Epicardial electrical heterogeneity after amiodarone treatment increases vulnerability to ventricular arrhythmias under therapeutic hypothermia

Chin-Yu Lin; Ting-Yung Chang; Yu-Feng Hu; Yu-Cheng Hsieh; Yi-Jen Chen; Hung-I Yeh; Yenn-Jiang Lin; Shih-Lin Chang; Li-Wei Lo; Tze-Fan Chao; Fa-Po Chung; Jo-Nan Liao; Ta-Chuan Tuan; Shih-Ann Chen

doi:10.1371/journal.pone.0282943

Peer Review History

Original SubmissionOctober 4, 2022
19 Dec 2022 Decision Letter - Tomohiko Ai, Editor PONE-D-22-27467Epicardial electrical heterogeneity after amiodarone treatment increases vulnerability to ventricular arrhythmias under therapeutic hypothermiaPLOS ONE Dear Dr. HU, Thank you for submitting your manuscript to PLOS ONE. Your study was evaluated by an expert in the field and myself. The reviewer is concerned with several significant points regarding study methods and designs. Please read the comments carefully and address the issues accordingly. Please submit your revised manuscript by Feb 02 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. 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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: No ****** 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: Using swine, authors　examined　the　electrophysiological　changes　and　proarrhythmic　risk　due　to　amiodarone　administration in therapeutic hypothermia (TH) and concluded that amiodaron and TH was associated with vulnerability to ventricular arrhythmias. The study is important in the use of amiodarone for the ventricular arrhythmias while TH, however, there are several comments to this study. 1. Why didn’t you check the effect of amiodaron at baseline temperature (BT)? Previous study by Piktel, et al. showed that amiodarone did not affect CV, DOR, or action potential duration during ischemia at 36°C. As you know, amiodaron changes various electrophysiological properties in the heart. If you could not examine the effect of amiodaron at BT by yourself, please cite adequate reference to compare the effect of amiodaron in BT and hypothermia. 2. You prepared 6 pigs for protocol I, however, there are only data from 3 pigs for the evaluation of vulnerability to ventricular arrhythmias. Why did you discard the data from other 3 pigs? 3. I could not find the detail of protocol II and III. In protocol I, you performed burst right ventricular pacing after amiodaron injection in TH. In protocol II, did you perform burst pacing just after TH, and in protocol III, you just performed burst pacing? Did you measure the baseline characteristics of 4 and 4 pigs in protocol II and III? Were the baseline electrophysiological characteristics of pigs in protocol II and III same with those in protocol I? Please revise the supplemental figure 1B and figure legend for the easy understanding of the readers. 4. Why did the inter-segmental wavelet propagation change from BT to TH and return to the same pattern with BT after amiodaron/TH? Please discuss the mechanism in the discussion section. 5. How did you obtain the cardiomyocytes for Immunofluorescent staining and western blotting, biopsy or after sacrifice? How did you treated the heart if you obtain the cardiomyocytes after sacrifice? Did you obtain the cardiomyocytes from endomyocardium or epicardium? 6. Please describe how to calculate the total and lateralization of connexin43 using image J software. 7. The sample numbers in graphs in figure 6A is 24. Did you obtain 6 specimens from each pig? Please add the explanation in the methods. 8. Did you check the connexin 43 levels in pigs assigned to protocol II and III? Conduction velocity was changed after TH or TH/amiodaron therapy. Showing connexin 43 levels in the BT in each segment are important to explain the relationship between conduction velocity change and the expression level of connexin 43. 8. In the cardiac conduction system, connexin 45 is highly expressed. Did you evaluate the expression level of connexin 45? ****** 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 ******** [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. https://doi.org/10.1371/journal.pone.0282943.r001
Revision 1
16 Jan 2023 Author Response Responses to Reviewer 1 # 1. Why didn’t you check the effect of amiodaron at baseline temperature (BT)? Previous study by Piktel, et al. showed that amiodarone did not affect CV, DOR, or action potential duration during ischemia at 36°C. As you know, amiodaron changes various electrophysiological properties in the heart. If you could not examine the effect of amiodaron at BT by yourself, please cite adequate reference to compare the effect of amiodaron in BT and hypothermia. Response: Thanks for your great comment. In our present study, we investigated the effect of hypothermia and the hypothermia + amiodarone and compared to the baseline temperature. The impact of amiodarone on the baseline temperature is important. Studies on amiodarone have revealed that intravenous amiodarone has minimal effect on the refractory periods of the ventricular muscle in baseline condition [Am Heart J. 1984;108(4 Pt 1):890-8; J Am Coll Cardiol. 1986;7(1):148-57] and might decrease conduction velocity in the ventricle. [Circulation. 