Fig. 4

HAND1/2 double knockout impeded the differentiation of cardiomyocytes and impaired their electrophysiological activity. A The phase and fluorescence images of WT and H1/H2-dKO cells at differentiation day 7. B The percentage of NKX2.5-eGFP⁺ cells in WT and H1/H2-dKO cells at different time points of cardiomyocyte differentiation (n = 3). Unpaired t test. C The beating onset of WT and H1/H2-dKO-derived cardiomyocytes (n = 10). Unpaired t test. D Expression of FHF and SHF markers in early cardiomyocyte differentiation of WT and H1/H2-dKO cells. Relative to GAPDH expression (n = 3). Unpaired t test. E Expression of ventricular, atrial and outflow tract cardiomyocyte (VCM, ACM and OFT) markers in WT and H1/H2-dKO-derived day 30 cardiomyocytes. Relative to GAPDH expression (n = 3). Unpaired t test. F Western blot analysis of CX43 and MYL2 expression in day 30 WT and H1/H2-dKO cardiomyocytes. Corresponding uncropped full-length gels and blots are presented in Additional file 8: Fig. S10. G The percentage of MYL2.⁺ cardiomyocytes in WT and H1/H2-dKO cells at differentiation day 30 (n = 3). H Immunofluorescence staining of NR2F2 in WT and H1/H2-dKO-derived day 30 cardiomyocytes. Scale bar = 100 μm. I The field potential and simulated action potential recorded by MEA in WT and H1/H2-dKO-derived day 30 cardiomyocytes. Black lines represented the field potential while red lines represented the action potential. J Comparison of corrected APD90, APD50 and APD90/APD50 ratio in WT and H1/H2-dKO-derived differentiation day 30 cardiomyocytes (n ≥ 6). Unpaired t test. K The action potential paced by 1Hz recorded by whole-cell patch clamp in WT and H1/H2-dKO-derived differentiation day 30 cardiomyocytes. L Comparison of APD90, APD50 and action potential amplitude in WT and H1/H2-dKO-derived day 30 cardiomyocytes (n ≥ 10). Unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001