathways, circadian rhythm, regulation of systemic arterial blood pressure, fatty acid metabolism and extracellular matrix organization.

I appreciate the effort put into this manuscript as indeed inhalation anesthetics, often used when performing experimental cardiovascular surgeries on rodents, may unintentionally influence (cardiac) cellular processes. However, in most of these cases, both controls/Shams and experimental intervention groups will receive the same (type of) anesthetic treatment. In this manuscript, the methods describe that control animals did not receive IHIH. The real comparison here is thus SHR + spontaneous breathing versus SHR + mechanical breathing on ISO. I believe it is thus not possible to discern between the effects of ISO vs IHIH on cardiac gene expression and thus impossible to attribute the findings to ISO alone.

Please find a more detailed overview of my comments below.

Major:

1) Regarding the experimental set-up: from the manuscript it becomes clear that the ISO but not the control group received IHIH. There is evidence that mechanical ventilation affects cardiovascular functioning (e.g. PMID: 2199000 and PMID: 10208959). So how sure are the authors that the induced changes in gene expression are due to ISO and not to IHIH? I think the fact that for instance the wound-healing pathways are activated, says more about the effect of introducing IHIH than the effect of ISO. Physical injury due to multiple intra-tracheal insertions could possibly explain a whole range of the DEGs and pathways found in this manuscript. In order to be really able to investigate the sole effect of ISO, experiments should be redone in SHR rats receiving free, spontaneous inhalation with ISO compared to air only.

2) There is indeed evidence that ISO inhalation results in altered cardiovascular function. Examples include concentration-dependent decreases in heart rate and blood pressure (PMID: 10622172) which may lead to more severe hypotension in hypertensive circumstances (PMID: 15574546). Others have also shown that vascular effects ex vivo and in vivo in hypertensive rats are more severe than in normotensive rats (PMID: 15574546). However, if cardiac gene expression is equally affected by ISO remains unknown. As outcomes in SHR are generally compared to e.g. WKY rats, authors should also consider taking into account the DEGs and pathways that RNA-seq yields for these animals on ISO versus control.

3) The methodological approach and pathway analyses based on the RNA-seq data looks sound.

Reviewer #2: Title: Longitudinal Impact on Rat Cardiac Tissue Transcriptomic Profiles due to Acute

Intratracheal Inhalation Exposures to Isoflurane

This is a remarkable study, with time points reaching 360 days. It is indeed uncommon to see studies go into that level of depth with their measurements. It is yet more remarkable that there is any effect at this time point. This study is very intriguing and will undoubtedly be impactful in the field, and I congratulate the authors on this monumental effort. However, I do think that several things need to be added to this paper to show individual gene measurements and plots of variance – for example showing some replicates for the later time points would reassure readers that these time points are real measurements and provide more convincing evidence of these striking findings.

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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.

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Reviewer #1: No

Reviewer #2: No

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Attachments

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PONE-D-21-09743R1

Longitudinal Impact on Rat Cardiac Tissue Transcriptomic Profiles due to Acute Intratracheal Inhalation Exposures to Isoflurane

PLOS ONE

Dear Dr. Park,

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.

Both reviewers find the revised manuscript improved. Note that reviewer #2 has submitted a separate file with comments.

Nevertheless, both still have some remaining comments that you must address to allow acceptance of this manuscript. 

Please submit your revised manuscript by Aug 27 2021 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. 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,

Jaap A. Joles, DVM, PhD

Academic Editor

PLOS ONE

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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: (No Response)

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: Partly

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: Aim of the study described in the manuscript by Park et al., is to elucidate the effect of the inhalation anesthetic isoflurane (ISO) on gene expression patterns of cardiac tissue. Short- and long-term effects on gene regulation by ISO, especially in models of cardiovascular disease, remain largely unexplored.

Park et al., investigated the role of ISO on SHR rats by 2 consecutive days of 2hour ISO (2%) intra-trachael inhalation (IHIH) versus air only inhalation. Hearts were explanted on D1, D30, D240 and D360 post-exposure. Their main finding was that ISO exposure leads to changes in cardiac gene expression. In their revised manuscript, the author place more emphasis on acute versus (novel) long-term effects, hereby discerning the differential pathways induced by ISO exposure.

I appreciate the additional effort put in the revised manuscript, both in writing and in data. The additional heatmaps, Volcano-plots and GO-pathway analysis give a more in-depth overview of the RNA-seq data.

