Extended Data Fig. 8: Suppression of mTORC1 improves the maturation of ACTN2 enh del hPSC-CMs.

a. Gene expression of RAPTOR and 3-phosphoinositidine-dependent protein kinase-1 (PDK1) as part of mTORC1 pathway were upregulated, while RICTOR levels which is part of the mTORC2 complex remained unchanged. (3 independent samples per group). b. Both ACTN2 enh del hPSC-CMs and isogenic controls showed very similar cell size after treatment with everolimus (total ~147 cells/group, 4 independent treatments). c. The morphology of ACTN2 enh del hPSC-CMs did not show any difference as compared to controls after treatment with everolimus. (4 independent samples per group) (bar graph: 25μm) d. ACTN2 enh del hPSC-CMs treated with everolimus (25 μM, mTORC1 inhibitor) normalized their calcium handling properties as compared to treated control hPSC-CMs. Time to peak, peak height and time to 50% to baseline are not different between control and ACTN2 enh del hPSC-CMs. (n = 8–10 batches per group). e. Seahorse analysis of cultured hPSC-CMs show reduced maximum oxygen consumption rate (OCR) and ECAR to OCR ratio (consistent with increased glycolysis and reduced oxidative phosphorylation) for ACTN2 enh del hPSC-CMs compared to isogenic controls. Treatment with everolimus, improved oxygen consumption for both for ACTN2 enh del hPSC-CMs and isogenic controls and normalized glycolysis for ACTN2 enh del hPSC-CMs. (4 independent samples per group). f. Both ACTN2 enh del hPSC-CMs and isogenic controls developed very similar gene expression of maturation related genes after everolimus treatment (3 independent samples per group). Data are presented as mean values +/− SEM. All replicates are biological. The Shapiro-Wilk test was performed to assess normal distribution, and the parametric student t-test or ANOVA with Bonferroni’s multiple comparisons test or the non-parametric Mann-Whitney (two-tailed) or Kruskal Willis with Dunn’s multiple comparisons tests were used as appropriate. Only P values < 0.1 are reported.