1995;91(2):451-61; J Am Coll Cardiol. 1985 Jul;6(1):179-85] Previous study by Piktel, et al. showed that amiodarone did not affect CV, dispersion of repolarization, or action potential duration during ischemia at 36°C. [J Am Heart Assoc. 2021 May 18;10(10):e016676, 33938226] We cited above-mentioned reference add revised the discussion (line 316-322) as follows, “ The impact of amiodarone alone without TH was not investigated in our present study. Studies on amiodarone have revealed that intravenous amiodarone has minimal effect on the refractory periods of the ventricular muscle in baseline condition31,32 and might decrease conduction velocity in the ventricle.33,34 A recent study by Piktel et al.4 showed that amiodarone did not affect CV, dispersion of repolarization, or action potential duration during ischemia at 36°C. Our study echoed the finding of the decrease in the CV after amiodarone infusion even under the TH. Reference 4. Piktel JS, Suen Y, Kouk S, Maleski D, Pawlowski G, Laurita KR, Wilson LD. Effect of Amiodarone and Hypothermia on Arrhythmia Substrates During Resuscitation. J Am Heart Assoc. 2021;10:e016676. doi: 10.1161/JAHA.120.016676 31. Ikeda N, Nademanee K, Kannan R, Singh BN. Electrophysiologic effects of amiodarone: experimental and clinical observation relative to serum and tissue drug concentrations. Am Heart J. 1984;108:890-898. doi: 10.1016/0002-8703(84)90451-4 32. Morady F, DiCarlo LA, Jr., Krol RB, Baerman JM, de Buitleir M. Acute and chronic effects of amiodarone on ventricular refractoriness, intraventricular conduction and ventricular tachycardia induction. J Am Coll Cardiol. 1986;7:148-157. doi: 10.1016/s0735-1097(86)80273-x 33. Nanas JN, Mason JW. Pharmacokinetics and regional electrophysiological effects of intracoronary amiodarone administration. Circulation. 1995;91:451-461. doi: 10.1161/01.cir.91.2.451 34. Morady F, DiCarlo LA, Jr., Baerman JM, Krol RB. Rate-dependent effects of intravenous lidocaine, procainamide and amiodarone on intraventricular conduction. J Am Coll Cardiol. 1985;6:179-185. doi: 10.1016/s0735-1097(85)80272-2 “ # 2. You prepared 6 pigs for protocol I, however, there are only data from 3 pigs for the evaluation of vulnerability to ventricular arrhythmias. Why did you discard the data from other 3 pigs? Response: Thanks for your great comment. Three of the pigs didn’t underwent the evaluation of vulnerability to ventricular arrhythmias. We revised the limitation as follows, (page 25, line 368-369) as follows,” The number in animals for vulnerability test for ventricular arrhythmias is small for a solid conclusion.“ # 3. I could not find the detail of protocol II and III. In protocol I, you performed burst right ventricular pacing after amiodaron injection in TH. In protocol II, did you perform burst pacing just after TH, and in protocol III, you just performed burst pacing? Did you measure the baseline characteristics of 4 and 4 pigs in protocol II and III? Were the baseline electrophysiological characteristics of pigs in protocol II and III same with those in protocol I? Please revise the supplemental figure 1B and figure legend for the easy understanding of the readers. Response: Thanks for your great comment. We performed burst pacing just after TH in the protocol II and in the BT in the protocol III. Baseline characteristics, electrocardiographic parameters, and electrophysiological parameters were not measured in the protocol II and protocol III. We revised the Supplemental Figure 1 accordingly as follows, “ Legend (A) In the first part of protocol I, epicardial window was created for the high-density epicardial mapping under sinus rhythm and right ventricular pacing rhythm by using multi-electrode mapping catheter in 6 pigs. Epicardial mapping was repeated after the induction of TH and after the infusion of amiodarone. (B) In the second part of protocol I, vulnerability to VA was performed in 3 swine. Another 4 and 4 swine underwent protocol II and protocol III respectively. In the protocol II and III, vulnerability to VA was examined under TH and BT respectively without 3D mapping /electrophysiological study. In the protocol II, induction of TH was performed and vulnerability to VA was conducted in 4 swine after an observation time of 60 minutes. In the protocol III, vulnerability to VA was conducted in 4 swine after an observation time of 120 minutes. OT = outflow tract; RV = right ventricle; LV = left ventricle; 3D = three-dimensional; VA = ventricular arrhythmias “ # 4. Why did the inter-segmental wavelet propagation change from BT to TH and return to the same pattern with BT after amiodaron/TH? Please discuss the mechanism in the discussion section. Response: Thanks for your great comment. We added discussion as follows (line 339-353), “ The inter-segmental wavelet propagation was determinate by CV of each segment. In our present study, we investigated the epicardial CV and electrogram of each segment. We observed a significant reduction in the CV after induction of TH in the anterior mid RV (segment 8), which was greater than that in the other segments, which caused the wavelet propagation change from BT to TH. After the infusion of amiodarone under TH, the CV of each segment