Application of retinoic acid (RA) to differentiating hiPSCs results in cardiomyocytes (CMs) that exhibit a robust acetylcholine-activated K+ current (IK,ACh). (A) Transcript expression of genes in RA-treated hiPSC-CMs relative to expression in DMSO-treated hiPSC-CMs. Data are mean±s.e.m. of three biological replicates, normalized to TBP. RA treatment enhanced expression of the genes KCNJ3, NPPA and NR2F2. *P<0.05 (two-sided t-tests). (B) Typical IK,ACh traces in DMSO- and RA-treated hiPSC-CMs initiated by fast application of 100 µmol l−1 carbachol (CCh). After reaching steady state, addition of 1 mmol l−1 atropine achieves instant removal of CCh from muscarinic receptors and deactivation of the current. In DMSO-treated hiPSC-CMs, IK,ACh was not detected, while RA-treated hiPSC-CMs all demonstrated a robust current (for average data, see Fig. 2). (C) Typical spontaneous action potentials (APs) of DMSO- and RA-treated hiPSC-CMs at baseline and upon addition of 10 µmol l−1 CCh. (D,E) Frequency of spontaneous APs (D: n=9, DMSO; n=10, RA) and maximal diastolic potential (MDP; E: n=9, DMSO; n=9, RA) of each cell at baseline and in the presence of CCh in DMSO- and RA-treated hiPSC-CMs. *P<0.05 (two-way ANOVA). CCh reduces the AP frequency and causes significant MDP hyperpolarization in RA-treated, but not in DMSO-treated, hiPSC-CMs.