PONE-D-21-15802Characterization of intracellular calcium mobilization induced by remimazolam, a newly approved intravenous anestheticPLOS ONE

Dear Dr. Miyoshi,

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.

In Your revision please pay special attention to the criticism formulated by Reviewer#1 regarding the detection of intracellular calcium concentration changes using a non-ratiometric dye. Please re-evaluate the data based on those comments, especially how the fluorescent values are calculated (background correction, use of F/F0, etc.).

Please submit your revised manuscript by Oct 26 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Laszlo Csernoch, Ph.D.

Academic Editor

PLOS ONE

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2. Please provide additional information about the source of the HUVEC cells. If the cells are a named cell line, please provide the name. If they were primary cells derived from clinical samples, please state whether the cells were isolated for the purposes of research and whether any identifying information was provided with them.

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7. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

-https://www.sciencedirect.com/science/article/abs/pii/S0014299920303952?via%3Dihub

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: No

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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: No

Reviewer #2: No

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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: This manuscript aimed to examine the source of increased Ca2+ in response to exposure to the drug remimazolam. The authors have done a lot of work and have endeavoured to pharmacologically block multiple cellular pathways that could account for the Ca2+ release observed. Ultimately, the authors suggest that the increased Ca2+ is likely mediated via a GPCR-IP3 pathway, although more work needs to be done to establish the specific pathway.

Major Points:

• What is the rational for the choices of cell lines used? One of the cell lines reported, but no data was shown, is from a monkey kidney line.

• The introduction is quite brief with minimal references provided throughout the submission.

• A high proportion of supportive data is not shown as figures. For example, it is reported as data not shown but how did the authors designate a “normal morphology” of the ER in the SHSY-5Y and HUVEC cells? Images would help substantiate this statement.

• What is the equivalent clinical dose for remimazolam? The authors state that the doses they use are higher than those used in clinical situations but seem to only get cellular effects at with high doses. How do these experimental doses correspond to the maximum dosage a patient would receive?

• A non-ratiometric, AM loading, Ca2+ indicating dye has been used. What is the justification for the ratio that was created for the Ca2+ trace analysis? Convention is to subtract the background intensity and then convert to F/F0 to take into account the differences in dye loading between individual cells and different cell lines. The summarised percentage change in fluorescence may not reflect a large change in Ca2+ concentration due to the use of a non-ratiometric dye. It is also not appropriate to apply statistics to percentage data.

• 30 mins after the TG treatment an agonist should be applied to show that there is no residual Ca2+ in the stores to further support the assumption that remimazolam is not causing a release of ER Ca2+.

• The authors state that Ca2+ release due to remimazolam is likely due to IP3 receptor activation. It would be beneficial to apply a cocktail of the inhibitors used in this study (minus the IP3 inhibitor) and see if the same increased Ca2+ response is seen with treatment with remimazolam.

• In Figure2 and 3, can the authors comment on why the same trends of Ca2+ release were not observed in the HUVEC cells but are conserved in the SHSY-5Y?

• Figure 8 A&B the data suggests that dantrolene was not providing a complete block of the RyR. Tetracaine would be a more efficient inhibitor.

Minor Points:

• Histogram formats should be changed to include individual data points.

• Figure 1, states “the rate” of calcium, is this a typo that should state ratio as the authors have not measured the rate of Ca2+ release/increase?

• Figure 4, it appears the data panel of HUVECS with 500uM remimazolam is absent, was the data unable to be gathered under these conditions?

Reviewer #2: Urabe et al. provide important information about the effect of remimazolam, a newly approved intravenous anesthetic. This work presents an empirical characterization of intracellular calcium alterations initiated by this new drug. The authors present results on calcium signaling using different mammalian cell lines such as neuronal-, fibroblast-like cells and human umbilical endothelial cells. Using calcium imaging, ionic substitution, calcium buffering, and pharmacological tools, the authors identified that supratherapeutic doses of remimazolam initiated intracellular calcium elevations from the ER. These Ca signals were GPCR and IP3 dependent when assessed in several cell types. The work is well-designed and presents empirical evidence of an interesting biological event that has a therapeutic interest.

