Dapagliflozin mitigates myocardial inflammation and metabolic stress in heart failure through STAT1 inhibition: Evidence from multi-omics analyses and experimental exploration

Lianqi He; Di Zhang; Shiwen Zhang; Zhichao Ren; Yihong Huangfu; Xiran Cheng; Zhanchun Song

doi:10.1371/journal.pone.0343296

Peer Review History

Original SubmissionOctober 11, 2025
11 Oct 2025 Author Response Attachments Attachment Submitted filename: Response to Reviewers(1).docx https://doi.org/10.1371/journal.pone.0343296.r001
26 Nov 2025 Decision Letter - Jian Wu, Editor Dear Dr. Song, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jan 10 2026 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. 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'. 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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? Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: In the present study authors performed comptehensive bioinformatic and experimental analysis of the role of STAT1 in the heart failure and the effect of dapagliflozin. The results show that heart failure induced by coronary artery ligation is associated with the up-regulation of myocardial STAT1 whereas dapagliflozin reduced its expression. Dapagliflozin reduced the abundance of circulating M1 and increased M2 macrophages in rats with heart failure. DAPA lso reduced the level of inflammatory cytokines and total bile acids. In vitro, STAT1 overexpression reduced viability of H9C2 cardiomyocytes; the effect suppressed by dapagliflozin. The topic and the results are of interest, however, there are also some important concerns to be addressed. 1. Only one SGLT inhibitor was used in the experiments. Could the results be extrapolated to the whole class of drugs or apply only do dapagliflozin? 2. The method of LAD ligation including anesthesia and recovery should be described in more details. 3. Sex of the animals used in the experiments should be specified. If only one sex was used, it should be discussed whether the results could be extrapolated to the other one. 4. The vehicle used to dissolve dapagliflozin should be specified. Were animals not treated with dapagliflozin receiving any vehicle? 5. Lines 194/195, the method of animal anesthesia and sacrifice for final tissue sampling should be described. 6. Section 2.8, which part of the heart was used for the analysis (infarcion area or non-infarcted area?) 7. The names, catalogue numbers and dilution rates of primary and secondary antibodies used for Western blotting should be specified. 8. Section 2.9, limits of quantification as well as intra- and inter-assay CV values for ELISA kits should be presented. 9. The principle of the assay of bile acid concentration should be described. 10. Line 262, what does it mean that each assay was performed at least three times? Were, for example, cytokines measured in three samples from each animals? 11. Statistical analysis, was normality of data distribution verified to justify using parametric tests? If so, what method was used? 12. Line 325, that STAT1 is the top contributor for distinguishing HF from control samples indicates only that its expression changes to the greater extent but not that it is the hub molecule in HF pathogenesis. 13. Line 424, where is the evidence that DAPA reduced fibrosis? How do you conclude about correction of metabolic abnormalities? 14. Was the effect of STAT1 overexpression on H9C2 cells examined at physiological conditions or under oxygen deprivation? 15. What is the evidence that STAT1 was involved in the effect of DAPA? Could down-regulation of STAT1 be the consequence rather than the underlying mechanism of its protective effect? Reviewer #2: I understand that this is the second version of the manuscript. I did not review the first version therefore my comments are based solely on the second version. The following points need to be addressed: 1) In the submission form, the authors inserted "N/A" in Ethics Statement section. Since they have undertaken animal studies, this section needs to be completed accordingly. 2) In the same form in Data availability section and the authors stated "Yes - all data are fully available without restriction" and "All relevant data are within the manuscript and its Supporting Information files" which are not entirely correct. If possible the authors should state "all raw data are available from the corresponding author upon request". If that is not possible, it should also be stated. This needs to be mentioned also in the manuscript. 3) Lines 55-57 is a very poor description of HF, please rephrase. 4) Lines 127-140: Human samples are mentioned here. Is this from available data set or did the authors analyse human cells? Please clarify. 5) Lines 341-368: "single-cell transcriptomic analysis of myocardial tissues" was this done in available data set or did the authors gather this data from tissues they had? Please clarify. 6) Lines 370-386: "we calculated enrichment scores for single-cell data" which dataset was this? Please clarify. 7) Line 409: "extensive fibrotic lesion" Figure 10A. No fibrotic lesion can be seen in this figure. It would be very difficult to assess fibrosis only with H&E staining. ECM staining should have been done. Also 4 days of HF would not cause much fibrosis formation. Since no specific markers of fibrosis were investigated and the duration of animal model was too short for fibrosis, in this reviewer's opinion the authors are making claims which are not justified by their results. I would strongly advise to remove any claims of fibrosis throughout the manuscript. 8) Lines 412-415: The WB is showing that DAPA prevents HF-induced increase in STAT1 expression. It is not showing that DAPA reduced STAT1 activation. A change in protein expression does not necessarily mean a change in activity of that protein. Please rephrase. 9) Line 431: The authors claim that STAT1 emerged as a key target for DAPA. They have no results to justify this conclusion. Changes in STAT1 expression by DAPA does not mean that STAT1 is a key target for DAPA. 10) Lines 438-439: "These in vitro findings reinforce the in vivo results, demonstrating that DAPA protects cardiomyocytes by inhibiting STAT1 activation". Again the authors do not have any data on STAT1 activity. Expression is not activity. 11) There are several lines in the discussion making claims about activity. The discission needs to be rewritten accordingly. ****** 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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0343296.r002
