2.2. Isolated Rat Heart Model of Ischemia Reperfusion Injury

Isolated mammalian heart model according to Langendorff was used for establishment of myocardial IRI [12]. The ex vivo method involved anesthetization of a rat (ketamine 80 mg/kg + xylazine 20 mg/kg) followed by the excision of heart and perfusion with Krebs-Henseleit buffer (118.0 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl₂, 1.2 mM MgSO₄, 25.0 mM NaHCO₃, 1.2 mM KH₂PO₄, and 10.1 mM glucose, pH 7.4), maintained at 37°C with continuous oxygenation (95% O₂+ 5% CO₂). The heart was stabilized for 20 min on the perfusion system (ADInstruments, Bella Vista, New South Wales, Australia) by maintaining a constant perfusate pressure of 70 mmHg. Hemodynamic changes were monitored using a pressure transducer connected to a latex balloon placed in the left ventricle. Electrical recordings were continuously made using a PowerLab data acquisition system (ADInstruments) and analyzed using the LabChart Pro 8 software (ADInstruments) [12].

The experimental groups included sham, fisetin + sham, I/R alone, and fisetin pretreatment, followed by I/R (fisetin + I/R). Fisetin (20 mg/kg; TOCRIS Bioscience, Bristol, UK) was injected intraperitoneally 1 h before the induction of ischemia. We performed pilot experiments to ascertain a suitable dosage regimen of fisetin, and our observations revealed that administration of fisetin at (20 mg/kg) consistently provided the optimal cardioprotection. Therefore, we adopted this dose for further experiments.

A typical I/R protocol consisted of 30 min of ischemia induced by stopping the buffer flow, followed by 60 min of reperfusion induced by resuming the flow. Throughout the duration of the experiment, hemodynamic parameters were continuously monitored and the perfusate was collected at the end of reperfusion for biochemical analysis. At the end of the experiment, the hearts were immediately frozen in liquid nitrogen and stored at −80°C until further analysis.