Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart

Manuela Santalla; Alejandra García; Alicia Mattiazzi; Carlos A. Valverde; Ronja Schiemann; Achim Paululat; Greco Hernández; Heiko Meyer; Paola Ferrero

doi:10.1371/journal.pone.0267156

Peer Review History

Original SubmissionSeptember 10, 2021
14 Oct 2021 Decision Letter - Thomas Preiss, Editor PONE-D-21-29388Interplay between SERCA, 4E-BP and eIF4-E in the Drosophila heartPLOS ONE Dear Dr. Ferrero, 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 carefully consider all comments by the reviewer and I would expect you to address each of the suggestions for improvement in your revision. Please submit your revised manuscript by Nov 28 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. 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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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 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 ****** 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 ****** 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: Santalla et all showed that there is a non-canonical role of the eukaryotic translation factor 4E-BP in modulating the heart function through SERCA. They showed that an overexpression of 4E-BP augmented calcium uptake and release in the freshly dissected fly heart. Authors attributed this effect to the activation of SERCA and the direct interaction of SERCA to eIF-4E proteins. Experiments are coherently presented and overall support their findings. Since the interaction of SERCA and eIF4Es was not expected, more supporting evidence is needed to establish the functional/physical connection. I recommend adding either or both of the following experiments: 1. A further dissection of the SERCA domain by Y2H to narrow down the interaction domain would validate the specific interaction between SERCA and eIF4Es. In the presented data, eIF4E-4 gave all positive results. Therefore, a simple technical error cannot be excluded. 2. Testing the effect of depleting eIF4E-4 on the heart contraction/relaxation to see if it has the same effect as 4E-BP overexpression Minor comments: 1. Please state that different "isoforms" of eIF4E are encoded by different genes. "Isoforms" imply transcripts coming from a single gene. 2. Figure panels benefit from having more captions and labels. For example, labelling "contraction" and "relaxation" on panels C and D in Figure 1 would help. 3. Please describe how exactly the parameters of calcium dynamics were calculated, for example, by showing examples of the raw data (including caffeine experiment). I understand what each parameter captures conceptually. But, windows and thresholds taken for calculations are also important to evaluate the robustness of the experiments. 4. In Figure 1, the relaxation decay constant tau did not change much upon over expression of 4E-BP in 7-day old flies while SERCA activity increased. Does this mean that the activity of Na/Ca exchanger decreased upon overexpression of 4E-BP? Is this likely? 5. Based on the tissue expression atlas available from the FlyBase, eIF4E-1/2 gene is most abundantly expressed among eIF4E genes in most tissues except that the testes abundantly express eIF4E-3 and eIF4E-4 genes. Therefore, isn't it possible that eIF4E-1/2 is more relevant as a potential effector of 4E-BP overexpression despite less strongly interacting with SERCA in Y2H? Did you find peptides of eIF4E-1/2 in the mass-spec data? 6. At least part of the effects of 4E-BP overexpression could be due to changes in translation, indirectly affecting the heart function. Could you comment on this possibility? ****** 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: Yes:** Rippei Hayashi [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". 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Revision 1
17 Mar 2022 Author Response Following, we add the response to the requirements and questions: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The file has been modified according to the style templates. 2. We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: Funding information has been removed from the manuscript. 3. 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. We add the raw gel image in Supporting Information file (S2 fig). 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. The data are not a core part of the research in this study therefore the phrase has been removed. 5. 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. References were revised and there are not retracted articles. 