Isolation of Adult Mouse Cardiomyocytes

The ALDH2 KO mice were provided by University of Occupational and Environmental Health (Fukuoka, Japan). The cardiomyocytes were isolated from adult male WT mice and ALDH2 KO mice (all were C57BL/6 background) as described previously (Ackers-Johnson et al., 2016 (link); Pang et al., 2016 (link)). Briefly, mice were sacrificed after anaesthetized with 2% isoflurane, and hearts were rapidly excised and mounted onto a temperature-controlled (37°C) Langendorff system (ADInstruments). The hearts were perfused retrogradely through the aorta with collagenase II (Sigma-Aldrich), collagenase IV (Sigma-Aldrich), and collagenase IV (Sigma-Aldrich) for about 20 min until digestion was apparent. The digested left ventricles were then cut into ~1 mm³ pieces and dissociated by pipetting 2 min. After four sequential rounds of gravity settling using three intermediate calcium reintroduction buffers to gradually restore calcium concentration to physiological levels, the cell pellet which was enriched with myocytes was collected and used for the experiments within 8 h after isolation.