Langendorff isolated heart experiments

Female offspring were anesthetized with sodium pentobarbital (150 mg/kg) when they reached 8 weeks of age. The heart was quickly isolated and mounted on a Langendorff isolated heart apparatus as previously described [8]. Krebs solution (in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 25 NaHCO₃, 1.2 KH₂PO₄, 0.5 Na₂EDTA, 11 glucose, and 2.5 CaCl₂, pH 7.4) was perfused at a constant pressure of 80 mm Hg through an aortic cannula. The contractile function of the left ventricle was measured using an intraventricular balloon connected to a pressure transducer and inflated to an end diastolic pressure of 4–8 mmHg. Cardiac contractile function was recorded using a Powerlab 4SP data acquisition system (AD Instruments, Colorado Springs, CO). The heart was maintained at a temperature of 37 °C throughout the experiment, and the temperature of the heart was continuously monitored using a thermocouple placed on the surface of the heart. Hearts were equilibrated for 25 min prior to the onset of 30 min of ischemia followed by 2 hours of reperfusion. Hearts were excluded after the 25 min equilibration period if developed pressure was less than 70 mmHg, coronary flow rate was greater than 15 ml/min (indicative of a leak or damaged vasculature), or if there were persistent arrhythmias.