Dear Dr. Alvarez-Lacalle,

Thank you very much for submitting your manuscript "Buffering and total calcium levels determine the presence of oscillatory regimes in cardiac cells" for consideration at PLOS Computational Biology.

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Sincerely,

Andrew D. McCulloch, Ph.D.

Associate Editor

PLOS Computational Biology

Daniel Beard

Deputy Editor

PLOS Computational Biology

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The manuscript by Marchena et al. explores mechanisms underlying calcium oscillations in cardiac cells using mathematical models. They use a detailed model and a minimal model and show how buffering and total calcium levels affect calcium oscillations. The manuscript is well written and the findings are interesting. I have a few questions and suggestions.

*In the minimal model, Po only depends on cd (dyadic calcium). Doesn’t luminal Ca regulation of RyRs play a role in oscillations?

*Some buffers are fast, and others are slow. How do affinities, kinetics, and concentrations of the different buffers affect your results. Does mobility affect your results?

*Pg 3: “distributed over the cell along the z-lines with a Gaussian distribution in both transversal and longitudinal axes.” Can you elaborate this sentence? What are the mean and the standard deviation? Is there any experimental evidence?

*Pg7: “Waves are normally initiated at different sites each time” This implies that the cell size is also important (i.e. larger cells have more waves). On the other hand, the minimal model is a model of a single CaRU. Can you justify the use of the minimal model to analyze the full subcellular model?

*Fig 5: The oscillation period is too short for cardiac cells.

*Pg15: “we consider that the inactivated states of the RyRs do not play a role in the mechanisms that produce oscillations”. This assumption needs a reference.

*Pg16: “The parameters of these equations are taken from the literature where, except for those of the RyR, are well documented.” References are missing.

*It would be helpful if authors could write all the equations and parameters in the appendix so that readers do not have to read multiple papers to reproduce models and results.

*In my opinion, “We find, for instance” in the abstract is not appropriate. We want to know all the novel findings in the paper. Please enumerate all the key findings in the abstract.

*CSUN is located in Northridge (not in Los Angeles).

Reviewer #2: This manuscript is well written and in principle is a nicely designed sequence of model reduction for describing the dynamics underlying calcium instabilities in cardiac myocytes. However, I have considerable concern that the novelty of the findings are not sufficient to warrant publication in PLoS Comp Biol.

Major comments: The authors have provided a coherent analysis of 2 models, which are both reductions of their recently published model of subcellular calcium signaling in cardiac myocytes. In general I support their efforts to condense the complexity of the complete model to its essential dynamics. Previously this has been done to great effect for understanding EAD dynamics, and several times prior (sometimes in very similar fashion) for defining the stability of the calcium resting state in cardiomyocytes (see for example Tveito et al. 2012, Nivala et al. 2012). While I support the objectives in this way, I do not see the novelty in the current study to support publication at this time. In particular, the role of reduced SR calcium buffering in promoting instability of the diastolic resting state has been fully established experimentally and clinically as the second (and most severe) form of catecholaminergic polymorphic ventricular tachycardia (CPVT2). Similarly, the role of increased whole-cell calcium load in this form of instability has been well known for several decades, and specific critical SR calcium thresholds have been measured in a number of species (see many studies by Eisner’s group). In sum, my major comment here is that, while there has been great importance in prior studies that have used reduced models for describing the essential dynamics of instability e.g EADs, the real value of those analyses was permitted because there was an important disagreement between experimental observation, and the reigning concepts of the time. In the case of EADs this was the observation that APD could be prolonged tremendously without observing the transition to instability. I do not see any additional level of insight generated by the current study, and while it need not be groundbreaking, I think that some new biological insight is necessary to justify consideration for publication in PLoS Comp Biol.

Specific comments:

1. Was there some matter of simplicity that made it easier to base most of the nullcline analyses around the assumption of rapid gating kinetics i.e. dO_Ryr/dt = 0, rather than the assumption of rapid diffusion as is assumed to assess model robustness? I ask, because this assumption (quasi-equilibrium gating) is similar to that used to construct an Eigen value analysis for describing resting calcium stability (Tveito et al. 2012). Conversely, it has generally been assumed (for a range of model reduction exercises that remain faithful for CICR), that diffusion from the cleft is approximately an order of magnitude more rapid than the gating events themselves (see papers from Hinch in 2004).

2. With respect to the above point. The dynamics shown in Fig 5 are somewhat surprising from an intuitive perspective. This is because the SR [Ca] reached under stable conditions (C(bar)_T = 32 uM) is so much larger than for the other conditions (even though there should be no fundamental differences in the RyR properties, or in the relationship between free and total SR [Ca]). This makes me skeptical that the assumption of rapid gating is reasonable for describing the determinants of stability in this model. Can the authors explain to me what I am missing here?

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

Figure 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. 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 us at figures@plos.org.

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Dear Dr. Alvarez-Lacalle,

Thank you very much for submitting your manuscript "Buffering and total calcium levels determine the presence of oscillatory regimes in cardiac cells" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the one remaining review recommendation from reviewer 2.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

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

Sincerely,

Andrew D. McCulloch, Ph.D.

Associate Editor

PLOS Computational Biology

Daniel Beard

Deputy Editor

PLOS Computational Biology

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

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: All of my comments have been addressed appropriately. I have no additional comments.

Reviewer #2: This revision has appropriately dealt with my comments and you have provided detailed responses and nicely reframed the paper.

I request one modification. My introductory comments (related to the value of reduced models in understanding the fundamental mechanisms of EADs) were simply to make a point about relating the core model behaviors to the missing information in the field. I suspect this may have driven you to include the section in the Discussion related to the involvement of these oscillatory behaviors on EADs. While I agree that the role of calcium cycling instability in EADs is clear, albeit in particular contexts, the involvement of the specific buffering-RyR gating regime that you have investigated here is not something that should be extended to that behavior without a full cell model incorporating detailed, and coupled, membrane dynamics. This would require a substantial investment that is beyond the scope of this paper. I recommend either removing the section relating this behavior to EADs, or to reduce its emphasis and make it very clear that such an involvement is highly speculative without verification in a comprehensive model of myocyte calcium and electrophysiology.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: No: There is no note of an available code repository for this model or simulations

**********

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: Andrew G Edwards

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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 http://journals.plos.org/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Alvarez-Lacalle,

We are pleased to inform you that your manuscript 'Buffering and total calcium levels determine the presence of oscillatory regimes in cardiac cells' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Andrew D. McCulloch, Ph.D.

Associate Editor

PLOS Computational Biology

Daniel Beard

Deputy Editor

PLOS Computational Biology

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