127 Metabolic maturation increases susceptibility to hypoxia-induced damage in human iPSC-derived cardiomyocytes 6 Significance Statement The immaturity of human iPSC-derived cardiomyocytes (iPSC-CMs) remains a roadblock for disease modelling. This study shows that only after metabolic maturation in low glucose, high oxidative substrate media, iPSC-CMs become susceptible to hypoxia-induced cellular damage. Inhibition of necroptosis prevented hypoxia-induced decrease in mitochondrial respiration and cell death in metabolically matured iPSC-CMs. Together, these findings suggest that metabolically matured iPSC-CMs are susceptible to hypoxia damage, representing a key step for establishing valid in vitro models of cardiac ischemia. INTRODUCTION Ischemic heart disease is a major cause of death worldwide.1 The decrease in oxygen and nutrient availability in the myocardium leads to cardiomyocyte (CM) death and therefore loss of cardiac contractile force.2 Current clinical therapies focus on early reperfusion of the ischemic tissue, thereby decreasing the tissue damage which occurs after myocardial infarctions. To develop improved therapeutic approaches to protect the heart from ischemic damage, both animal models and in vitro disease modelling platforms have been used frequently. However, although cardioprotective factors have shown promising therapeutic effects in in vitro cell models and in animal experiments, they often failed in showing clear beneficial effects in clinical trials.3 The roles of comorbidities, ageing, and the use of medication, often neglected in preclinical models, have been considered as reasons for translational failure.4 Moreover, existing models do not robustly reflect the human CM-specific pathophysiology due to marked differences between CMs from humans compared to other species5, e.g., with respect to calcium handling6, electrophysiology6, myofilament composition7,8, maturation expression profile9, and metabolism.10 The development of human induced pluripotent stem cell (iPSC) technology11 and their differentiation to cardiomyocytes (CMs)12 opened doors for more suitable human-based cardiac disease modelling by the generation of patient-specific CMs13 and pre-clinical screening of therapeutics.14,15 Despite the mentioned advantages linked to their human origin, iPSC-CMs, typically derived from a 20-day differentiation protocol, display a foetal rather than adult CM phenotype.16,17 Adult human CMs generate 90% of their energy from mitochondrial oxidative phosphorylation while neonatal rat CMs and iPSC-CMs use glycolysis as their main energy source18, as reflected in a lower expression of TCA cycle and fatty acid β-oxidation markers.19,20 Thus, human iPSC-CMs can have limited clinical validity and predictive value as models of pathophysiological processes, especially when linked to oxidative metabolic processes.19,21 Several studies focussed on increasing the maturation of iPSC-CMs by stimulating the post-natal shift from anaerobic glycolysis-dependent metabolism to aerobic β-oxidation.18,22–24 Recently, a study showed increased cell death of iPSC-CMs metabolically matured for 8 days in glucose deprived, fatty acid rich media, upon submission to in vitro ischemia-reperfusion injury by applying two hours of complete oxygen deprivation (0% O2) followed by four hours of reperfusion (20% O2). 25 In a similar attempt, we
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