*2.2. Heart Isolation*

The animals were anesthetized by the intraperitoneal injection of 100 mg/kg ketamine along with 1000 U heparin to prevent blood clotting. After a negative response to a noxious stimulus, the animals were euthanized by decapitation followed by thoracotomy. The aorta was cannulated distal to the aortic valve, and the heart was perfused retrograde with oxygenated Krebs solution (4 ◦C) containing (in mM) 148 Na<sup>+</sup>, 4.7 K<sup>+</sup>, 1.2 Mg<sup>2</sup>+, 1.6 Ca2+, 127 Cl<sup>−</sup>, 27.8 HCO3<sup>−</sup>, 1.2 H2PO4<sup>−</sup>, 1.2 SO4<sup>2</sup><sup>−</sup>, 5.5 glucose, 2 pyruvate, 0.026 EDTA, and 5 U/l insulin. Both the venae cavae were ligated, and the heart was rapidly placed into a Langendorff support system and perfused at a constant pressure of 70 mmHg at 37 ◦C. The perfusate was equilibrated with ~95% O2 and ~5% CO2 to maintain a constant pH of 7.40 and filtered in-line (5 μm pore size). The isovolumetric left ventricular pressure (LVP) was measured with a saline-filled latex balloon (Radnoti LLC, Monrovia, CA, USA) inserted into the left ventricle. The diastolic LVP was initially adjusted to 10 mmHg at baseline (bl) so that any subsequent pressure increases reflected diastolic contracture. Systolic, diastolic, and developed (systolic–diastolic) LVP, and its maximal and minimal first derivatives (dLVP/dtmax and dLVP/dtmin) were calculated as indices of ventricular contractility and relaxation, respectively. Electrodes attached to the right atrial and ventricular walls monitored atrial and ventricular electrocardiograms to calculate the spontaneous heart rate (HR) and identify arrhythmias. The rate-pressure product (RPP) as the product of developed pressure and HR was calculated to correct for HR-dependent changes in developed pressure. The coronary flow was measured in-line with an ultrasonic flowmeter (model T106X; Transonic Systems, Ithaca, NY, USA). All the analog signals were digitized (PowerLab/16 SP, AD Instruments; Castle Hill, Australia) and recorded at 200 Hz (Chart & Scope version 5.6.6, AD Instruments) for later analysis.
