Modeling Hypoxia-Reoxygenation Injury in Metabolically Matured Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Myocardial infarction (MI) remains a leading cause of death and heart failure (HF). Despite advancements in percutaneous coronary intervention, about 5-20% of patients die, and further 20-30% develop HF within one-year post-MI. The infarct size and the extent of tissue damage are critical predictors for mortality, hospitalization for HF, and left ventricular remodeling after MI. However, although numerous compounds have successfully reduced infarct size in animal models, no drug is yet clinically available that promotes the survival of cardiac cells—particularly cardiomyocytes—during the hypoxia or reperfusion phases of a MI. Thus, human models of hypoxia-reperfusion (H/R) injury are urgently needed to identify novel cardioprotective targets and mechanisms.

In this study, we aimed to establish a human H/R injury model based on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Modelling hypoxia-induced injury using iPSC-CMs remains challenging due to their immature phenotype and distinct metabolic profile compared to adult CMs. We have previously demonstrated that culturing iPSC-CMs in lipid-supplemented maturation medium (MM) and under electrostimulation (ES) strongly enhances their metabolic and functional phenotype. RNA-seq analysis revealed a strong upregulation of mitochondrial and metabolic pathways in iPSC-CMs cultured under MM+ES compared to MM, including mitochondrial biogenesis, cristae formation, TCA cycle and electron transport chain. Additionally, flow cytometry measurements confirmed an increased Tom20 mean fluorescence intensity in iPSC-CMs matured under MM+ES compared to MM, further supporting their mitochondrial maturation.

To investigate whether the metabolic maturation state of iPSC-CMs affects their hypoxia-sensitivity, we compared the viability of iPSC-CMs cultured under MM and MM+ES following incubation under hypoxia (1% O₂) and glucose-starvation. Strikingly, we observed substantial cell death of iPSC-CMs that were matured under MM+ES already after 4 hours of hypoxia (1% O₂), as evidenced by strong changes in cell morphology and significantly elevated lactate dehydrogenase (LDH) activity in medium supernatant. Live-dead staining and flow cytometry confirmed a significant increase in dead cells under these conditions. Importantly, incubation of iPSC-CMs in starvation medium under normoxia did not affect cell viability, demonstrating that cell death was induced by hypoxia. In contrast, iPSC-CMs cultured in MM showed only minor changes in cell morphology and a small increase in LDH activity in supernatant even after 24 hours of hypoxia. To simulate H/R injury, iPSC-CMs matured under MM+ES were subjected to 4 h of hypoxia and rapid reoxygenation was induced by exchanging the hypoxic medium with fresh medium containing glucose and fatty acids. We detected further release of LDH in the supernatant of iPSC-CMs 6 hours after reoxygenation, suggesting continuous death of iPSC-CMs during the reoxygenation period.

Taken together, our findings demonstrate the crucial role of metabolic maturation and mitochondrial development for modelling of H/R injury using human iPSC-CMs. In ongoing studies, we are investigating the metabolic and molecular changes in iPSC-CMs during hypoxia and hypoxia-reoxygenation injury.