Fig 2: Nkx2-5 mutant heart organoids displayed functional and lineage defects.a Diagram of the strategy to generate an isogenic line carrying an EA-associated mutation (PM28, PM52) and a control line with a large deletion on Nkx2-5 (Del33). b Representative organoids differentiated from the two mutants at day 30. c Nkx2-5 expression significantly reduced in the Del33 line derived organoids. Scale bar = 100 μm. d The percentages of beating and non-beating organoids in the control and mutant at RA- and RA+ groups. n = 3 biological independent organoid differentiation. e The beating rates of control and mutant heart organoids. n = 20 organoids in controls, n = 20 and 10 organoids in Del33 RA+ and A-, n = 10 and 11 organoids in PM28 RA+ and A-, respectively. ***p < 0.001, ****p < 0.0001, Student’s t test against WTC. f (i) Representative plots of Ca2 + transients in mutant and control organoid CMs at day 30. (ii) Quantification of the transient duration 50, transient duration 90 and time to 50% decay in WT and mutant organoid CMs. n = 81 cells in WT RA-, n = 208 cells in WT RA+, n = 77 cells in Del33 RA-. ****p < 0.0001, Student’s t test against WT RA-. g High expression of atrial genes and low expression of ventricular genes in RA- mutant CMs. Scale bar = 100 μm. h Defective sarcomere structures were identified in mutant heart organoids and sarcomere length was quantified based on the ACTN2-eGFP signal. n = 23 sarcomeres in each group, data was displayed as mean ± SEM. ****p < 0.0001, Student’s t test against WTC. Scale bar = 20 μm.