Hypoxia-Reoxygenation in Cardiac Myocytes

Cardiac myocytes were subjected to hypoxia using 1× Esumi lethal ischemic media, pH 6.2 (containing 117 mmol/L NaCl, 12 mmol/L KCl, 0.9 mmol/L CaCl₂, 0.49 mmol/L MgCl₂, 4 mmol/L HEPES, 20 mmol/L sodium lactate, and 5.6 mmol/L L-glucose) by sealing the myocytes in humidified hypoxic chambers (Billups-Rothenberg, Inc) 16 (link), flushing each chamber with a gas mixture consisting of 5% CO₂ and 95% N₂ for fifteen minutes, and incubating the hypoxic chamber in a modular incubator at 37°C for three hours. Following hypoxia, the media was changed to 1× Esumi control media (pH 7.4, 137 mmol/L NaCl, 3.8 mmol/L KCl, 0.9 mmol/L CaCl₂, 0.49 mmol/L MgCl₂, 4 mmol/L HEPES, and 5.6 mmol/L D-glucose) and culture dishes reoxygenated for one or six hours in the modular incubator or on the fluorescent microscope during imaging, as appropriate. Similarly treated NRCMs were subjected to four or 9 hours of normoxia in 1× Esumi control media to serve as normoxic/aerobic controls.