was further decreased and alleviated the difference between the anterior mid RV (segment 8) and other segments. This change in CV resulted in the reverse of inter-segmental wavelet propagation inter-segmental wavelet propagation. The mechanism might be related to the change of distribution of connexin 43 or the heterogenous change of CV in the endocardium/transmural myocyte.37 The amiodarone could provide heterogeneous effect on the myocardium and decrease the ventricular repolarization dispersion.38 Our study indicated the amiodarone might provide heterogenous effect on the myocardial CV under TH. Further study investigating the electrophysiological parameter with simultaneous endocardial and epicardial mapping to clarify the is warrant. “ Reference 37. Dietrichs ES, McGlynn K, Allan A, Connolly A, Bishop M, Burton F, Kettlewell S, Myles R, Tveita T, Smith GL. Moderate but not severe hypothermia causes pro-arrhythmic changes in cardiac electrophysiology. Cardiovasc Res. 2020;116:2081-2090. doi: 10.1093/cvr/cvz309 38. Drouin E, Lande G, Charpentier F. Amiodarone reduces transmural heterogeneity of repolarization in the human heart. J Am Coll Cardiol. 1998;32:1063-1067. doi: 10.1016/s0735-1097(98)00330-1 # 5. How did you obtain the cardiomyocytes for Immunofluorescent staining and western blotting, biopsy or after sacrifice? How did you treated the heart if you obtain the cardiomyocytes after sacrifice? Did you obtain the cardiomyocytes from endomyocardium or epicardium? Response: Thanks for your great comment. We obtained the tissue after sacrifice. A fixation procedure was performed immediately in both 20% formalin and liquid nitrogen to prevent sample degradation. We obtained the cardiomyocytes from both endomyocardium and epicardium. We revised the supplemental file (line 45-49) as follows, “ Tissue sampling We obtained the tissue from the pre-specified 13 segments after sacrifice. A fixation procedure was performed immediately in both 20% formalin and liquid nitrogen to prevent sample degradation. We obtained the cardiomyocytes from both endomyocardium and epicardium. “ # 6. Please describe how to calculate the total and lateralization of connexin 43 using image J software. Response: Thanks for your great comment. Assisted by image analysis software, the percentage of total tissue area occupied by connexin 43 immunoreactive signals, and the percentage of connexin 43 signal located outside the end-to-end cell junctions (lateralization). The percentages of lateralization in specific segment were means of the 24 slides (6 slides from segment 5, 8, and 12 of each pig) from the specific segment. Please see the revised supplemental file, line 66-70. # 7. The sample numbers in graphs in figure 6A is 24. Did you obtain 6 specimens from each pig? Please add the explanation in the methods. Response: Thanks for your great comment. Please see the response to the #7. # 8. Did you check the connexin 43 levels in pigs assigned to protocol II and III? Conduction velocity was changed after TH or TH/amiodaron therapy. Showing connexin 43 levels in the BT in each segment are important to explain the relationship between conduction velocity change and the expression level of connexin 43. Response: Thanks for your great comment. We didn’t check the connexin 43 levels in pigs assigned to protocol II and III. We only examined the vulnerability to ventricular arrhythmias in the protocol II and III. # 9. In the cardiac conduction system, connexin 45 is highly expressed. Did you evaluate the expression level of connexin 45? Response: Thanks for your great comment. We realized that Cx45 is one of the most important connexins expressed in the heart and is localized to the sinoatrial node, atrioventricular node, and His-Purkinje system, but has also been shown to be expressed in atrial and ventricular myocytes. [Curr Opin Cardiol, 30 (2015), pp. 33-39] In our present study, we didn’t evaluate the expression levels of connexin 45. The further study investigating both the expression of Cx45 and Cx43 may enrich out study and improve the quality. We will work on this. Thanks for your valuable comments! https://doi.org/10.1371/journal.pone.0282943.r002
28 Feb 2023 Decision Letter - Tomohiko Ai, Editor Epicardial electrical heterogeneity after amiodarone treatment increases vulnerability to ventricular arrhythmias under therapeutic hypothermia PONE-D-22-27467R1 Dear Dr. HU, 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, Tomohiko Ai, M.D., Ph.D. 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: Authors replied to my previous comments adequately. I have no additional comments to this manuscript. ****** 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 ******** https://doi.org/10.1371/journal.pone.0282943.r003
Formally Accepted
11 Apr 2023 Acceptance Letter - Tomohiko Ai, Editor PONE-D-22-27467R1 Epicardial electrical heterogeneity after amiodarone treatment increases vulnerability to ventricular arrhythmias under therapeutic hypothermia Dear Dr. Hu: 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 Dr. Tomohiko Ai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0282943.r004

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