135 Metabolic maturation increases susceptibility to hypoxia-induced damage in human iPSC-derived cardiomyocytes 6 Although iPSC-CMs are continuously exposed to an atmospheric concentration of 21% O2 during cell culture conditions, in myocardial tissue, normoxic oxygen concentrations can be substantially lower.33 We therefore assessed whether a decrease from 21% O2 to a more physiological cardiac normoxia of 5% O2 would already induce damage in MM iPSC-CMs (Figure 3a). Although hypoxia did not change the fraction of ACTN1-positive MM iPSC-CMs in the surviving population (ACTN1+: 92,7 ± 3,35% [24 hours, 21% O2] vs. 93,8 ± 1,7%, P = 0,74 [24 hours, 1% O2]; Supplemental figure 2), both, incubation in 5% O2 and 1% O2, decreased myofibrillar organization, as shown by immunostaining for ACTN1 and cardiac troponin T (cTnT) (Figure 3b, c). An increased ratio of apoptotic (TUNEL+) iPSC-CMs was observed in MM after short periods (4 hours) of exposure to 5% O2 (25,5% ± 1,2%), similar to 1% O2 (23,7% ± 1,6%), compared to atmospheric 21% O2 (15,4% ± 0,83%; p<0,05; Figure 3d, e). Longer hypoxia periods (24 hours) increased percentage of apoptotic iPSC-CMs to 41,5% ± 4,5% (5% O2) and 37,6% ± 3,5% (1% O2) compared to 12,0% ± 1,5% (21% O2; p<0,001; Figure 3d, e). We did not observe a significant difference in the ratio of apoptotic iPSC-CMs between 5% and 1% O2, both after short- (4 hours) or long-term (24 hours) incubation. We collected media after hypoxia for 4 and 24 hours to assess glucose, lactate and lactate dehydrogenase concentrations and found significantly increased glucose consumption after 24 hours at both 5% and 1% O2, reducing glucose levels below the detection limit of 0,6 mM (Figure 3f).
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