For this reviewer, the work is novel and original, necessary for the general understanding of the additional (side) effects of remimazolam. The experiments are, in general, well-conducted and the paper is well written and easy to follow; however, as far as this reviewer concerns, some issues require attention.

*Specific Comments

Abstract:

Briefly mention that remimazolam causes an elevation in intracellular calcium and why this may be of interest.

Introduction:

Line 75. Propose a mechanism by which propofol (or remimazolam) could cause hypotension or vascular pain. This mechanism could be used to justify the study of calcium mobilization and the use of neuronal-like cells and endothelial cells.

Methods:

Lines 126-130. For reproducibility purposes, please provide more details about the apparatus and software used to measure calcium signals. Was a photodiode, PMT, or camera used (include manufacturer and model)? Also, add details about excitation light source, excitation, and emission filters, and objective used. What was the sampling rate?

Statistical: Please clarify whether the normality test was assessed and how. When reporting results (significant or non-significant), please give the exact p-value, adjusted significance level (when appropriate), and the test used.

Results:

Lines 194-204. “Observation of remimazolam…” Delete this section or show the results, a negative results still a result. Data could be show as supplementary information.

To further characterize the involvement of GPCR, the authors could use GDPbetaS, to prevent non-specific activation of GPCR. If remimazolam effect depend on GPCR activation GDPbetaS should prevent its effects.

Discussion:

Lines 262. “ a newly marketed” delete this, it has been already mention few lines above.

The authors wrote: “Remimazolam concentrations used in this study were considerably higher than those used clinically.”

You could add something like: However, concentrations like the ones used in this study or higher could occur locally near the injection site.

It will be worth mentioning somewhere in the discussion that while remimazolam does not seems to affect RyR1 directly, the IP3-dependent elevation in Ca2+ mediated by remimazloam could play a role in sensitizing RyR1 in an individual with HM-susceptibility.

Figure 1:

Traces in panel B appear identical to those previously published by the authors (see Ref #6 Urabe T et al. 2020, Fig. 1B). This issue is very problematic and needs to be fixed or request permission to reuse already published data. Also, in the same panel, how comes that the background signal is not flat. Would you please indicate the region of interest used to measure both the cell and background on the image?

Figures 2, 3, 4, 6-8.

Use box plot with data overlap instead of plunger bars (show the data).

Figures 2 and 3 add the number of observations to boxes as in figures 4,6-8.

Minor Comments:

Figure 2-8. Adding labels to the figure panels (i.e., Control solution for Fig 2, Zero Ca2+ for figure 3) may help the reader quickly identify the results.

Figure 3A, remove data for DMSO 0.05-0.3 as in panel B.

Figure 4. Increase Font size for BAPTA-AM and symbols

Figures 6-8. Increase the font size for x- and y-axis labels

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-15802R1Characterization of intracellular calcium mobilization induced by remimazolam, a newly approved intravenous anestheticPLOS ONE

Dear Dr. Miyoshi,

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. In line with the the comments of Reviewer#1, please redraw all figures where necessary using box plots.

Please submit your revised manuscript by Feb 10 2022 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,

Laszlo Csernoch, Ph.D.

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.

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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

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: The authors have done much to address reviewer comments and improve the manuscript.

However, Figures 1 and 2 are still displayed as summary histograms and have not been altered to show the spread of the individual data points. While the subsequent use of box plots is a positive adjustment, as the data is stated in the figure legends as mean and SEM this is still are not showing the spread of the data. The box plots could be altered to include the individual data points or to show mean with SD.

Reviewer #2: The authors have addressed all my queries, I have no further comments. I find the authors' responses and the revised manuscript satisfactory and I congratulate the authors for the work done.

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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: Erick O. Hernández-Ochoa

[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.

Characterization of intracellular calcium mobilization induced by remimazolam, a newly approved intravenous anesthetic

PONE-D-21-15802R2

Dear Dr. Miyoshi,

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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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,

Laszlo Csernoch, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: