Dear Dr Hilfiker-Kleiner,

Thank you for submitting your manuscript entitled "Increased prostaglandin-D2 in male but not female STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Apr 22 2020 11:59PM.

Login to Editorial Manager here: https://www.editorialmanager.com/pbiology

During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Given the disruptions resulting from the ongoing COVID-19 pandemic, please expect delays in the editorial process. We apologise in advance for any inconvenience caused and will do our best to minimize impact as far as possible.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Di Jiang, PhD

Associate Editor

PLOS Biology

Dear Dr Hilfiker-Kleiner,

Thank you very much for submitting your manuscript "Increased prostaglandin-D2 in male but not female STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.

The reviews of your manuscript are appended below. You will see that the reviewers find the work potentially interesting. However, based on their specific comments and following discussion with the academic editor, I regret that we cannot accept the current version of the manuscript for publication. We remain interested in your study and we would like to consider resubmission of a comprehensively revised version that thoroughly addresses all the reviewers' comments. Importantly, we would like to see evidence that the PGD2-Zfp423 axis is a critical pathway driving adipogenesis in vivo (reviewer 2), and more in-depth analyses of the human tissue samples (reviewer 1, point 3). We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript would be sent for further evaluation by the reviewers.

We appreciate that these requests represent a great deal of extra work, and we are willing to relax our standard revision time to allow you six months to revise your manuscript.We expect to receive your revised manuscript within 6 months.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Resubmission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this resubmission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Di Jiang, PhD

PLOS Biology

*****************************************************

REVIEWS:

Reviewer #1: The study by Stelling et al. presents interesting results on how STAT3 deficiency drives left ventricular dysfunction during aging in male mice. The authors present clear evidence that a shift from differentiation of progenitor cells from endothelial cells towards adipocytes occurs in aged male STAT3 deficeint mice mainly driven by increased release of prostaglanin D2 due to increased activity of cyclooxygenase 2 and less activity of HPGD. The data are further supported by extensive in-vitro work on progenitor cells which most interestingly keep their phenotype even after long term cultivation. The experiments are well conducted and I have only a few points the authors might want to consider:

1. The release of PGD2 from isolated cardiomyocytes was normalized to total RNA. Why did the authors choose total RNA rather than a cardiomyocyte specific protein (troponin, alphaMHC) to account for cardiomyocyte number? Total RNA could still be derived from contaminating other cells.

2. Did the authors verify the changes obeserved in the immunohistology (figure 1) on capillary density and fibrosis by other techniques (Western blot: CD31, collagen etc)? Both the changes in capillary density and fibrosis (which appears to be very small (1-3%)) are not very impressive and most likely does not account for heart failure development.

3. The human data are interesting but very superficial. I am aware that obtaining human tissue might be complicated but at least the tissue available should be analyzed further: markers for endothelial cell content, adiopocyte content etc. Even more, some data from female heart failure patients would be of interest to proof that these parameters might differ depending on sex.

4. The authors demonstrate that COX2 might be a key protein in age-depnendent or post-partum triggered heart failure development in STAT3-deficiency. Does this mean that the phenotype can be rescued by COX2 inhibition? Did the authors try to treat aged male mice with COX2 inhibitors? In this context, the discussion on COX2 (PMID: 31734378; PMID: 31380437; PMID: 31278427 to name only a few recent studies) on ischemia-reperfusion related cardiac effects (which involves remodelling and heart failure development as well) needs to be extended.

Reviewer #2: Reduced expression and activity of cardiac STAT3 is linked to dilatative cardiomyopathy or peripartum cardiomyopathy; however, the mechanisms underlying pathologic cardiac remodeling triggered upon cardiomyocyte STAT3 inactivation has remained unclear. Here, the authors propose that cardiomyocyte-specific STAT3 deficiency leads increased PGD2

secretion from male cardiomyocyte but not from female cardiomyocytes. Increased PGD2 in the microenvironment then leads to suppression of EZH2 in cardiac progenitor cells (defined here as Sca-1+ positive cells) and subsequent activation of Zfp423, a transcription factor driving adipocyte differentiation. The authors propose that PGD2 driven adipogenesis from the Sca-1+ positive cells drive the ectopic adipocyte accumulation and loss of vascular density.

