Anne-Marie Koop

158 METHODS Animal experiments 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, in the Netherlands approved the experimental protocol (permit number: AVD105002015134-2). MaleWistarWU rats (160-180 gram) were randomly subjected to pressure load by means of pulmonary artery banding (PAB, n=25) or sham surgery (control, n=13), and were checked daily for clinical signs of RV failure according to ABCDE criteria, as previously described. 31 Animals were terminated at 2 (n= 5 vs. 4 ), 5 (n= 11 vs. 4 ), and 12 weeks (n= 9 vs. 5) following surgery. Hemodynamic measurements Echocardiographic assessment of PAB-gradient, LV cardiac output (LV CO), stroke volume (SV) eccentricity index end diastolic (EI ED), eccentricity index end systolic (EI ES), and tricuspid annular plan systolic excursion (TAPSE)was performed at 2, 5 and 12 weeks according to previous described protocol. 31 Invasive pressure measurements performed by heart catheterization including end diastolic pressure (EDP), RV contractility (dP/dt max ), RV stifness (dP/dt min ) and cardiac power were performed before termination, whereafter blood and organs were taken out and preserved. For detailed description of hemodynamic measurements, see supplemental methods. Histology Ventricular remodelling was characterized by cardiomyocyte cross-sectional area (wheat germ agglutinin), fibrosis (Masson Trichrome) and capillary density (Lectin) in the RV free wall, as described previously. 4 Perivascular fibrosis greater than 200 μm was excluded for analysis of the section. Gene expression Gene expression ofmarkers of cardiomyocyte stress, hypertrophy, fibrosis, metabolic regulators, substrate transporters, inflammation, oxidative stress, and cardiolipin synthesis and remodelling were assessed at mRNA level measured with standard qPCR as described in detail in the supplemental methods.