141 Metabolic maturation increases susceptibility to hypoxia-induced damage in human iPSC-derived cardiomyocytes 6 for Seahorse metabolic profiling analysis. Cell damage from reperfusion is caused by the release of reactive oxygen species upon the sudden increase in oxygen availability42. Hypoxia and reoxygenation have previously been shown to induce only limited damage and prolonged hypoxia without reoxygenation did not affect beating frequency in immature iPSC-CMs36. This is in line with our results of maintained beating frequency in immature, non-MM iPSCCMs after prolonged hypoxia. A previously observed limited effect of hypoxia compared to reperfusion injury on calcium overload related cellular damage in immature iPSC-CMs43, potentially explains why our seahorse results do show a response of iPSC-CMs in RPMImedia to hypoxia. During late human embryonal development, the glycolytic metabolism is insufficient to generate adequate ATP levels for cardiac contractions leading to the metabolic shift towards oxidative phosphorylation.39 The increased oxygen availability around birth destabilizes the HIF1 transcription factor complex, resulting in the mitochondrial shift towards more energy-efficient oxidative phosphorylation-based metabolism.38,44 With this increased availability of ATP, CMs mature with a concomitant increase in ploidy, myofibrillar organization, and number of mitochondria providing for increased energy demands of cardiac contractility.39 However, these necessary changes in functional and structural phenotype seem to limit metabolic flexibility. We observed that MM iPSC-CMs, as opposed to non-MM iPS-CMs were sensitive to hypoxia-induced cell death and showed decreased mitochondrial respiration after 4 hours of hypoxia. Additionally, lactate production plays a vital role in glycolysis and was not observed in normoxic conditions or in short-term hypoxia, but did increase in long-term hypoxia, indicating that MM iPSC-CMs eventually shift towards oxidative phosphorylation of fatty acids. This observation was in line with the drop in glucose levels in these conditions without an increase in lactate production. Interestingly, we showed that 5% O2, considered physiological normoxia in most tissue33, induced a similar degree of cell death as 1% O2 for MM iPSC-CMs. This suggests, that in our model hypoxia-induced cell death pathways are already induced at a decrease from atmospheric 21% O2 to 5% O2, in contrast to previous studies suggesting 0,5-2% O2 as effective cellular hypoxia43. This difference in physiological tissue normoxia and cell culture normoxia is an important factor to take into account when further developing in vitro hypoxia models. Within this study, differentiation of iPSC to iPSC-CMs is conducted at atmospheric O2, whereas during cardiac development in vivo CMs differentiate and mature at lower physiological concentrations (5-8% O2). 33,37 We have shown, while glycolytic non-MM iPSC-CMs were insensitive to hypoxia, MM iPSC-CMs showed increased sensitivity to hypoxia and even physiological O2 concentrations. Using a monolayer of a single cell-type in an in vitro setting alongside hyperoxia during cardiac differentiation are likely to contribute further to sensitivity to O2 concentration considered physiological. Further investigation could determine thresholds of specific O2 concentrations resulting in activation of hypoxia signalling (e.g., HIF1α stabilization) in iPSC-CMs. This could be followed up by hypoxia studies using viable cardiac tissue slices45 or engineered heart tissues46 to circumvent limitations of monolayer single cell-type models, although obviously
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