a Ca2+ transients in the HOs. Ca2+ oscillations were analyzed using the Ca2+-binding fluorescent dyes Fluo8 AM. Representative images of HOs (n = 7, two experiments, each top left panels, bright field and bottom left panels: fluorescent images). Three fluorescent images were extracted from every ten frames of sequential images (each right column, see also Supplementary Fig. 7). Scale bar: 100 μm. b Ca2+ transients in the 11-day cultured HOs, pre- and post administration of the β adrenergic agonist isoproterenol (1 μM). Beating rate was increased and time constant of Ca2+ decay was decreased after treatment of isoproterenol (left, n = 6, P = 0.0055 and 0.0046 by two-sided paired t test). The representative traces of changed Ca2+ signal (F/F0) before and after the treatment of isoproterenol (right). c IK1 immunostaining in a representative HO (left, n = 10 out of n = 12, two independent experiments), embryonic hearts (E9.5, E10.5 and E12.5; middle) and a postnatal day 1 (P1) mouse heart (right). Inset indicates the whole image of P1 mouse heart. Scale bar: 100 μm (HO), and 1 mm (P1 mouse heart).