207 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 INS+ medium was replaced by the maturation medium for a minimum of 3 weeks, which metabolically matured hiPSC-CMs. Next, to study the metabolic capacity of hiPSC-CMs, they were continuously cultured in a glucose-rich medium or lipid-rich medium for another 3 weeks before the Seahorse assays and the immunofluorescence analyses. An overview of culture media is shown in Table S6. RNA isolation and transcriptome analysis of hiPSC-CMs using RNA-seq. Total RNA was isolated from cells using TriZol (Ambion) according to the manufacturer’s instructions. DNase treatment was carried out using the RNase-Free DNase Set (Qiagen). The quality of total RNA was first examined using Bioanalyzer 6000 Pico Kit (Agilent), and mRNA was selected. Libraries were prepared and sequenced as indicated before. Raw counts per gene per sample were imported to the Galaxy server and differentially expressed genes (Table S7) between PLNR14del and control hiPSC-CMs were identified using DESeq2 as indicated above.14 Metabolic measurements of iPSC-CMs using Seahorse assay. Metabolic activity of purified hiPSC-CMs was determined by mitochondrial fatty acid β-oxidation (FAO) and cytosolic glycolysis using the Seahorse XFe24 Extracellular Flux Analyzer (Seahorse Bioscience). Briefly, long-term cultured hiPSC-CMs (77-159 days old) were first seeded to Matrigel-coated Seahorse XFe24 assay plates at a density of 50,000 cells/well and cultured in the standard culture medium for 2 days. Afterwards, the standard culture medium was replaced by a glucose-rich or lipid-rich medium and refreshed every other day. To measure the oxygen consumption rate (OCR, pmol/min/µg protein) and extracellular acidification rate (ECAR, mpH/min/µg of protein), hiPSC-CMs were first washed three times with 0.5 ml fresh Seahorse medium (Agilent Technologies/Seahorse Bioscience) supplemented with 2% B27, 1% Chemically Defined Lipid Concentrate (Gibco), 4mM L-glutamine (Gibco), and 10mM glucose (Agilent, 103577-100) and then kept in the same medium for 1 hour in a non-CO2 incubator at 37 °C. Afterwards, 100 µM etomoxir (ETO, a specific irreversible inhibitor of carnitine palmitoyltransferase 1, Agilent) was added to determine FAO dependency by measuring the oxygen consumption rate (OCR, pmol/min/µg protein), followed by the addition of 50 mM 2-Deoxy-D-glucose (2DG competitive glycolytic inhibitor, Sigma Aldrich) to determine cytosolic glycolysis by measuring extracellular acidification rate (ECAR, mpH/min/µg of protein).17 Three measurements were taken before and after each injection and mixing cycle. Both OCR and ECAR were compared between PLN-R14del and wild-type hiPSC-CMs. Besides, immunofluorescent staining with alpha-actinin antibody, DAPI, and Nile-red was performed to demonstrate the purity and the accumulated lipids of both PLN-R14del and wild-type hiPSC-CMs included in the Seahorse assay (Fig.S9). Seahorse mitochondrial FAO assay by measuring OCR: Each parameter for % OCR from baseline we assessed and calculated as described below: (1) Baseline: the last measurement of basal
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