Bardoxolone methyl triggers Ferroptosis in cardiomyocytes

Introduction: Bardoxolone methyl (Bardo), a Kelch-like ECH-associated protein 1 (Keap1)–Nrf2 pathway activator, has demonstrated efficacy in slowing eGFR decline in diabetic kidney disease (DKD). However, its Phase 3 trial in stage 4 DKD was terminated owing to unexpected cardiovascular complications. Methods: To explore the underlying mechanisms, the human cardiomyocyte cell line AC16 was subjected to various concentrations of Bardo. Nuclear translocation of Nrf2 and the expression of its downstream antioxidant factors, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1), were quantified. Cell injury was assessed using microscopy, crystal violet staining, and lactate dehydrogenase release assays. The research employed ferroptosis, apoptosis, and necrosis inhibitors to identify the mechanisms of cell death. Additional analyses included measurements of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), reactive oxygen species (ROS), ferrous ions, and malondialdehyde (MDA), while mitochondrial ultrastructure was evaluated by transmission electron microscopy. Results: Bardo induced dose-dependent Nrf2 activation and increased AC16 cell death, which was attenuated by the ferroptosis inhibitor Ferrostatin-1 (Fer-1) but not by apoptosis or necrosis inhibitors. Mechanistically, Bardo suppressed SLC7A11 and GPX4 expression while elevating ROS, ferrous ions, and MDA levels. Ultrastructural analysis further revealed mitochondrial volume reduction, disrupted cristae, and increased membrane density. Discussion: These findings establish that Bardo induces ferroptosis in cardiomyocytes, potentially explaining the cardiotoxic effects observed in clinical trials.