Anne-Marie Koop

5 215 failure occurs within the duration of PAB, as measured by decreased cardiac output, RV systolic pressure will decrease, biasing results. Such biases can be avoided or minimized by assessing RV pressure load at 2 weeks after PAB surgery, instead of at termination. By means of echocardiography, assessment of RV afterload at this time point can be performed reliably and safely. This allows grouping of the mice into groups with equal pressure load, which could be helpful for intervention studies. Also, repeated measurements are easily feasible. Themost critical step in the surgical protocol is the separation of the arteria pulmonalis from the aorta and the subsequent placement of the suture loop. This has to be performed gently in order not to cause any rupture, because this would result in fatal bleeding. PAB in mice requires that well-trained microsurgeons perform the actual banding, including knotting the suture, which should be done very carefully. The current model aims to generate chronic RV pressure load, resulting in RV remodelling, RV dysfunction, and eventually RV failure. Therefore, adequate tightening of the PAB is important. During the development of the model, it has become apparent that small differences in tightness of the banding significantly affected the profile of RV adaptation: e.g., the use of a 25 G needle appeared to be “too tight”, as it induced high rates of mortality during surgery. Needles <23 G were “too loose”, as they did not induce the desired phenotype of RV remodelling and dysfunction. The most critical step in the echocardiographic examination is adequate measurement of pulmonary flow velocity (step 3.3.7). One has to make sure that the angle of the probe is correct: the pulmonary artery has to be exactly vertically visible within the image. Otherwise, flow velocity, and therefore the PAB gradient, are underestimated. It is important to try to limit the length of time of the procedures during the experiment, especially CMR. Furthermore, when analyzing the CMR images with postprocessing software, the researcher must become familiar with the manual segmentation and postprocessing guidelines before reproducible results can be obtained. Using CMR as in the current protocol does not enable assessment of flow velocities over the PAB. Therefore, additional echocardiographic measurements using the Doppler mode are inevitable. Due to the PAB and the subsequent marked increase in PA flow, the signal is very clear, making determination of the PAB gradient by echocardiography convenient and reproducible. Notwithstanding, the extra echocardiographic measurements may involve more logistical arrangements. In general, inclusion or exclusion of papillary muscles and trabeculae affects volumes and subsequent functional parameters. Here, we chose to include papillary muscles and trabeculae in blood volumes (and thus exclude from myocardial mass) which