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 ________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ________________________________________ 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 ________________________________________ 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 ________________________________________ 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: Santalla et all showed that there is a non-canonical role of the eukaryotic translation factor 4E-BP in modulating the heart function through SERCA. They showed that an overexpression of 4E-BP augmented calcium uptake and release in the freshly dissected fly heart. Authors attributed this effect to the activation of SERCA and the direct interaction of SERCA to eIF-4E proteins. Experiments are coherently presented and overall support their findings. Since the interaction of SERCA and eIF4Es was not expected, more supporting evidence is needed to establish the functional/physical connection. I recommend adding either or both of the following experiments: 1. A further dissection of the SERCA domain by Y2H to narrow down the interaction domain would validate the specific interaction between SERCA and eIF4Es. In the presented data, eIF4E-4 gave all positive results. Therefore, a simple technical error cannot be excluded. 2. Testing the effect of depleting eIF4E-4 on the heart contraction/relaxation to see if it has the same effect as 4E-BP overexpression We proceed to carry out the experiment suggested by the reviewer on point 1. A new figure 4 is added to the manuscript. This figure includes the analysis of two shorter fragments of SERCA, namely amino acids 1-250 and 200-250. As we detailed in Results Section in the new version of this manuscript, the SERCA constructs 1-250 and 51-250, but not construct 200-250, interacted with eIF4E-4. This result suggests that the SERCA eIF4E-binding motif is located within region encompassing amino acids 1-200. Please, see the new figure 4. Pages 13 and 14 contains the rewritten results highlighted (lines 364 to 373). Figure legend was modified according to the annexed experiment (lines 375-383). In the section method, page 6 shows the description of the experiment. Minor comments: 1. Please state that different "isoforms" of eIF4E are encoded by different genes. "Isoforms" imply transcripts coming from a single gene. All sentences were revised and reformulated according to the reviewer suggestion. We conserved the term isoform for referring to eIF4E-1 and 2 with results from alternative splicing. 2. Figure panels benefit from having more captions and labels. For example, labelling "contraction" and "relaxation" on panels C and D in Figure 1 would help. We greatly thank the reviewer for the suggestion. Labeling has been improved in panels C, D and E in Figures 1, 2 and 3. 3. Please describe how exactly the parameters of calcium dynamics were calculated, for example, by showing examples of the raw data (including caffeine experiment). I understand what each parameter captures conceptually. But, windows and thresholds taken for calculations are also important to evaluate the robustness of the experiments. We added information in the section methods as follow. “The Ca2+ transients were recorded during 30 seconds resulting in a 1024-pixel image. The images obtained were interpreted to graph sequentially in time the intensity of fluorescence (S1 Fig), mediating a customized algorithm using the Jupiter notebook program, based on Python programming language, with Matplotlib and NumPy libraries from Anaconda (Balcazar et al 2018). Values expressed as relative change of fluorescence along time, calculated according to the formula: Fmax-F0/F0 and expressed in arbitrary units of fluorescence (AU), were analyzed with LabChart software (AD Instruments, CO, USA). Measurements included: peak Ca2+ transient amplitude (Fmax-F0/F0) (AU), maximal rates of fluorescence increasing and decreasing (+dΔF/dt) (sec), (-dΔF/dt) (sec). Relaxation was measured by calculating the Tau constant (in seconds) of the transient exponential decay of Ca2+ (S1 Fig).” Please, see pages 5 and 6, lines 135 to 149). Moreover, we included one supporting information figure that shows an image of the fluorescence, a digitalized recording, and the measured parameters. 4. In Figure 1, the relaxation decay constant tau did not change much upon over expression of 4E-BP in 7-day old flies while SERCA activity increased. Does this mean that the activity of Na/Ca exchanger decreased upon overexpression of 4E-BP? Is this likely? Thank you for this important question. Actually, the first bar of Figure 1 was drawn incorrectly. We apologize for this error. However, and as the reviewer indicated this difference, although greater than before, did not reach statistical difference. The activity of the NCX did not decrease. The reason for the lack of correlation between the time constant of transient decay in 4E-BP flies and the increase in SERCA activity might result from the comparison of independent preparations. To test this possibility and with the aim of clarifying this point and verifying a direct impact of SERCA activity on calcium transient decay, we analyzed the constant of relaxation Tau for a subset of data in which SERCA activity were simultaneously measured. An increase in SERCA activity was correlated with an