Langendorff Experimental Protocols and I/R

The I/R experiments were performed on isolated rat hearts as described previously. 22 , 23 The hearts were rapidly removed and cannulated through the aorta in a modified Langendorff apparatus and perfused at the constant flow of 10 mL/min with KHB solution: in mmol/L: NaCl 118; NaHCO₃ 25; KCl 4.7; KH₂PO₄ 1.2; MgSO₄ 1.2; CaCl₂ 1.25, and glucose 11, at 37C and equilibrated with a gas mixture of 95% O₂ and 5% CO₂ (pH 7.4). The perfusion temperature was held constant by a heat exchanger located next to the aortic cannula. A fluid‐filled latex balloon was inserted through the left atrium into the left ventricle and connected to a pressure transducer and the PowerLab System (AD Instruments, Australia) for continuous left ventricular pressure recording. The left ventricular end‐diastolic pressure (LVEDP) was set to 10 mm Hg by balloon inflation; during the experiment, the hearts were continuously immersed in 37C warm buffer to avoid hypothermia. Hearts were allowed to stabilize for 20 minutes before a protocol was started. All hearts were subjected to 30 minutes of basal recordings. The ischemia protocol was induced by a full stop of retrograde perfusion for 30 minutes. The reperfusion was performed by full reestablishment of retrograde perfusion during 10 minutes for mitochondria analysis or 60 minutes for infarct size measurements.

No I/R: Hearts were perfused with KHB for 70 minutes (only for mitochondria isolation); I/R: Global ischemia was induced for 30 minutes by complete perfusion arrest followed by 10 minutes (for mitochondrial function assessment) or 60 minutes (for infarct size measurements) of reperfusion with KHB solution; CDNF preconditioning (preCDNF): The hearts were perfused with the KHB solution containing CDNF (1 µmol/L) for 5 minutes before I/R protocol; CDNF postconditioning (postCDNF): After 30 minutes of global ischemia, CDNF (1 µmol/L) was perfused during the first 5 minutes of reperfusion, followed by 5 or 55 minutes of reperfusion with KHB solution without CDNF. In these 2 groups the antagonists to previously reported cardioprotective pathways were used and the substances were perfused for 5 minutes before CDNF and along with CDNF (1 µmol/L). The antagonists used were wortmannin 0.3 µmol/L (PI3K‐AKT inhibitor), chelerythrine 10 µmol/L and rottlerin 10 µmol/L (PKC inhibitors), and AG490 10 µmol/L (JAK‐STAT3 inhibitor).