Cardiomyocyte isolation from adult mice

Ventricular myocytes were enzymatically isolated from the hearts of C57BL/6 mice using a protocol modified from that described previously (Zhang et al., 2013). Briefly, the animal was euthanized by cervical dislocation. The heart was immediately removed from the chest, raised and perfused with oxygenated modified Ca2+ free-Tyrode's solution for 5 min. Then the heart was perfused with 50 ml low Ca2+ solution containing 300 U/ml collagenase II + 0.5 mg/ml hyaluronidase at 37°C for 20–30 min. The ventricle was cut into small pieces and further digested under gentle agitation. Rod-shaped adult cardiomyocytes were collected by settling down of cells and plated in 24 well plates for XF24e Extracellular Flux Analyzer analysis (Seahorse Bioscience) or on glass coverslips for confocal imaging.