Fig. 2: RA signaling modulation guides differentiation into distinct cardiomyocyte subtypes.

a mRNA expression of markers of outflow tract CMs (OFT-CMs), atrial CMs (A-CMs) and ventricular CMs (V-CMs) relative to TNNT2 and GAPDH at day 30 of the indicated differentiation conditions. Data are mean ± SEM. n = 4 independent experiments except for: RA− LTBP3 n = 7; MYL2 and FHL2 n = 5; RSPO3 and KCNJ3 n = 6; RA+ except for RA+ (1 µM 10d) LTBP3, RSPO3, MYL2, KCNJ3 n = 5; RA+ (1 µM 4d) FHL2 n = 5; RA+ (0.5 µM 4d) n = 6. Exact p-values of unpaired two-tailed t-test are shown. b Representative immunofluorescence images after staining for MLC2v (magenta) and MLC2a (turquoise) at day 45 of indicated differentiations conditions. Nuclei were counterstained with Hoechst-33258 (blue). Scale bar = 25 µm. c Percentage of cells stained positive for NR2F2 at day 30 of indicated differentiation conditions as determined by flow cytometry. Data are mean ± SEM. n = 3 independent experiments. d Percentage of cardiomyocyte subtypes at day 30 of differentiation with 1 µM RA for 4d or without RA (RA−) as defined by the ratio of action potential duration at 50% and 90% repolarization determined by whole-cell voltage clamp recording. I-CMs intermediate ventricular cardiomyocytes, V-CMs ventricular cardiomyocytes, A-CMs atrial cardiomyocytes, N-CMs nodal cardiomyocytes. e Representative action potential traces of indicated cardiomyocyte subtypes at day 30. f Amplitude (left) and maximum diastolic potential (MDP, right) of action potentials in cardiomyocytes at day 30 of differentiation with 1 µM RA for 4d or without RA (RA−). Data are mean ± SEM. For 1 µM RA for 4d n = 18 cells; for RA− n = 13 cells examined over two independent experiments. Source data are provided as a Source Data file.