2.4. Langendorff Isolated Rat Heart Perfusion

The Langendorff technique for perfusion of isolated rat hearts has been described in a previous study [27]. All rats were anesthetized with thiopentone (65 mg/kg intraperitoneally (i.p.)); each of the hearts was quickly excised and placed in ice-cold Krebs–Henseleit (K-H) buffer. Every heart was gently pressed to remove residual blood and then mounted on the Langendorff apparatus. The heart was subjected to retrograde perfusion at a constant flow with K-H buffer (aerated with 95% O₂ and 5% CO₂) at 37°C and 70 mmHg. A water-filled polyethylene balloon was inserted into the left ventricle (LV) through the left atrium and connected to a pressure sensor via a fluid-filled tube to measure cardiac hemodynamics. Left ventricular end-diastolic pressure (LVEDP) was maintained at 5–10 mmHg by adjusting the volume of the water-filled balloon. Cardiac hemodynamic parameters were recorded using a biological function acquisition system (Chengdu Taimeng Technology Co., Ltd., China) that measured LVEDP, left ventricular systolic pressure (LVSP), heart rate (HR), maximum change rate of left ventricular pressure (±dP/dt_max), and coronary flow (CF).

Isolated rat hearts were randomly divided into seven groups (n = 10 per group): vehicle control, I/R, VT+I/R, I/R+8-CPT, VT+I/R+8-CPT, I/R+ESI-09, and VT+I/R+ESI-09. The drug concentrations used were 10 μM VT, 30 μM 8-CPT, and 1 μM ESI-09. In the control group, the rat hearts received a 90 min constant infusion of KH buffer alone. In the I/R group, the rat hearts were perfused with KH buffer for 30 min, followed by a 30 min cessation of perfusion, and finally 30 min of washout with buffer alone. In the I/R+drug groups, the rat hearts were perfused with KH buffer for 10 min, then perfused with KH buffer containing corresponding drugs for 20 min, followed by cessation of perfusion for 30 min, and finally resumption of KH drug perfusion for 30 min.