Anne-Marie Koop

2 51 7 system and 10S-transducer (GE Healthcare, Waukesha, WI, USA). Rats were anesthetized with isoflurane (5% induction; 2-3% maintenance; a pulse-oxymeter (Nonin Medical, Plymouth, MN, USA) was used to monitor adequacy of anesthesia). We used apical 3- and 4- chamber views and parasternal short and long axis views to measure RV dimensions, tricuspid insufficiency, TAPSE, and gradient across the PAB. Cardiac output was calculated using systolic aorta diameter and pulsed wave Doppler of aorta flow as (aorta diameter) 2  × 3.14 × velocity time integral (VTI) x heart rate. The mean of measurements from 6-12 consecutive beats with a proper signal was taken to average out beat-to-beat variation. Pressure-volume analysis At termination, hemodynamic characterization of the RVwas performed by pressure- volume analysis, obtained by RV catheterization according to a previously described protocol. 4 Rats were anesthetized with isoflurane (5% induction; 2-3% maintenance; a pulse- oxymeter (Nonin Medical, Plymouth, MN, USA) was used to monitor adequacy of anesthesia), intubated and ventilated. Analgesiawas applied using buprenorphine 0.01 mg/kg s.c. at the start of the procedure. Subsequently the rat was positioned supine under a stereomicroscope (Zeiss, Hamburg, Germany) and fixated on a temperature- controlledwarmingpad.The right jugularveinwasdissectedandcannulated facilitating hypertonic saline infusions. Following bilateral thoracotomy and pericardiotomy a combined pressure-conductance catheter (SPR-869, Millar Instruments Inc., Houston, TX, USA) was introduced via the apex into the RV and positioned in the RV outflow tract. RV pressures and conductance were recorded using a MPVS 400 processor at a sample rate of 1.000 Hz with Chart 5 (Millar Instruments Inc., Houston, TX, USA). Subsequently, via the dissection in the neck, the right carotid artery was exposed and the catheter was introduced via the right carotid artery and ascending aorta into the LV to measure LV pressures. Blood loss during the procedure was minimal (<0.5mL). Analyses were performed offline using custom-made software (CircLab 2012, P. Steendijk). Steady-state pressure-conductance data were obtained by averaging the values of 3 steady-state recordings (at least 7 loops each). Parameters obtained from pressure-volume loops included heart rate, peak pressure, end diastolic pressure and maximal and minimal first time-derivative of pressure (dP/ dtmax and dP/dtmin). The relaxation time constant (tau) was calculated as the time constant of monoexponential decay of RV pressure during isovolumic relaxation. Stroke volume (in arbitrary units) derived from the conductance signalwas calibrated, using stroke volume (in mL) measured by echocardiography. End systolic and end diastolic elastance were determined using the single-beat method; 5,6 vena cava