Anne-Marie Koop

50 SUPPLEMENTARY MATERIAL Animal care and experiments were conducted according to the Dutch Animal Experimental Act and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The Animal Experiments Committee of the University of Groningen, the Netherlands approved the experimental protocol. Symptoms and signs of clinical RV failure (ABCDE system) Rats were daily checked for symptoms of RV failure, as described before. 1,2 The A-symptoms were considered present when the animal had a ruffled fur, red discoloration of head and neck (due to decreased cleaning-behaviour) or was less active than previously, despite stimulation. Bodyweight in RVF can either decrease due to low intake or steeply increase due fluid retention in chest and abdomen. The bodyweight-symptom was therefore considered present if there was a change in bodyweight of more than 15 grams in <48 hours. Cyanosis was checked at exposed skin on head, paws and tail. Hampered peripheral circulationwas considered present if both front paws and hind legs/tail were pale and markedly colder than normally. Dyspnea and tachypnea were defined as markedly increased breathing-effort and, -frequency, respectively. Edema and effusions were defined as fluid collection in thorax and/or abdomen, palpable (ascites) and confirmed at termination (pleural/ pericardial effusion and ascites). Voluntary exercise To measure voluntary exercise, 3,4 running wheels were mounted in the rat cages. Five days before PAB/sham surgery, 5 days before the 5-wks time point and 5 days before sacrifice (for those that reached the 11-wks time point; due to the sudden onset of clinical RVF (<48u), exercise testing could not be performed in PAB- and PAB+CF rats at end point), rats were allowed to run in the cage wheel. Running distance was recorded daily using a digital magnetic counter (Commodoor Cycle Odometer, Commodoor, the Netherlands) and used as a measure of voluntary exercise. Because of large inter-individual variation, the percentage change in run distance versus baseline was used as outcome. Echocardiography Echocardiography was performed at 5 weeks and at termination in all animals. Echocardiography was performed as described previously 4 using a Vivid Dimension