Fig. 4

Fatty acid challenged HADHA Mut CMs displayed elevated cytosolic calcium levels leading to increased beat rate irregularities. a Seahorse mitostress assay to analyze maximum oxygen consumption rate after oligomycin and FCCP addition. MiMaC treated CMs showed a significant increase (2.2-fold change) in maximum OCR compared to control EV CMs. *p < 0.05, one-way ANOVA on ranks vs WT-CM EV was performed. n = 1–14 biological replicates. b Representative trace of the mitostress assay. c Seahorse analysis of fatty acid oxidation capacity showed that MiMaC treated hiPSC-CMs matured to a point where they could oxidize palmitate for ATP generation while controls cells were not able to utilize palmitate. MiMaC hiPSC-CMs had a significant increase in OCR due to palmitate addition. Both MiMaC treated Mut and KO hiPSC-CMs were unable to oxidize palmitate. *p < 0.05, one-way ANOVA on ranks vs WT-CM EV was performed. n = 1–14 biological replicates. d Representative trace of the change in fluorescence during calcium transient analysis. e Quantification of the maximum change in fluorescence during calcium transients. Mut CMs as compared to WT CMs after 12D of Glc + FA media treatment had a statistically significantly lower change in calcium. WT CM: 2.03, Mut CM: 1.55. ***p < 0.001, t-test was performed. n = 28–30 cells measured. f Quantification of the tau-decay constant. Mut CMs as compared to WT CMs after 12D of Glc + FA media treatment had a higher tau-decay constant. WT CM: 0.63 s, Mut CM: 0.76 s. n = 28–30 cells measured. g Representative trace of the change in cell membrane potential during whole-cell patch clamp analysis. h Quantification of the APD90 in WT and HADHA Mut CMs after 12D of Glc + FA media. WT 541 ms, Mut 1068 ms. *p < 0.05, t-test was performed. n = 9–10 cells measured. i Quantification of the tau-decay constant in WT and HADHA Mut CMs after 12D of Glc + FA media. p = 0.066, t-test followed by a Mann-Whitney rank sum test. n = 9–10 cells measured. j Time to wave duration 50% is significantly longer in Mut CMs as compared to WT CMs after 12D of Glc + FA media treatment. ***p < 0.001, t-test was performed. n = 18–36 cells measured. k Representative beat rate trace of Mut CM in Glc or Glc + FA media. l Quantification of the change in beat interval (ΔBI). Mut CMs in Glc + FA media as compared to Mut CMs in Glc media had a statistically significant higher ΔBI. *p < 0.05, t-test followed by a Mann-Whitney rank sum test. n = 13–16 cells measured. m Poincaré plot showing ellipses with a 95% confidence interval for each group. The more rounded ellipse of the Mut Glc + FA condition shows that these cells had a greater beat to beat instability as compared to Mut Glc CMs. Box plot middle line represents the median, x represents mean, bottom line of the box represents the median of the bottom half (1st quartile) and the top line of the box represents the median of the top half (3rd quartile). The whiskers extend from the ends of the box to the non-outlier minimum and maximum value. Source data are provided as a Source Data file