Simulated Ischemia-Reperfusion Injury

Following 48 h in serum-free DMEM, cardiomyocytes plated on 12-well tissue plates were subjected to simulated ischemia, induced by replacement of culture media with sterile-filtered Krebs buffer (118 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 50 mM EDTA. 11.0 mM glucose, 1.75 mM CaCl₂), before incubation for 6 h at 37°C under hypoxia (95% N₂-5% CO_2, using a hypoxia chamber, QNA International, Melbourne, VIC, Australia), with subsequent 48 h reoxygenation as described previously (Qin et al., 2017b (link)). Agonists were dissolved in DMEM or Krebs buffer, and were present for the full duration of hypoxia (H) and reoxygenation (R). At the end of 48 h reoxygenation, culture supernatant aliquots were collected on ice and stored at -80°C for assessment of cardiomyocyte injury via levels of lactate dehydrogenase (LDH) released (Cyto-Tox-One^TM Homogeneous Membrane Integrity Assay, Promega Inc, Dane County, WI, United States). Levels of cardiac troponin (cTnI) released from cardiomyocytes were also determined using a high-sensitivity rat cTnI ELISA kit (Life Diagnostics Inc, West Chester, PA, United States) as per manufacturer’s instructions.