Extended Data Figure 9. Intensity training upregulates cardiac maturation within early-stage tissues through enhanced calcium handling.
a,b Intensity training promotes t-tubule formation within early-stage hiPS-CM tissues as demonstrated by immunofluorescence imaging of ryanodine 2 receptor (RyR2, green), di-8-ANEPPs/t-tubules (red), and bridging integrator 1 (BIN1, blue) (scale bar = 10 μm). c Gene expression, normalized to GAPDH, of (i) ATP2A2 and (ii) SLC8A1, responsible for maintaining proper calcium homeostasis, within early-stage tissues as determined by qPCR over 4 weeks of culture for the designated stimulation regimen (independent biological replicates per group: FCT (n=8), Control/Constant (n=6), Intensity (n=14), Adult (n=1)), Mean ± 95% CI, *p<0.05 versus FCT group at week 4 by ANOVA with Tukey’s HSD test and —p<0.05 compared to other training regimens by 2-way ANOVA with Tukey’s HSD test. d Relaxation times within early-stage tissues as characterized by the Full-Width Half-Max (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 and — 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 5mM caffeine in early-stage tissues and FCTs treated with f 1 mM verapamil or g 2 μM thapsigargin. h Representative traces of calcium release after stimulation with 5 mM caffeine for early-stage tissues and FCTs. i Calcium spikes (by immunofluorescent calcium dyes) in early and late-stage tissues (C2A line) after 4 weeks of culture at two [Ca2+] levels. j Intensity-trained early-stage but not late-stage tissues (C2A line) after 4 weeks of culture respond to ryanodine (1μmol/L). k The force-frequency relationship of early-stage intensity-trained cardiac tissues (C2A line) after 4 weeks of culture treated with the ryranodine blocker ryanodine (1μM) or the SERCA2a blocker thapsigargin (1μM) (directly measured force data, n=13 biologically independent samples for Intensity group and n=3 biologically independent samples for other groups), Mean ± 95% CI, —P<0.05 by two-way ANOVA.