Please find a more detailed overview of my comments below.

1) Regarding the experimental set-up: the authors have shared their thoughts and considerations on choosing this specific approach (eg. IHIH vs free-breathing). I would suggest to include part of this information in the Materials and Methods sections and discussion. This can be done to both emphasize and discuss the differences in acute (eg <d30) and="" long-term="">D240) DEG patterns.

2) In general, the authors have made a clearer distinction in representing and discussing the acute and long-term DEG patterns. This gives the reader a better insight between the types of pathways regulated. Indeed, I believe the long-term, newly manifested, effects are the most interesting; regulation of angiogenesis, extracellular matrix organization, (OXPHOS and FA) metabolism and cell signaling. While I understand that the authors will dedicate a separate manuscript on how these changes may lead to heart function alterations, lacks information on how the SHR model relates to previous work in healthy animals, which links to point 3) and the question of adding a WKY control. If the authors primary goal is to share information on routine ISO employment in a variety of clinical settings, patients at risk should be identified. Now all previous models and spatio-temporal changes are equally discussed.

3) The discussion lacks connections between existing literature and the current study on potential mechanisms of ISO to induce these DEG patterns. Specifically by now separating the acute and long-term phase, some explanation should be included how these transient changes come into play.

4) Novel text addition lines 531 – 536 seems to have overlapping message with lines 537 – 541. The distinction between these statements is better described in line 604 – 611.)

Reviewer #2: This is an interesting work. Overall I support the publication of these data as an important addition to the scientific record. Please see my attached detailed comments for minor revisions to improve the communication of these findings to the reader and clarify several issues I feel may be important for the reader.

**********

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: Yes: Matthew R. Sapio

[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.

PONE-D-21-09743R2

Longitudinal Impact on Rat Cardiac Tissue Transcriptomic Profiles due to Acute Intratracheal Inhalation Exposures to Isoflurane

PLOS ONE

Dear Dr. Park,

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.

Reviewer #1 has a few additional remarks that should not form a problem.

Please submit your revised manuscript by Oct 02 2021 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. 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,

Jaap A. Joles, DVM, PhD

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 (if provided):

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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: 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 #1: No

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: Aim of the study described in the manuscript by Park et al., is to elucidate the effect of the inhalation anesthetic isoflurane (ISO) on gene expression patterns of cardiac tissue. Short- and long-term effects on gene regulation by ISO, especially in models of cardiovascular disease, remain largely unexplored.

The additional changes from this second round have further improved the content and message of the manuscript. My comments therefore primarily concern writing to improve clarity and readability.

1) Line 122 - 125 repetition of materials and methods with lines 136 – 137 but final concentration of ISO in not consistent (2% vs 2,5%)

2) It is common that the total amount of animals, eg animals per timepoint per group are described in the materials and methods (line 114 onwards) and not just the figure legends. I assume the Figure legend 1A means n=6 or n=5-6 animals per group PER TIMEPOINT. Ideally, use of animals is repeated for each Figure.

3) Materials and methods from line 126: text explaining the use of ITIH because of the WTC-dust experiments confuses me. Do the authors mean that SHR animals on ITIH were part of a control for WTC-dust trials? I think it must be explicitly mentioned that the animals in this manuscript were not exposed to WTC-dust themselves.

4) Caption Table 6, Line 463; addition of ‘Late onset’ DEG

5) Line 562 – 553: ‘Kouki et al. (2016) reported strain-specific isoflurane (…)’

6) Line 639 – 640: missing references about comparisons to other inhalant anesthetics

7) Line 654: Copy-paste of response to reviewer. I would remove ‘respectfully’

8) Line 659: Copy-paste of response to reviewer. I would remove ‘ -and still do-‘ and change to ‘Thus we believe’

9) Insertion of lines 657 – 664 largely overlapping in content with following lines 667 – 673.

Reviewer #2: I have no further revisions. I recommend the publication of this manuscript if there are no other comments from other reviewers or the editor.

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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 #1: No

Reviewer #2: Yes: Matthew R. Sapio

[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.

Longitudinal Impact on Rat Cardiac Tissue Transcriptomic Profiles due to Acute Intratracheal Inhalation Exposures to Isoflurane

PONE-D-21-09743R3

Dear Dr. Park,

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.

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Kind regards,

Jaap A. Joles, DVM, PhD

Academic Editor

PLOS ONE

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Reviewers' comments: