217 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 Figure 4: Examination of metabolic activity between PLN-R14del and control hiPSC-CMs. A) Tetrazolium assay showed a significantly lower general metabolic activity in PLN-R14del hiPSC-CMs when compared to the controls. B) A simple diagram illustrating utilization pathways of fatty acids and glucose in cardiomyocytes and two commonly used compounds, etomoxir (ETO) and 2-deoxyglucose (2-DG), to study the capacity of these pathways by blocking their utilization, respectively. C) An overview illustrating Seahorse XF24 Extracellular Flux assay, which measures the oxygen consumption rate (OCR) to study the activity of the fatty acid oxidation (FAO) in PLN and wild-type hiPSC-CMs by manipulating the FAO and glucose metabolism using etomoxir (ETO) and 2-deoxyglucose (2-DG). D) An overview illustrating Seahorse XF24 Extracellular Flux assay, which measures extracellular acidification rate (ECAR) to study the activity of the glycolytic pathway in PLN-R14del and wild-type hiPSC-CMs by manipulating the FAO and glucose metabolism using ETO and 2-DG. E-G) OCR of hiPSC-CMs cultured in the maturation medium, the glucose-rich medium, and the lipid-rich medium, respectively (grey arrow indicates a switch in energy substrates). Quantification of OCR values at the basal level, after ETO injection, and after 2-DG injection, were normalised to nuclei count. The degree of FAO dependency is determined by the reduction of mitochondrial function after ETO injection, and the degree of metabolic flexibility is determined by the OCR after 2-DG injection. H-J) ECAR of hiPSC-CMs cultured in the maturation medium, the glucose-rich medium, and the lipid-rich medium, respectively (grey arrow indicates a switch in energy substrates). Quantification of ECAR values at the baseline level, after ETO injection, and after 2-DG injection, were normalised to nuclei count. The degree of glycolysis dependency is determined by the total glycolysis minus the non-glycolytic acidification (after 2-DG injection). The glycolytic reserve ability is determined by ECAR after ETO injection minus ECAR after 2-DG injection. Data are expressed as mean ±SD, biological replicates are 3 individual differentiations with each N= 5-12 wells. One-way ANOVA with Tukey's post-hoc comparison or Student's t-tests were used, P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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