137 Metabolic maturation increases susceptibility to hypoxia-induced damage in human iPSC-derived cardiomyocytes 6 Metabolic profiling of non-MM and MM iPSC-CMs after hypoxia exposure To investigate whether hypoxia affects mitochondrial function of iPSC-CMs, we compared the oxygen consumption rates (OCR) of MM iPSC-CMs and non-MM iPSC-CMs 40 days after differentiation in normoxia and hypoxia (1% O2) using Seahorse metabolic analysis. To study the role of glucose in hypoxia-mediated mitochondrial damage in the non-MM iPSCs, we used RPMI-Glu/B27 as high glucose medium and RPMI-Lac/L as low glucose medium (Figure 1). In normoxia, MM iPSC-CMs showed the highest OCR, indicating increased used of oxidative phosphorylation (Figure 4a-c). Under hypoxic conditions, non-MM iPSC-CMs cultured in RPMI-Glu/L maintained responsiveness, while non-MM iPSC-CMs cultured in RPMI-Lac/L lost responsiveness to the mitochondrial respiratory chain inhibitors (Figure 4a, b). Glucose deprived (RPMI-Lac/L ) non-MM iPSC-CMs showed decreased OCR in normoxia compared to non-MM iPSC-CMs in glucose-rich medium (RPMI-Glu/B27) illustrating metabolic starvation caused by the absence of glucose (Figure 4a, b). While MM iPSC-CMs showed the highest OCR in normoxic conditions and similar responses to the mitochondrial inhibitors as the non-MM iPSC-CMs in glucose medium, 24 hours of hypoxia caused a massive drop in OCR and complete loss of mitochondrial flexibility in MM iPSC-CMs as non-MM iPSC-CMs maintained responsiveness to oligomycin, FCCP and antimycin A and rotenone. 24 hours hypoxia significantly decreased basal respiration, ATP production and maximal respiration in both non-MM and MM iPSC-CMs where the strongest decrease in OCR was observed in MM iPSC-CMs. Spare respiratory capacity and non-mitochondrial respiration was only significantly decreased in MM iPSC-CMs and not in non-MM iPSC-CMs. Additionally, comparative analysis of non-MM and MM-iPSC-CMs cultured in the same plate showed an increased ratio of apoptotic cells (TUNEL+ ) in MM iPSC-CMs (4 hours, 1% O2: 23,8% ± 1,5%; 24 hours, 1% O2: 93,0% ± 2,4% vs. 21% O2: 7,92% ± 1,09%; P<0,001), while no increase in apoptosis was observed for non-MM iPSC-CMs (4 hours, 1% O2: 1,18% ± 0,2%; 24 hours, 1% O2: 10,6% ± 6,6% vs. 21% O2: 1,1% ± 0,23%; P>0,05; Figure 4d, e). This showed metabolic maturation in MM is responsible for increased sensitivity to hypoxia. All together, these results provide further support for our findings that MM iPSC-CMs depend on oxidative phosphorylation and consequently are more sensitive to hypoxia than non-MM iPSC-CMs cultured in glucose-rich or low glucose/lactate media. These results strongly suggest that iPSC-CMs, cultured conventionally and in the presence of glucose, resemble the immature embryonic CM phenotype with respect to their energy substrate utilization, active metabolic pathways, and survival upon low oxygen exposure. In contrast, MM iPSC-CMs cultured with lipids as the primary energy source, more closely resemble adult CMs, consistent with previous reports18. We found that MM iPSC-CMs are more sensitive to hypoxia, leading to minimized mitochondrial function, DNA fragmentation, and ultimately apoptosis.
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