Anne-Marie Koop

32 occlusion caused immediate fatal deterioration of cardiac function in the majority of PAB+CF rats precluding this method of determining elastance. Echocardiography was performed at 5 weeks and at termination as described before. 18 Apical 3- and 4-chamber views and parasternal short- and long axis views were used to measure ventricular and atrial dimensions and tricuspid annular plane systolic excursion (TAPSE) and to assess tricuspid insufficiency. The gradient across the PAB was measured using continuous wave Doppler. Cardiac output was calculated as (aorta diameter) 2 * 3.14 * velocity time integral * heart rate, using systolic aorta diameter and pulsed wave Doppler measurements of aorta flow. Beat-to-beat variation was accounted for by averaging measurements from 6 to 12 consecutive beats. Voluntary exercise was measured using running wheels mounted in the cages for 5 consecutive days at baseline and after 5 weeks as described previously 6,16,18 and in the online-only Data Supplement. Histological analyses Ventricular remodelling was studied using RV free wall tissue. We used using standard histological techniques for measurement of RV cardiomyocyte cross- sectional area, fibrosis, capillary density and macrophage infiltration, as described in the online-only Data Supplement. Gene expression Changes in gene expression in the RV myocardium were investigated using transcriptome-wide expression profiling and qRT-PCR for specific genes. Total RNA was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA); high quality was confirmed (RQI 9.3) using Experion (Bio-Rad, Veenendaal, the Netherlands). For the array-studies, RNA was purified for individual rats (n=7/4/5 CON/PAB-/PAB+CF) using theQiagen RNeasymini kit (Venlo, TheNetherlands); RNA quality was verified (RIN >9) (Agilent, Amstelveen, the Netherlands). Biotin-labeling, hybridization, washing, scanning of GeneChip Rat Gene 1.1 ST arrays (Affymetrics) and processing in the MADMAX pipeline (Nutrigenomics Consortium, Wageningen, The Netherlands) 19 using Bioconductor software packages were all performed according to standard Affymetrix protocols at expert labs. Extensive quality control is described in the online-only Data supplement. Array data are deposited at the Gene Expression Omnibus (GEO) database (GSE46863). Significantly regulated genes were related to biological processes using DAVID. 20 Gene set enrichment analysis, 21 which includes expression of all genes (not just