Revision 1
15 Dec 2025 Author Response Journal Requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We have carefully reviewed and ensured that our revised manuscript fully complies with PLOS ONE's style requirements. 2. To comply with PLOS One submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. We thank for this important reminder. We have now expanded our Methods section to include comprehensive details on animal welfare and experimental procedures, like: "Specifically, rats were anesthetized with isoflurane and fixed in the supine position after hair removal. Electrocardiogram (ECG) was continuously monitored throughout the procedure. After sterile preparation, a thoracotomy was performed along the left sternal border at the 3rd–4th intercostal space using tissue scissors to expose the heart. The left atrial appendage was gently lifted with sterile toothed forceps to expose the aortic root. A surgical suture was passed around the proximal segment of the left anterior descending coronary artery near the superior margin of the heart, and three secure knots were tied to achieve complete ligation. The thoracic wall was then rapidly closed with sutures. Control rats underwent the same thoracotomy procedure without LAD ligation (sham operation). Post-operatively, all rats were placed in a warm recovery chamber and monitored until full consciousness was regained. Analgesics and antibiotics were administered as needed to minimize discomfort and prevent infection..." 3. Please note that PLOS One has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. 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We have amended our financial disclosure statement as requested. The complete and correct statement is: "This study was supported by the talent Program of Fushun City of China (No. FSYC202107012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." We have updated this statement in our cover letter accordingly. 6. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). 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This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. We confirm that the corresponding author's ORCID iD has been validated in the Editorial Manager system as required. The ORCID iD is now properly linked and authenticated in our submission. 8. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. We thank the editor for this important reminder regarding blot/gel reporting requirements. We have now prepared and uploaded the original, uncropped, and minimally adjusted images for all Western blot results reported in our manuscript. All original blot images are now available in the Supporting Information file S1_raw_images.pdf. And we have added the description to the cover letter. 9. We notice that your supplementary figures are uploaded with the file type 'Figure'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. We have now amended the file type of all supplementary figures from 'Figure' to 'Supporting Information' in the submission system. Additionally, we have ensured that each Supporting Information file has a corresponding legend listed in the manuscript after the references list, as required by PLOS ONE guidelines. 10. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. We have carefully reviewed all reviewer comments and confirm that no specific previously published works were recommended for citation by the reviewers. Therefore, no additional citations based on reviewer recommendations have been added to the revised manuscript. Reviewer #1: In the present study authors performed comptehensive bioinformatic and experimental analysis of the role of STAT1 in the heart failure and the effect of dapagliflozin. The results show that heart failure induced by coronary artery ligation is associated with the up-regulation of myocardial STAT1 whereas dapagliflozin reduced its expression. Dapagliflozin reduced the abundance of circulating M1 and increased M2 macrophages in rats with heart failure. DAPA lso reduced the level of inflammatory cytokines and total bile acids. In vitro, STAT1 overexpression reduced viability of H9C2 cardiomyocytes; the effect suppressed by dapagliflozin. The topic and the results are of interest, however, there are also some important concerns to be addressed. Dear Reviewer #1, We sincerely thank you for your thorough and insightful review of our manuscript. Your comprehensive evaluation and constructive suggestions have substantially improved the quality and rigor of our work. We are particularly grateful for your attention to methodological details, statistical considerations, and interpretative nuances that we had not adequately addressed in the original submission. Your thoughtful comments regarding the generalizability of our findings to the SGLT2 inhibitor class, the importance of sex differences in heart failure research, and the distinction between biomarker identification and mechanistic validation have prompted us to think more critically about the scope and limitations of our study. These insights have not only strengthened our current manuscript but have also deepened our understanding of the research questions and guided our thinking about future experimental directions. We have carefully addressed each of your comments point-by-point and made corresponding revisions throughout the manuscript. The detailed responses to your specific comments are provided below. We believe these revisions have significantly enhanced the scientific rigor, transparency, and interpretative accuracy of our work. Once again, we greatly appreciate the time and expertise you devoted to reviewing our manuscript. Your rigorous evaluation has been invaluable in refining our research. 