208 Chapter 8 respiration before ETO injection (time point 3) was normalised to 100%, and the base measurement was defined by combining three measurements normalised to time point 3. (2) FAO: the three measurements after ETO injection normalised to time point 3. (3) Glycolysis: the three measurements after 2-DG injection normalised to time point 3. (4) FAO dependency: the percentage of fatty acids dependence was calculated by quantifying the change in basal OCR after ETO-inhibited FAO compared to the total mitochondrial function from other substrate oxidation. (5) Metabolic flexibility: the maximal respiration due to the utilisation of other substrates was determined by the OCR after 2-DG-inhibited glycolysis. Seahorse glycolysis assay by measuring ECAR: Each parameter for % ECAR from baseline was measured and processed as follows: (1) Baseline: the last measurement of baseline before ETO injection (time point 3) was normalised to 100%, and the base measurement was defined by combining three measurements normalised to time point 3. (2) Glycolysis: the three measurements after ETO injection normalised to time point 3. Glycolysis was quantified as the maximum percentage increase of ECAR over baseline. (3) Glycolysis inhibition: the three measurements after 2-DG injection normalised to time point 3. (4) Glycolysis dependency: the degree of glycolysis dependency was determined by the reduction of glycolysis after 2-DG injection. (5) Glycolytic reserve was determined by the average ECAR level after ETO injection subtracted from the average ECAR level after 2-DG injection. Statistical analysis of OCR and ECAR measurements: The Seahorse experiments were performed using 3 biological replicates, with each replicate consisting of 5–12 technical repeats per condition. OCR and ECAR rates were normalised to the non-glycolytic acidification rate per well and all values were further normalised to the in situ nuclear staining cell counts (Mean ± SD, n=3 counts/well). To compare OCR and ECAR between PLN-R14del and wildtype hiPSC-CMs, an unpaired t-test or one-way ANOVA (biological replicates n=3, n=5-12 wells per replicate) was used. All data was represented as Mean ± SD and significance was displayed by *p < 0.05; **p < 0.01; ***p < 0.001, 0.001 and ****P ≤.0001. All ANOVA tests were subsequently analysed using Tukey’s post hoc test. Statistical analysis was performed using the GraphPad Prism 8.3 software. Bezafibrate application. Bezafibrate, a PPAR-agonist, was applied to control and PLNR14del hiPSC-CMs. Briefly, all hiPSC-CMs were cultured in the maturation medium for 120 days. Bezafibrate was dissolved in dimethyl sulfoxide (DMSO) and added to hiPSC-CMs at 100 μM. After 24hr incubation, all hiPSC-CMs with and without treatment were fixed for immunofluorescence staining purposes as described below. Optical Ca2+ transient analysis. Ca2+ transient analysis was performed to evaluate Ca2+ handling property between control and PLN-R14del hiPSC-CMs with and without bezafibrate
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