Fig. 6

Fatty acid challenged HADHA Mut CMs displayed swollen mitochondria with severe mitochondrial dysfunction. a Representative confocal images of WT and Mut CMs in 12D of Glc + FA media. b Quantification of mitotracker and ATP synthase β colocalization and intensity. ***p < 0.001, t-test followed by a Mann-Whitney rank sum test. n = 40 cells per group measured. c Quantification of Rhod-2 and ATP synthase β colocalization and intensity. ***p < 0.001, t-test followed by a Mann-Whitney rank sum test. n = 53–66 cells measured. d Transmission electron microscopy images of WT and Mut CMs after 12D of Glc + FA media showing sarcomere and mitochondria structure. e Histogram of mitochondria circularity index for WT and HADHA Mut CMs after 12 days of Glc + FA media showed HADHA Mut CMs mitochondria are rounder. n = 81–251 mitochondria measured. f Histogram of mitochondria area for WT and HADHA Mut CMs after 12 days of Glc + FA media showed HADHA Mut CMs mitochondria are smaller. g Quantification of maximum OCR from mitostress assay. Mut and KO CMs as compared to WT CMs after 12D of Glc + FA media had a significantly lower max OCR. WT CM: 359 pmoles/min/cell, Mut CM: 190 pmoles/min/cell, KO CM: 125 pmoles/min/cell. ***p < 0.001, one-way ANOVA was performed vs WT 12D Glc + FA. n = 6–19 biological replicates. h Quantification of ATP production from mitostress assay, calculated as the difference between baseline OCR and OCR after oligomycin. Mut and KO CMs as compared to WT CMs after 12D of Glc + FA media had significantly lower ATP production. WT CM: 93 pmoles/min/cell, Mut CM: 51 pmoles/min/cell, KO CM: 43 pmoles/min/cell. ***p < 0.001, one-way ANOVA was performed vs WT 12D Glc + FA. n = 6–18 biological replicates. i Quantification of proton leak from mitostress assay, calculated as the difference between OCR after oligomycin and OCR after antimycin & rotenone. Mut and KO CMs as compared to WT CMs after 12D of Glc + FA media had significantly higher proton leak. SS-31 treated Mut CMs after 12D of Glc + FA had a significantly lower proton leak and non-treated Mut CMs. WT CM: 3.64 pmoles/min/cell, Mut CM: 7.66 pmoles/min/cell, KO CM: 10.52 pmoles/min/cell. **p < 0.01, ***p < 0.001, one-way ANOVA was performed vs WT 12D Glc + FA. n = 4–19 biological replicates. j Schematic of patient harboring the point mutation, c.1528G > C, in the gene HADHA and the process of obtaining patient skin cells, reprograming them into iPSCs and then differentiating them into iPSC-CMs. k Representative confocal images of HADHA c.1528G > C CMs treated for 12-days of Glc or Glc + FA media. l Quantification of mitotracker and ATP synthase β colocalization and intensity. ***p < 0.001, t-test followed by a Mann-Whitney rank sum test. n = 31–35 cells measured. Box plot middle line represents the median, x represents mean, bottom line of the box represent 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