3 Hz) through a pair of Ag/AgCl electrodes attached to the upper region of the right ventricle. Mechanical activity was investigated by measuring developed left ventricular isovolumic systolic pressure (LVISP). To evaluate contractility the rate of rise of LVISP (dP/dt) was used because it is highly sensitive to changes in contractility ( Gleason and Braunwald, 1962). These parameters were measured

with a pressure transducer connected to an amplifier (MP 100 Biopac Systems: Inc.; CA) and recorded with a data acquisition click here system (BIOPAC MP100WSW, including a software Acqknowledge III, Goleta, CA). The isovolumic pressure derivative (dP/dt) was gotten offline by the same software (digital filter Blackman −61 dB, 25 KHz of cut frequency and sample rate of 1000/s). All measurements began 30 min after mounting to allow the beating preparation to adapt to the in vitro conditions. The coronary perfusion pressure (CPP) was continuously registered by connecting a pressure transducer (TDS 104A) to the inflow of the aortic pressure tube. Since coronary flow was kept constant (10 mL/min),

changes of the CPP were dependent on changes of coronary resistance. Protocols were performed beginning with a constant diastolic pressure of 5 mm Hg by adjusting the volume of the balloon. Ventricular function curves were obtained by measuring the left ventricular isovolumic systolic pressure (LVISP) developed while diastolic pressure was increased from 0 to 30 mm Hg in steps of 5 mm Hg. Balloon volume was kept

constant during experiments involving other protocols; Metformin purchase this permitted changes Amrubicin in diastolic and systolic pressures to be measured. Initially, recordings were taken under control conditions in both groups. In order to analyze inotropic response, a single dose of isoproterenol (Sigma, St Louis, MO, USA) in bolus (100 μl, 10 μM) was administered to evaluate β-adrenoceptor response. Some animals were killed at the end of hemodynamic measurements. The hearts were rapidly frozen in liquid nitrogen and kept at − 80 °C until the day of analysis. Briefly, as previously reported (Moreira et al., 2003), myosin was prepared from minced and homogenized left ventricles extracted with KCl-phosphate buffer (0.3 M KCl and 0.2 M phosphate buffer [pH 6.5](Klotz et al., 1975)). Myosin ATPase activity was assayed according to previous reports (Klotz et al., 1975 and Cappelli et al., 1989) by measuring inorganic phosphate (Pi) liberation from 1 mM ATP in the presence of 50 mM HEPES (pH 7), 0.6 M KCl, 5 mM CaCl2, or 10 mM ethylene glycol-bis (β-amino ethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) in a final volume of 200 μL. Samples were assayed in duplicate or triplicate and corrected for non-enzymatic hydrolysis by using controls assayed in the same conditions, except that the protein sample was added after the interruption of the reaction by using 200 μL of 10% trichloroacetic acid.