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. 2020 Jan 7;11:75. doi: 10.1038/s41467-019-13868-x

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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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Fig. 2 — a Schematic representation of the process of engineered heart tissue (EHT) generation. Chamber-specific human embryonic stem cell-derived cardiomyocytes (hESC-CMs, scale bar: 10 μm) were combined with collagen, solidified in circular molds, and placed on a silicon stretcher for creation of the EHTs (scale bar: 100 μm). b Real-time qPCR analysis of the atrial (n = 4, biologically independent samples) and ventricular (n = 4, biologically independent) EHTs as well as of control adult human atrial and ventricular tissue samples (n = 3, biologically independent samples). Shown are the expression levels of the general cardiac-specific markers (TNNI1, TNNT2, KCJ2, and GJA1), atrial-specific markers (GJA5, KCNA5, KCNJ3, NPPA, MYL7, and NR2F2), and ventricular-specific markers (MYL2, MTH7, and HEY2). Expression values were normalized to GAPDH. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; unpaired two-tailed t test. c, d Co-immunostaining of 30d atrial and ventricular EHTs for cardiac troponin I (cTnI) and either the ventricular-specific marker MLC2v (c) or the atrial marker sarcolipin (SLN) (d). Nuclei were stained with DAPI. Scale bars: 20 μm. All eight additional immunostaining images were similar to the representative image shown. e Western blot densitometry of Cx40 and Cx43 protein expression in the atrial and ventricular EHTs (n = 8, biologically independent samples). All values are expressed as mean ± SEM. **p < 0.01, ***p < 0.001, Mann–Whitney test is used for comparison.