139 Metabolic maturation increases susceptibility to hypoxia-induced damage in human iPSC-derived cardiomyocytes 6 Preconditioning with Nec-1 protects metabolically matured iPSC-CMs from hypoxic injury To determine the utility of our damage model as a platform to screen for protective agents, we preconditioned the cells with the necroptosis inhibitor necrostatin-1 (Nec-1) and assessed whether the previously found protective effects34,35 could be reproduced (Figure 5a). Supplementation of MM-iPSCs with Nec-1 24 hours prior to hypoxia, indeed resulted in a significant decrease in cell death compared to control (fold change EthD1/calcein AM ratio: 0,47 ± 0,06 with Nec-1, P<0,01; Figure 5b, c). MM iPSC-CMs preconditioned with Nec-1 showed a significant increase in OCR (Figure 5d, e), as opposed to non-MM iPSC-CMs (Supplemental figure 3). Furthermore, Nec-1 preconditioning increased the expression of the mitochondrial outer membrane protein TOMM20 compared to control, indicating increased cellular mitochondrial content (Figure 5f, g). DISCUSSION In the present study, we confirmed that the conventional method for differentiation of iPSCCMs using RPMI/B27-based medium (RPMI-Glu/B27) generates cells with low sensitivity to hypoxia and consequential cell death. We furthermore demonstrated that applying metabolic maturation of iPSC-CMs increased sensitivity to hypoxia, rendering these matured cells better models for in vitro models of cardiac ischemia. The observed ability of conventionally cultured, non-MM iPSC-CMs to increase the glycolytic flux in anaerobic conditions is in line with the characteristics of foetal immature CMs having a higher threshold for oxygen insufficiency.36–38 During development, arterial blood oxygen saturation fluctuates around 3% O2, which would be considered a hypoxic condition in the adult human heart, with respect to activation of hypoxia-induced gene expression.37 However, foetal immature CMs are conditioned to low oxygen pressures during development and thus rely on anaerobic energy pathways for metabolism and cardiac growth. Mechanistically, it has been shown that transcription factor hypoxia-inducible factor 1 (HIF1 ) is stabilized in foetal CMs under low oxygen pressures and plays a key role in maintaining and enhancing glycolytic metabolism via LDH-A regulation in the compact myocardium and counteracting CM maturation.23,38,39 Also in iPSC-CMs the inhibition of HIF1 has been shown to lead to CM maturation and a metabolic shift from aerobic glycolysis towards oxidative phosphorylation.24 Frequently used “high glucose” cell culture media containing glucose at concentrations of ~15 mmol/L do not relate to human physiology, with local glucose concentrations of ~3 mmol/L, and have been considered obstructive for iPSC-CM maturation.40 Furthermore, a four-fold postnatal increase in concentration of circulating fatty acids enables and drives increased activation of fatty acid β-oxidation pathways in adult cardiomyocytes.40,41 Despite relying mainly on metabolization of glucose, we did observe a decrease in mitochondrial respiration rate in non-MM iPSC-CMs following hypoxia. This could be related to the short intermittent reoxygenation step required
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