Overall, the manuscript is well written and the hypothesis is certainly novel and of general interest. The major concern lies in whether the states hypothesis is sufficiently supported by the data on hand. The authors demonstrate convincingly in vitro that PGD2 can activate Zfp423 expression in progenitor cells and promote adipogenesis. What is missing is evidence that this is the critical pathway driving adipogenesis in vivo in this model. In fact, any number of secreted factors from STAT3-deficient cardiomyocytes might influence adipogenesis. As such, the current observations establish associations, but not a clear sense of causality of the phenotype in vivo. For example, does inactivation of PGD2 receptor in Sca-1+ cells rescue the phenotype? Further evidence that this pathway is mediating the phenotype in vivo would strengthen the conclusions.

Dear Dr Hilfiker-Kleiner,

Thank you for submitting your revised Research Article entitled "Increased prostaglandin-D2 in male but not female STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation" for publication in PLOS Biology. I have now obtained advice from the two original reviewers and have discussed their comments with the Academic Editor.

We're delighted to let you know that we're now editorially satisfied with your manuscript. However before we can formally accept your paper and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication. Please also make sure to address the data and other policy-related requests noted at the end of this email.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)

- a track-changes file indicating any changes that you have made to the manuscript.

*Copyediting*

Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Please do not hesitate to contact me should you have any questions.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD,

Senior Editor,

ialvarez-garcia@plos.org,

PLOS Biology

------------------------------------------------------------------------

ETHICS STATEMENT:

- Please include the full name of the IACUC/ethics committee that reviewed and approved the animal care and use protocol/permit/project license. Please also include an approval number.

------------------------------------------------------------------------

DATA POLICY: IMPORTANT - PLEASE READ

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Fig. 1B, D-L; Fig. 2A, E-L; Fig. 3A-K; Fig. 4B-L; Fig. 5A, B, D-H; Fig. 6A-F; Fig. S1B, C, D, E; Fig. S2A-H; Fig. S3B-E; Fig. S4; Fig. S5C-J; Fig. S6B-I; Fig. S7A, B, E-H; Fig. S8A, B, D-G

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on WHERE THE UNDERLYING DATA CAN BE FOUND, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

Thanks for depositing the sequencing data for epigenetic analyses in SRA - please make sure the data is made publicly available before the manuscript is accepted for production.

For figures containing flow cytometry data, we ask that you provide FCS files and a picture showing the successive plots and gates that were applied to the FCS files.

----------------------------------------------------------

Reviewers’ comments

Rev. 1:

None.

Rev. 2:

The authors have made a good effort to address the concerns with new data. The manuscript is now much better suited for publication.

Dear Dr Hilfiker-Kleiner,

On behalf of my colleagues and the Academic Editor, Cecilia W Lo, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology.

PRODUCTION PROCESS

Before publication you will see the copyedited word document (within 5 business days) and a PDF proof shortly after that. The copyeditor will be in touch shortly before sending you the copyedited Word document. We will make some revisions at copyediting stage to conform to our general style, and for clarification. When you receive this version you should check and revise it very carefully, including figures, tables, references, and supporting information, because corrections at the next stage (proofs) will be strictly limited to (1) errors in author names or affiliations, (2) errors of scientific fact that would cause misunderstandings to readers, and (3) printer's (introduced) errors. Please return the copyedited file within 2 business days in order to ensure timely delivery of the PDF proof.

If you are likely to be away when either this document or the proof is sent, please ensure we have contact information of a second person, as we will need you to respond quickly at each point. Given the disruptions resulting from the ongoing COVID-19 pandemic, there may be delays in the production process. We apologise in advance for any inconvenience caused and will do our best to minimize impact as far as possible.

EARLY VERSION

The version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.

Kind regards,

Erin O'Loughlin

Publishing Editor,

PLOS Biology

on behalf of

Ines Alvarez-Garcia,

Senior Editor

PLOS Biology