PONE-D-24-21187Hydrogen Decreases Susceptibility to Ang II-induced Atrial Fibrillation and Atrial Fibrosis via the NOX4/ROS/NLRP3 and TGF-β1/Smad2/3 Signaling PathwaysPLOS ONE

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Reviewers' comments:

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Comments to the Author

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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: Zhang et al. studied the role of hydrogen in Ang II-induced Atrial Fibrillation and Atrial

Fibrosis and showed that the NOX4/ROS/NLRP3 and TGF-β1/Smad2/3 Signaling Pathways are involved in the pathogenesis. Although they provided strong evidence for hydrogen’s role in improving atrial fibrillation and fibrosis, the data must be improved. I have the following comments and suggestions.

Line 55, please provide the references.

Figure text is not clean; please improve them.

The images in Figure 2 G are unclear, making it difficult to identify any cells. Please enhance the image quality to make the cells more visible.

In Figure 4, quantification is not visible.

Figures 5A and B are unclear; please provide better images where cardiac tissue is recognizable.

All the images in Figure 6 must be improved. The current images are not clear.

Same with Figure 7 D.

Reviewer #2: The study “Hydrogen Decreases Susceptibility to Ang II-induced Atrial Fibrillation and Atrial Fibrosis via the NOX4/ROS/NLRP3 and TGF-β1/Smad2/3 Signaling Pathways” by Zhang et al. investigated the ability of hydrogen in prevention of Atrial Fibrillation and atrial Fibrosis. The study used selective administration of hydrogen and angiotensin II in rats and cell cultures, and employed echocardiography, electrophysiological studies, and biochemical assays to collect pertinent data. Their findings showed that hydrogen reduces susceptibility to Angiotensin II (Ang II)-mediated AF and atrial fibrosis in rats. Rats treated with Ang II and hydrogen showed decreased AF susceptibility, reduced atrial fibrosis, lower reactive oxygen species levels, inhibited NLRP3 inflammasome activation, improved electrical remodeling, and reduced proliferation and migration of cardiac fibroblasts. Hydrogen also enhanced Ca2+ handling and ion channel function by inhibiting NOX4 activity. Furthermore, hydrogen inhibited Ang II-induced atrial fibrosis through the TGF-β1/Smad2/3 pathway. The findings suggest that hydrogen may be an effective treatment for preventing and managing Ang II-mediated AF and atrial fibrosis.

This is a good study, however, to improve its readability and credibility, I suggest the following;

Minor comments

1. The authors should ensure consistent use of abbreviations and terminology, for instance, "AngII" should be consistently written as "Ang II" or "AngII" throughout the manuscript.

2. The authors should work on grammatical errors, and ensure that all abbreviations are in place to improve readability

Major comments

1. The authors should consider specifying the concentrations and passage number of cells used in experiments to ensure consistency and reproducibility of their work since every study is a foundation for the next, clarity is paramount. The following article could offer guidance https://www.ahajournals.org/doi/full/10.1161/01.HYP.27.4.897

2. The protocols for intracellular ROS measurement, intracellular Ca2+ content detection, fibroblast migration, and proliferation assays lack sufficient detail. For reproducibility, the authors should consider including specific details such as concentrations, incubation times, and steps for washing and preparation.

3. The authors should specify the preparation of samples, dilutions used, and steps taken to ensure accuracy and consistency in the ELISA assay. Also, clarify how the GEN5 CHS 3.03 program software was used in the analysis of their findings.

4. The description of the qRT-PCR procedure should include details on the thermal cycling conditions.

5. The statement by the authors that animal care and use protocols were followed is appropriate, however, should consider emphasizing ethical considerations and how animal welfare was maintained throughout the study.

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Reviewer #1: No

Reviewer #2: No

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Hydrogen Decreases Susceptibility to Ang II-induced Atrial Fibrillation and Atrial Fibrosis via the NOX4/ROS/NLRP3 and TGF-β1/Smad2/3 Signaling Pathways

PONE-D-24-21187R1

Dear Dr. Yang,

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.

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Kind regards,

Sepiso K. Masenga, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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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

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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: Zhang et all have addressing my all comments. I do not have any further comments. Thank you. Good luck.

Reviewer #2: (No Response)

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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

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