a, b, Intensity training promotes T-tubule formation in early-stage hiPS-CM tissues, as demonstrated by immunofluorescence of ryanodine 2 receptor (RYR2, green), bridging integrator 1 (BIN1, blue) and T-tubule staining (di-8-ANEPPs, red). Scale bar, 10 µm. c, Expression of ATP2A2 and SLC8A1 genes, which are responsible for maintaining proper calcium homeostasis, in early-stage tissues as determined by RT–PCR and normalized to GAPDH over four weeks of culture with the designated stimulation regime. Independent biological replicates per group: FCT, n = 8; control, n = 6; constant, n = 6; intensity-trained, n = 14; adult, n = 1. Mean ± 95% CI, *P < 0.05 versus FCT group at week four by ANOVA with Tukey’s HSD test. Line over graph indicates P < 0.05 compared to other training regimes by two-way ANOVA with Tukey’s HSD test. d, Relaxation times in early-stage tissues as characterized by the full-width half-maximum (FWHM) values and the decay time (90% of the time from the maximal peak of the calcium transient). Independent biological replicates per group: FCT, n = 8; C2A, n = 12; WTC11, n = 6; IMR90, n = 6. Mean ± 95% CI; *P < 0.05 versus FCT group by ANOVA with Tukey’s HSD. Line over graph indicates P < 0.05 between cell lines by two-way ANOVA. e, Representative calcium traces of early-stage tissues treated with 1 µM nifedipine. f, g, Representative traces of calcium release after stimulation with 5 mM caffeine in early-stage tissues and FCTs treated with 1 mM verapamil (f) or 2 µM thapsigargin (g). h, Representative traces of calcium release after stimulation with 5 mM caffeine for early-stage tissues and FCTs. i, Calcium spikes detected by fluorescent calcium dyes in early and late-stage tissues (C2A line) after four weeks of culture at two calcium concentrations. j, Intensity-trained early-stage but not late-stage tissues (C2A line) after four weeks of culture respond to ryanodine (1 µmol l−1). k, The force–frequency relationship of early-stage intensity-trained cardiac tissues (C2A line) after four weeks of culture, treated with the RYR2 blocker ryanodine (1 µM) or the SERCA2a blocker thapsigargin (1 µM). Directly measured force data; n = 13 biologically independent samples for intensity group and n = 3 biologically independent samples for other groups. Mean ± 95% CI, line over graph indicates P < 0.05 by two-way ANOVA.