acceleration of relaxation (S1 table). However, an action of 4EBP on other targets associated with intracellular calcium handling could not be ruled out. For example, it has been shown in cell lines that 4EBP modulates mitochondrial biogenesis and activity (Morita et al, 2013). Mitochondria are a slow calcium removal system in vertebrates (Eisner et al, 2017), but their contribution to intracellular calcium removal in the Drosophila heart is unknown. Moreover, intracellular calcium handling is influenced by the sensitivity of contractile proteins to calcium. During the relaxation phase, calcium must be released from troponin C to promote dissociation of actin and myosin filaments. If the calcium sensitivity of contractile proteins is incremented, contractility also increases, but relaxation may be prolonged because calcium dissociates more slowly from protein C (Chung et al, 2016). Although we did not explore this aspect, changes in the sensitivity of contractile proteins to calcium could counteract the relaxant effect produced by increased SERCA activity in the analyzed individuals (Fig 1E). These aspects were incorporated in results (page 11, lines 291 to 295) and discussion section (pages 16 and 17 lines 447 to 462). S1 table is provided in the supporting information file. 5. Based on the tissue expression atlas available from the FlyBase, eIF4E-1/2 gene is most abundantly expressed among eIF4E genes in most tissues except that the testes abundantly express eIF4E-3 and eIF4E-4 genes. Therefore, isn't it possible that eIF4E-1/2 is more relevant as a potential effector of 4E-BP overexpression despite less strongly interacting with SERCA in Y2H? Did you find peptides of eIF4E-1/2 in the mass-spec data? We detected peptides belonging to eIF4E 1/2 in our mass spectra. However, modulation of 4E-BP on eIF4E1/2 might not affect the intracellular calcium handling. Our results indicate that overexpression of TOR, the 4E-BP inhibitory kinase, did not produce changes in intracellular calcium dynamics in the individuals analyzed (Fig. 2). Therefore, it is possible that changes observed in the calcium transient in flies overexpressing 4E-BP, were not associated with global translation modulation even when eIF4E1/2 is present. This was added to the discussion section. Please, see page 20 lines 551 to 558. 6. At least part of the effects of 4E-BP overexpression could be due to changes in translation, indirectly affecting the heart function. Could you comment on this possibility? We agree with the reviewer. The following paragraphs have been incorporated in the discussion section. Please, see pages 20- 21. Lines 567 to 575. In mammals, the activation of mTOR signalling is associated with the development of cardiac hypertrophy (Kemi et al., 2008; Maillet et al., 2013). Thus, mTOR phosphorylates 4E-BP and reduces its inhibitory effect on eIF4E by stimulating protein synthesis (Kemi et al., 2008). Under normal conditions, cardiomyocytes from the adult mammalian heart have a low capacity to proliferate (Soonpaa et al., 1996, Bergmann et al., 2015), and heart protein synthesis levels are low (Gray and Gray 2017). However, there is possible translation regulation of specific proteins. Recent studies confirm the ability of 4E-BP to repress the translation of specific target mRNAs (Jin et al, 2020). We did not measure translation or hypertrophy in the adult Drosophila heart. One possibility is that it behaves similarly to the mammalian heart in that overall levels of translation are not elevated. In addition, we observed that overexpression of TOR kinase did not produce changes in the calcium transient relative to wild-type flies (Fig. 2). This suggests that changes observed in the calcium transient in flies overexpressing 4E-BP were not associated with global translation modulation. However, there cannot be ruled out that the translation of specific proteins is affected. As mentioned before, aging might affect translation of several proteins (Basisty et al, 2018). This aspect must be further explored. ________________________________________ https://doi.org/10.1371/journal.pone.0267156.r002
4 Apr 2022 Decision Letter - Thomas Preiss, Editor Interplay between SERCA, 4E-BP and eIF4-E in the Drosophila heart PONE-D-21-29388R1 Dear Dr. Ferrero, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Thomas Preiss, 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 carefully addressed all of the reviewer's comments. The manuscript is ready to be published. ****** 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: Yes:** Rippei Hayashi https://doi.org/10.1371/journal.pone.0267156.r003
Formally Accepted
28 Apr 2022 Acceptance Letter - Thomas Preiss, Editor PONE-D-21-29388R1 Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart Dear Dr. Ferrero: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof Thomas Preiss Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267156.r004

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