Inhibition of Prolyl-Hydroxylase Domain Enzymes Protects From Reoxygenation Injury in Engineered Human Myocardium
Authors: Elif Levent, Claudia Noack, Laura C. Zelarayán, Dörthe M. Katschinski, Wolfram-Hubertus Zimmermann, Malte Tiburcy
Journal: Circulation (2020)

reperfusion ◼ stem cells ◼ tissue engineering R eperfusion after revascularization of an acute myocardial infarction can contribute up to 50% of the resulting infarct damage. 1 Protection from reperfusion injury is an important strategy to prevent excessive damage.To date, clinical studies on reducing lethal reperfusion injury have been disappointing, despite promising preclinical data. 2 To improve preclinical drug development and close the apparent translational gap, human preclinical models of ischemia/reperfusion injury may be useful.To address this challenge, we have developed a novel model system of hypoxia followed by reoxygenation in engineered human myocardium (EHM).Hypoxia-inducible factor 1 (HIF-1) is the major switch for cellular adaptation to hypoxia.Its α-subunit HIF-1α is hydroxylated by prolyl-hydroxylase domain enzymes (PHDs) at 2 conserved proline residues that are located in the oxygen-dependent degradation domain in a strictly oxygen-susceptible manner leading to rapid proteasomal degradation (Figure [A]).Hypoxia or inhibition of PHDs stabilizes the HIF-1α protein that dimerizes with ARNT (aryl hydrocarbon receptor nuclear translocator) to activate target gene transcription-promoting metabolic adaptation, cell survival, mitochondrial function, erythropoiesis, and angiogenesis.In rodent models of myocardial infarction, inhibition of PHDs has been shown to stabilize HIF-1α, conferring not only protection when given preinsult but also in a more clinically relevant scenario when given immediately post insult. 3Whether similar effects can be achieved in human myocardium is not known.To monitor the effect of hypoxia and PHD inhibitors (PHDi) in human cardiomyocytes, we established a transgenic human embryonic stem cell line (Institutional Review Board approval obtained; ref. number: 1710-79-1-4-16) expressing the oxygen-dependent degradation (ODD) domain of HIF-1α fused to Firefly Luciferase (Luc) under the control of the CAG promoter.ODD-Luc reporter human embryonic stem cells responded to decreasing levels of ambient oxygen with an oxygen-dependent increase in bioluminescence (Figure [B]).High purity ODD-Luc cardiomyocytes (92±6% sarcomeric actinin-positive) were obtained by directed differentiation and metabolic selection as previously described. 4ODD-Luc cardiomyocytes responded to the addition of the PHDi FG-2216 (EC 50 : 250±3 µmol/L) and FG-4592 (EC 50 : 110±3 µmol/L) in a concentration-dependent manner, demonstrating that tested PHDis are biologically active in human cardiomyocytes (Figure [C]).Consecutive experiments were performed with FG-4592 (Roxadustat), because this drug is a promising first-in-class PHD inhibitor for renal anemia patients and has recent regulatory approval in China.In line with the Luciferase reporter data, FG-4592 stabilized HIF-1α protein (Figure [D]).HIF-1α stabilization was followed by increased expression of prototypical HIF-1 target genes in human cardiomyocytes (Figure [E]) indicative of metabolic adaptation (GLUT1 [glucose transporter 1], LDHA [lactate dehydrogenase A]), angiogenic signaling (VEGFA [vascular endothelial growth factor]), and intact autoregulation (PHD2 [Prolyl hydroxylase domain-containing protein 2]).