1. Only one SGLT inhibitor was used in the experiments. Could the results be extrapolated to the whole class of drugs or apply only do dapagliflozin? Thank you for raising this insightful question regarding the generalizability of our findings. We agree that since only dapagliflozin (DAPA) was used in our experimental setup, the direct conclusion that DAPA mitigates heart failure through STAT1 inhibition strictly applies to this specific agent. However, based on extensive clinical evidence and proposed mechanistic overlap, we believe there is a strong rationale for a potential class effect concerning this pathway. The SGLT2 inhibitor class (including DAPA and Empagliflozin) has consistently demonstrated a class effect in major clinical trials (such as DAPA-HF and EMPEROR trials), significantly reducing heart failure hospitalization and cardiovascular death across different patient populations, irrespective of diabetes status. This suggests that the cardioprotective benefits are driven by mechanisms common to the class. Given that STAT1 is a crucial mediator of inflammation and metabolic stress—both universal pathological features of heart failure targeted by SGLT2 inhibitors—we hypothesize that STAT1 suppression represents a shared, pleiotropic, downstream target for the entire SGLT2 inhibitor class. Nonetheless, we acknowledge this as a limitation of our current study, and we have added a statement to the Discussion section to address this point and suggest future comparative studies to definitively confirm the proposed class effect: “The robust evidence of a class effect for SGLT2 inhibitors in reducing HF hospitalization and cardiovascular mortality[37, 38] suggests that the fundamental mechanisms, such as STAT1 inhibition and subsequent immune/metabolic modulation, may be shared across the class. Future studies directly comparing the effects of different SGLT2 inhibitors (e.g., Dapagliflozin vs. Empagliflozin) on the STAT1 pathway are warranted to definitively confirm this proposed class effect.” 2. The method of LAD ligation including anesthesia and recovery should be described in more details. We thank the reviewer for this important suggestion. We have now expanded the Methods section to provide more detailed descriptions of the LAD ligation procedure, including anesthesia protocols and post-operative recovery procedures: "Myocardial infarction–induced heart failure (HF) was created in the experimental group by ligation of the left anterior descending (LAD) coronary artery. Specifically, rats were anesthetized with isoflurane and fixed in the supine position after hair removal. Electrocardiogram (ECG) was continuously monitored throughout the procedure. After sterile preparation, a thoracotomy was performed along the left sternal border at the 3rd–4th intercostal space using tissue scissors to expose the heart. The left atrial appendage was gently lifted with sterile toothed forceps to expose the aortic root. A surgical suture was passed around the proximal segment of the left anterior descending coronary artery near the superior margin of the heart, and three secure knots were tied to achieve complete ligation. The thoracic wall was then rapidly closed with sutures. Control rats underwent the same thoracotomy procedure without LAD ligation (sham operation). Post-operatively, all rats were placed in a warm recovery chamber and monitored until full consciousness was regained. Analgesics and antibiotics were administered as needed to minimize discomfort and prevent infection." 3. Sex of the animals used in the experiments sho Attachments Attachment Submitted filename: Response to Reviewers V2.docx https://doi.org/10.1371/journal.pone.0343296.r003
5 Feb 2026 Decision Letter - Jian Wu, Editor Dapagliflozin mitigates myocardial inflammation and metabolic stress in heart failure through STAT1 inhibition: evidence from multi-omics analyses and experimental exploration PONE-D-25-51193R1 Dear Dr. Song, 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. For questions related to billing, please contact billing support . 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, Jian Wu, M.D, Ph.D Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed Reviewer #3: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No ****** Reviewer #1: The manuscript has been revised according to the reviewers' comments. All concerns raised by the reviewers have been adequately addressed by the authors. Reviewer #2: no further comments, all issues have been addressed xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Reviewer #3: Although the manuscript addresses a potentially relevant topic in papillary thyroid carcinoma, the overall quality of data presentation and methodological rigor is insufficient. Key figures are low-resolution and heavily cropped, uncropped original blots and raw data are not provided, and the Data Availability statement does not comply with PLOS ONE policies. In addition, experimental design and statistical reporting lack essential details, and the mechanistic conclusions are overstated relative to the supporting evidence. Addressing these issues would require substantial reanalysis and additional work beyond a reasonable revision; therefore, I recommend rejection. ****** 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 Reviewer #3: No ******** https://doi.org/10.1371/journal.pone.0343296.r004
Formally Accepted
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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 .