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. 2020 Jul 14;10:11551. doi: 10.1038/s41598-020-68200-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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A decreased activation of cardiomyocyte proliferation and dedifferentiation after multiple cryoinjuries. (A) Cross sections of transgenic zebrafish hearts at 7 dpci following 1, 2, 3 or 6 CIs expressing nuclear DsRed in cardiomyocytes. Proliferating cells are detected by immunostaining against Minichromosome Maintenance Complex Component 5 (MCM5; green). Proliferating cardiomyocytes are observed (white arrows) by colocalization between cmlc2:DsRed2-nuc and MCM5 in the vicinity of the wound (encircled with a dash line). Scale bars = 50 µm. (B) Cross sections of adult zebrafish hearts at 7 dpci following 1, 2, 3 or 6 CIs, immunostained for embryonic cardiac myosin heavy chain (EmbCMHC; N2.261; green). The cardiac muscle is detected by F-actin staining (Phalloidin, red). EmbCMHC-positive cardiomyocytes are detected in the peri-injury zone within an area of 100 µm from the injury border (dashed line). (C) Analysis of the percentage of MCM5-positive nuclei of cardiomyocytes (CMs) at 4 and 7 dpci, following multiple cryoinjuries. N ≥ 4. *P < 0.05; **P < 0.01. Scatter plot of the data with a large bar indicating the mean and smaller bars representing the SEM. (D) Analysis of the percentage of embCMHC-positive cardiomyocytes (CMs) at 7 and 14 dpci, within the peri-injury zone. N ≥ 4. **P < 0.01. Scatter plot of the data with a large bar indicating the mean and smaller bars representing the SEM.