Analysis of mitochondrial respiration

The Seahorse™ XF96 extracellular flux analyzer was used to assess mitochondrial respiration as previously described (Mosqueira et al, 2019), using the Mito Stress Kit (Agilent Technologies). Briefly, cryopreserved isogenic sets of hiPSC‐CMs were seeded into Matrigel™‐coated (BD #356235) XF96‐well plates at a density of approximately 5000 cells/mm². HiPSC‐CMs were cultured for 2 days in RPMI1640 (US Biological Life Sciences #R9010‐01) supplemented with B‐27 with insulin (Life Technologies #0080085‐SA), 2 mM L‐glutamine (Life Technologies #25030‐081), 10% fetal bovine serum (Gibco #16000044), and 0.6 mM CaCl₂. After 2 weeks, medium was exchanged for XF basal medium (Agilent Technologies #102353), supplemented with 10 mM glucose (Sigma #G7528), 1 mM sodium pyruvate (Sigma #S8636), and 2 mM l‐glutamine (Life Technologies #25030‐081) 1 h prior to conduction of the assay. Selective inhibitors were sequentially injected during the measurements (1.5 μM oligomycin, 0.4 μM FCCP, 1 μM rotenone; Agilent Technologies), following the manufacturer’s instructions. The measured oxygen consumption rate (OCR) values were normalized to the number of cells in each well, quantified by 1:400 Hoechst 33342 staining (Sigma #B2261) in PBS (Gibco #14190‐094) using fluorescence at 355 nm excitation and 460 nm emission in an automated imaging platform (CellaVista, Synentec). Statistical analysis was performed by using GraphPad Prism (v7, La Jolla, CA, USA) software, evaluated by unpaired one‐way ANOVA test followed by Tukey post hoc test for correction of multiple comparisons, and differences were considered significant when *P < 0.05.