PONE-D-23-27842Somatostatin receptors in fibrotic myocardiumPLOS ONE

Dear Dr. Gupta,

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 Nov 30 2023 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: https://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,

Ismaheel Lawal, MD,

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. Thank you for stating the following financial disclosure: 

 [This work was supported, in part, by a National Heart, Lung and Blood Institute of the National Institutes of Health (R01-HL131872) (to G.F). S.S. received funding from the Thoracic Surgery Foundation Resident Research Award ].  

Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. We note that Figure 1, 3, 4, 5, 6, 7, S1 and S2 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

A. You may seek permission from the original copyright holder of Figure 1, 3, 4, 5, 6, 7, S1 and S2 to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an ""Other"" file with your submission. 

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

B. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

[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: Somatostatin receptor is expressed in sarcoid granulomas, and preliminary clinical studies have shown that myocardial sarcoidosis can be identified on somatostatin receptor (SSTR)-targeted PET and 68Ga-DOTATATE PET/CT for diagnosis and response assessment in cardiac sarcoidosis is clinical tools. The present study was aimed to test whether SSTR could be upregulated in cardiac fibrotic scar, and found that using IHC and gene analysis STR1 and SSTR2 are occasionally upregulated in end-stage cardiac fibrotic areas without increased inflammatory cell presence. The manuscript is well written, although parts of introduction and discussion should be more focused and structured, and the patients data are very limited.

Major concerns:

1) In order to compare the relative expression of SSTR and make unbiased comparison, the controls group demographic data have to be presented as well. The SSTR may change due to sex and age, please discuss that.

2) Only 50% of HF patients have previous MI, do they have similar magnitude in changes of SSTR in compared to patients with no previous MI. Do LVEF and SSTR show positive correlation?

3) Case report - its not really fit into the global scope of the MS, the patient had a relative fresh MI, scar/healing process is completed? In addition, inflammation was detected? Similar Suppl Fig 2 is unnecessary to show.

4) It will be ideally and more supportive if the authors would provide 68Ga-DOTATATE PET/CT data; including IM cardiomyopathy patients. The n numbers are extremely low, this is not enough for proper conclusion and interpretations.

5) mRNA expression levels, SSTR show no significant changes between the group, but was a large differene in protein expression levels, please discuss that.

6) Figure 4 - SSTR2 seems to be expressed in cardiomyocytes as well, however the authors stated that "We showed, in a small cohort of ICM patients, that SSTR1 tends to be upregulated at the expression and protein level in end-stage fibrotic myocardium with ongoing cardiac remodeling and stress but without overly active pro-fibrotic remodeling". In order to clarify that the investigators should also apply IHC staining for cardiomyocytes.

Reviewer #2: The authors performed a hard work. I assume that to recruit 8 suitable patients they had to analyze a large number of patients, with a large dropout rate, which is indirectly evidenced by the time interval for the selection of 11 years (from 2010 to 2021).

A unique material was obtained - tissue samples of healthy hearts from donors (coincidentally, turned out to be unclaimed), tissue samples of hearts of patients with terminal CHF who underwent heart transplantation or implantation of left ventricular mechanical support devices.

The study of heart tissue samples from these patients by PCR and determination of somatostatin receptor (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5) mRNA concentrations was performed.

In the introduction and discussion, the authors refer to the latest most significant works in this field, including their previous works on this topic, the authors are well familiar with the research topic and are deeply immersed in the problems of the discussed area. The analysis of the premise of this paper is well done.

But when analyzing the work, I have questions for the IHC block, which need to be clarified if we want to be sure of the accuracy of the results obtained and their correct interpretation.

For a more correct interpretation and to determine the quality of the IHC staining performed and to ensure the reproducibility of the results, the manuscript lacks information about the IHC technique itself, namely the IHC protocol, what buffer solution was used, the imaging system used and the secondary antibodies (avidin-biotin imaging method or other, horseradish peroxidase or alkaline phosphatase, what reagents were used and how long the peroxidase block was performed).

There are no questions to the interpretation of Figures 2-5, in my opinion, the authors have evaluated and described them correctly.

In Figure 7, the presented SSTR1, SSTR2 preparations show intense positive staining of cardiomyocytes (CMC), weak staining of fibrosis areas, and intense staining of individual cells in the fibrosis area (presumably macrophages).

