Langendorff isolated perfused heart.

The methodology of Langendorff's perfused heart preparation and measurement of ventricular function has been described previously in detail (25, 26, 28). Briefly, male wild-type C57BL/J6 mice (n = 7) and PRAK^−/− mice (n = 11) were anesthetized with a lethal intraperitoneal injection of pentobarbital sodium (120 mg/kg). Hearts were rapidly excised and arrested in ice-cold Krebs-Henseleit buffer. They were then cannulated via the ascending aorta for retrograde perfusion by the Langendorff method using Krebs Henseleit buffer containing the following (in mM): 110 NaCl, 4.7 KCl, 1.2 MgSO₄·7H₂O, 2.5 CaCl₂·2H₂O, 11 glucose, 1.2 KH₂PO₄, 25 NaHCO₃, and 0.5 EDTA. The buffer, aerated with 95% O₂–5% CO₂ to give a pH of 7.4 at 37°C, was perfused at a constant pressure of 55 mmHg. For experiment protocol, hearts were subjected to 20 min of equilibration and 30 min of ischemia followed by 30 min of reperfusion. A water-filled latex balloon, attached to the tip of polyethylene tubing, was then inflated sufficiently to provide a left ventricular end-diastolic pressure (LVEDP) of ∼10 mmHg measured by means of a disposable Gould pressure transducer. LV functional analysis was recorded using software and a computer-based recording system (MP100A; BIOPAC Systems, Goleta, CA). These parameters included left ventricular systolic pressure (LVSP), LVEDP, heart rate, and cardiac contractile function. The rate pressure product (RPP) was calculated as the product of left ventricular developed pressure and heart rate, where developed pressure is systolic pressure minus LVEDP.