Fig 2: Stage-specific causal role of ERR? in regulating TNNI3-driven cardiac maturation.a Fluorescence images of ERR?KO hiPSC-CMs treated with DMSO, T112, or T623 for TNNI1EmGFP and TNNI3mCherry expression. Scale bars: 500 µm. b Flow cytometric analysis of day-16 CMs derived from ERR?KO hiPSCs for TNNI3mCherry expression. n = 4 independent experiments per group. Data are the mean ± SEM; Statistical analysis was compared to DMSO using one-way ANOVA followed by Dunnett’s test. NT no treatment. c Mitochondrial respiration rates of ERR?KO hiPSC-CMs treated with either DMSO or T112 (O Oligomycin; F FCCP, phenylhydrazone; AR Antimycin A and Rotenone). n = 5 biologically independent samples per group. d Basal and maximal OCR and ATP production of ERR?KO hiPSC-CMs treated with either DMSO or T112. Statistical analysis was done using unpaired two-tailed t-tests. n = 5 biologically independent samples per group. Data are the mean ± SEM; Statistical analysis was using unpaired two-tailed t-test.

Fig 3: Metabolic and structural functionality of hiPSC-CMs.a Mitochondrial respiration rates of hiPSC-CMs treated with DMSO (n = 4), T112 (n = 5), T623 (n = 5), or Combo (n = 5) (O, Oligomycin; F, FCCP, phenylhydrazone; AR, Antimycin A and Rotenone). n = biologically independent samples per group. Data are the mean ± SEM. b Basal and maximal OCR and ATP production of hiPSC-CMs treated with DMSO (n = 4), T112 (n = 5), T623 (n = 5), or Combo (n = 5). n = biologically independent samples per group. Data are the mean ± SEM. Statistical analysis was compared to DMSO using one-way ANOVA followed by Dunnett’s test. ****P < 0.0001. c top: Representative western blots of cTnI, ssTnI, and GAPDH proteins in hiPSC-CMs treated with DMSO, T112, T623, or Combo. For ssTnI, two bands were detected: the shorter one is wild type ssTnI and the longer one is ssTnI with F2A peptides. Bottom: cTnI/ssTnI relative ratio. n = 3 independent experiments per group. Data are the mean ± SEM. Statistical analysis was compared to DMSO using one-way ANOVA followed by Dunnett’s test. d Cell area of hiPSC-CMs treated with DMSO, T112, or T623 measured by high content imaging. For each group, n = 300 cells over three independent experiments. e Roundness of hiPSC-CMs treated with DMSO, T112, T623, or Combo. Each group, n = 3 independent experiments. Data are the mean ± SEM. Statistical analysis was done using one-way ANOVA followed by Tukey’s HSD test. f Sarcomere length in hiPSC-CMs treated with DMSO, T112, T623, or Combo. Boxes represent 25th–75th percentiles; whiskers represent the minimum and maximum ranges; horizontal lines indicate the median values. n = 15, one measurement per TEM image from 15 TEM images over two consecutive experiments; ****P < 0.0001 compared to DMSO, ‡P < 0.0001 compared to T623 using one-way ANOVA followed by Tukey’s HSD test. g Contractile properties of hiPSC-CMs treated with DMSO (n = 6), T112 (n = 7), T623 (n = 5), or Combo (n = 9). Top: Contraction velocity. Middle: Relaxation velocity. n = biologically independent samples examined over three independent experiments. bottom: Maximum conduction velocity. n = 3 independent experiments. Statistical analysis was compared to DMSO using one-way ANOVA followed by Dunnett’s test. ****P < 0.0001. h Representative AP recorded from hiPSC-CMs treated with DMSO or T112. i The fraction of APs with a notch (black). j Electrophysiology data of APA, Vmax and MDP in hiPSC-CMs treated with DMSO (n = 21) or T112 (n = 18) over 3 independent experiments. Boxes represent 25th–75th percentiles; whiskers represent the minimum and maximum ranges; horizontal lines indicate the median values. Statistical analysis was done using unpaired two-tailed t-test. k Typical high magnification TEM images of hiPSC-CMs treated with T112 or Combo. Scale bars: 500 nm. I: I-band; TT: T-tubule; Z: Z line.All results are from #8-5-3.