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. 2018 Jan 25;9:368. doi: 10.1038/s41467-017-02796-3

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© The Author(s) 2018

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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Fig. 6 — Endothelial cells support myocardial growth in the absence of blood flow in an Ino80-dependent manner. Heart ventricle and sinus venosus (SV) explants cultured for 5 days and immunostained for myocardium and endothelial cells. a Confocal images of ventricular explants show that expansion of myocardium (orange brackets) and endocardial-derived cells is decreased when Ino80 is deleted using Nfatc1Cre. Images are representative of the following number of replicates: control, n = 8 heart explants; mutant, n = 5 heart explants. Scale bars: 100 μm (low and high magnification). b–e Quantifications show that endothelial sprouting (b), myocardial outgrowth (control, n = 8 heart explants; mutant, n = 5 heart explants) (c) and cardiomyocyte proliferation (control, n = 6 heart explants; mutant, n = 8 heart explants) (d, e) are significantly reduced. Images are representative of the following number of replicates: control, n = 6 heart explants; mutant, n = 8 heart explants. Scale bars: 100 μm (d). Error bars in graphs are standard deviation. ***P < 0.001; ****P < 0.0001, evaluated by Student’s t-test (d). f–i Inhibiting endothelial cell growth through targeting VEGFR with SU1498 recapitulates the effect of Ino80 mutation (f), significantly decreasing vessels sprouting (g), myocardial expansion (control, n = 6 heart explants; SU 1498 treatment, n = 6 heart explants) (h), and cardiomyocyte proliferation (control, n = 10 heart explants; SU 1498 treatment, n = 8 heart explants) (i). Error bars in graphs are standard deviation. ***P < 0.001; ****P < 0.0001, evaluated by Student’s t-test. Scale bars: 100 μm (low and high magnification). Images are representative of the following number of replicates: control, n = 6 heart explants; SU 1498 treatment, n = 6 heart explants (f). j, k Images of human induced pluripotent cell-derived cardiomyocytes (iPSC-CMs) cultured either alone or with mouse ventricle explants. Proliferating NKX2.5⁺EdU⁺ iPSC-CMs are turquoise and indicated by arrowheads. Images are representative of the following number of replicates: control n = 10 heart explants, mutant n = 4 heart explants, iPSC-CM only control n = 7. Scale bar: 25 μm (j); 100 μm (k; low magnification) and 25 μm (k; high magnification). l Quantification of iPSC-CM proliferation within three cells lengths from endothelial cells reveals a significant increase over regions distant from vessels or iPSC-CM cultures only (control n = 10 heart explants; mutant n = 4 heart explants; iPSC-CM only control n = 7). This increase is significantly dampened when endothelial cells are depleted of Ino80. Error bars in graphs are standard deviation. *P < 0.05; **P < 0.01; ****P < 0.0001, evaluated by Student’s t-test. Scale bars: 100 μm