Isolated, perfused heart experiments.

Ex vivo cardiac function was assessed in isolated mouse hearts perfused in the Langendorff mode. The heart was excised and perfused with a constant pressure of 80 mmHg (33). A water-filled balloon was inserted into the LV to record isovolumic ventricular function with a data-acquisition system (PowerLab, AD Instruments, Colorado Springs, CO). The KH buffer perfusate contained the following (in mM): 118 NaCl, 25 NaHCO₃, 5.3 KCl, 2 CaCl₂, 1.2 MgSO₄, 0.5 EDTA, and 10 glucose, equilibrated with 95% O₂-5% CO₂ (pH 7.4). After an equilibration period, the heart was subjected to isoproterenol (0.05 μM) with KH buffer containing glucose as the sole oxidative energy substrate for 30 min. The heart was then perfused with a solution containing 100 μM iodoacetate (IAA) (Sigma-Aldrich) for another 30 min. Temperature was maintained at 37.5°C throughout the protocol.