4HNE Treatment of Cardiomyocytes

HL1 cardiomyocytes were cultured in a humidified 5% CO₂, 37°C incubator with complete Claycomb medium (51800C; Sigma‐Aldrich; St. Louis, MO) containing 10% FBS, 2 mmol/L GlutaMAX (35‐050‐061; ThermoFisher Scientific; Waltham, MA), 100 µmol/L norepinephrine (A0937; Sigma‐Aldrich), 300 nmol/L ascorbic acid (A7506; Sigma‐Aldrich), and 100 U/mL penicillin‐streptomycin (P4333; Sigma‐Aldrich). The HL1 cells were treated for 1 hour with 50 µmol/L of 4HNE (32100; Cayman Chemical; Ann Arbor, MI), where ethanol was used as the vehicle. 100 and 200 µmol/L 4HNE treated cells demonstrated decreased cell viability by 3,[4,5‐dimethylthiazol‐2‐ yl]‐2,5‐diphenyl‐tetrazolium bromide assay (Figure S4A) and mitochondrial swelling suggestive of imminent cell death while 50 µmol/L 4HNE treated cells did not (Figure S4B). Thus, to investigate the effect of 4HNE at a sublethal dose, 50 μmol/L 4HNE was used. The effect of 4HNE treatment on HL1 cardiomyocytes was similar to that seen in human induced pluripotent stem‐cell derived cardiomyocytes as we have previously shown. 37 We therefore used HL1 cardiomyocytes in this study since it is a readily accessible source of cardiomyocyte mitochondria of consistent quality.