138 Chapter 6 D Figure 4 21% O2 24h 1% O2 4h 1% O2 24h non MM iPSC-CM MM iPSC- CM TUNEL ACTN1 DAPI E MM-iPSC-CM (MM) non-MM iPSC-CM (RPMI-Glu/L) non-MM iPSC-CM (RPMI-Lac/L) 0 25 50 75 100 TUNEL+ CMs (%) 21% O2 24h 1% O2 4h 1% O2 24h *** *** P=0,2 A 1 10 20 30 40 50 60 70 80 90 100 110 0 50 100 150 Time (min) Mitochondrial Respiration OCR (pMol/min) Oligomycin FCCP Antimycin A & Rotenone B 21% O2 24h 1% O2 24h RPMI/Glu/L 1 10 20 30 40 50 60 70 80 90 100 110 Time (min) RPMI-Lac/L Oligomycin FCCP Antimycin A & Rotenone 0 50 100 150 Mitochondrial Respiration OCR (pMol/min) C 1 10 20 30 40 50 60 70 80 90 100 110 Time (min) 0 50 100 150 Mitochondrial Respiration OCR (pMol/min) Oligomycin FCCP Antimycin A & Rotenone MM 21% O2 24h 1% O2 24h 21% O2 24h 1% O2 24h Spare Respiratory Capacity Maximal Respiration Basal Respiration ATP production Proton Leak Non mitochondrial Respiration ** ** **** * 0 50 100 150 Mitochondrial Respiration OCR (pMol/min) Spare Respiratory Capacity Maximal Respiration Basal Respiration ATP production Proton Leak Non mitochondrial Respiration **** **** **** 0 50 100 150 Mitochondrial Respiration OCR (pMol/min) Spare Respiratory Capacity Maximal Respiration Basal Respiration ATP production Proton Leak Non mitochondrial Respiration **** **** **** **** **** 0 50 100 150 Mitochondrial Respiration OCR (pMol/min) Figure 4. Metabolic profiling of non-MM iPSC-CMs and MM iPSC-CMs exposed to hypoxia A-C. Normalized real-time oxygen consumption rate (OCR) of non-MM iPSC-CMs in RPMI-Glu/B27 (A), RPMI-B27 (B) and MM iPSC-CMs (C) in normoxia (21% O2, 24 hours), short-term hypoxia (1% O2, 4 hours) or long-term hypoxia (1% O2, 24 hours) by Seahorse extracellular flux analyser. Cells were treated with oligomycin, FCCP, and antimycin A and rotenone to measure mitochondrial respiration. D. Comparative images of TUNEL staining of cardiomyocytes used in metabolic profiling. E. Quantification of TUNEL staining. A-C: 2 cell lines, 2 biological replicates, 1-2 technical replicates (n=5). Scale bar: 200 μm. Data was analysed using one-way ANOVA and Dunnett multiple comparison. *P<0,05; **P<0,01; ***P<0,001; ****P<0,0001. Data are shown as mean ± SEM.
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