Based on previously published works (5) (https://pubmed.ncbi.nlm.nih.gov/31197742/ ), it was shown that the positive expression of SSTR1 and SSTR2 by activated macrophages in the area of sarcoid granulomas is well detected by IHC, while the rest of the heart tissue, in particular, CMC are negative in staining. Positive staining of macrophages by IHC is expected. Intense staining of SMCs is an unexpected result that requires additional verification and caution in interpreting the results.

Nonspecific staining should be ruled out for a more reliable interpretation of the results.

One possible reason for nonspecific staining of cardiomyocytes is the use of polyclonal antibodies; the presence of antibodies to multiple epitopes may increase the likelihood of cross-reactivity with other proteins. There is less risk of cross-reactivity when monoclonal antibodies are used.

If possible, if paraffin blocks with tissue samples are preserved, it makes sense to make additional slices from the same blocks and stain them with monoclonal antibodies to SSTR1, SSTR2.

Neuroendocrine tumor tissue (NEO) was used as a positive control for IHC. The authors' decision to use NEO as a control is understandable; this tumor can serve as a link between two methods - PET and IHC, if in the same patient this tumor demonstrated 68Ga-DOTATATE accumulation in PET in vivo, and the same tumor after tumor removal and biopsy demonstrates positive staining for somatostatin receptors in IHC.

However, NEO may have angstigenic heterogeneity, in combination with polyclonal antibodies, this may reduce the reliability of the result obtained.

At least for SSTR2, it makes sense to use pancreatic tissue as an additional control (positive - islets of Langerhans, and negative - the rest of the pancreatic tissue).

Another possible reason for nonspecific staining of cardiomyocytes when using the immunoperoxidase staining system is insufficient inhibition of endogenous peroxidases. All samples containing hemoproteins, including myoglobin in muscle cells, hemoglobin in erythrocytes, and cytochrome in granulocytes and monocytes, may show unsuppressed endogenous peroxidase activity. There is experimental evidence that myoglobin concentration in cardiomyocytes may increase during prolonged hypoxia (https://pubmed.ncbi.nlm.nih.gov/19005161/ ; https://pubmed.ncbi.nlm.nih.gov/20675543/ ).

Intense positive staining of cardiomyocytes appeared in patients with severe heart failure, it is known that tissues of such patients suffer from hypoperfusion and experience hypoxia, which, theoretically, can lead to increased expression of myoglobin and increased activity of endogenous peroxidases. To eliminate the influence of this factor in IHC staining, change the peroxidase block or increase the tissue incubation time with it or choose a different tagging enzyme such as alkaline phosphatase.

The above factors may change the answer to the main question: What exactly is the tissue substrate for PET imaging when using somatostatin-directed radiopharmaceuticals (68Ga-DOTATATE)? Previous work suggests that this substrate is very likely to be inflammatory cells, particularly macrophages. This work claims to identify a fundamentally new substrate - directly the SMCs themselves, under certain conditions (terminal CHF). Since this work claims to validate fundamentally new knowledge, this requires additional careful scrutiny to avoid unpleasant mistakes. This is the basis for my recommendations.

I believe that the paper can be considered for publication after the above points have been elaborated or clarified.

Minor issues requiring clarification/correction:

1) If the mean mRNA content of SSTR1 and SSTR2 did not differ between the study group and the control group, how to explain this difference with respect to the IHC pattern, where differences in staining intensity are clearly visible?

2) A clinical case of a patient with a combination of NEO and ischemic cardiopathy is given as background. Unfortunately, I could not understand from the text of the article whether any of the patients in the main group (n=8, with ischemic cardiopathy) was diagnosed with HEO, or whether all patients in the main group were without HEO? I think this point is important; it may be better to spell it out more clearly. If the patients were without NEO, what were the indications for PET with 68Ga-DOTATATE?

3) Numbering of figures is confused - Figure 1 in the text corresponds to Figure 2 in the appendix, Figure 2 in the text corresponds to Figure 3 in the appendix, etc.

**********

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.

Somatostatin receptors in fibrotic myocardium

PONE-D-23-27842R1

Dear Dr. Gupta,

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. If you have any questions relating to publication charges, 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,

Ismaheel Lawal, MD,

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: