Anne-Marie Koop

256 and i13L-transducer (GE Healthcare, Waukesha, WI, USA) from a short-axis view at aortic level, at day 5 and week 6 after sham or PAB surgery. MRI was performed by using a vertical 9.4T 89-mm diameter bore scanner (Bruker BioSpin, Etllingen, Germany) equipped with 1,500 mT/m gradienset (Bruker BioSpin GmbH, Ellingen, Germany). Respiratory and heart rate were derived using a pressure pad placed under the chest of the mouse. The longitudinal axis of the RV was determined with two and four chamber scout scans, where after axes were adjusted to actual axes. Slices of longitudinal axis, four chamber view, and ten or eleven slices of the short- axis of one millimeter and no slice gap were obtained. Slices were derived including complete apex and base of the right ventricle. Cine imaging was performed with a retrospectively-triggered (self-gated) gradient-echo sequence (Paravision 4.0 and IntraGate, Bruker Biopspin GmBH) with the following settings: TR=6.8 ms, TE= 1.9 ms, number of movie frames= 15, slice thickness= 1 mm, matrix= 256 x 256 and field of view= 30 x 30. The myocardium was manually segmented by drawing the epicardial and endocardial contours, excluding the papillary muscles using QMass (version MR 7.6, Medis Medical Imaging Systems, Leiden, The Netherlands). Semiautomatic segmentation was used to determine end-diastolic volume (EDV), end-systolic volume (ESV), and wall thickness (WT). Stroke volume (SV) was calculated as EDV- ESV. Ejection fraction (EF) was calculated as (EDV - ESV)/EDVX100. Cardiac output (CO) was calculated manually as SV x mean observed heart rate. Septal flattening is expressed by the eccentricity index, both end diastolic and systolic, which was calculated by dividing the diameter of the left ventricular diameter parallel to the intraventricular septum by the diameter perpendicular to the intraventricular septum derived from short-axis at the mid-papillary level. RNA isolation, cDNA conversion and Real-time RT-PCR Total RNA was isolated from mouse heart tissue using Direct-zol™ reagent (ZYMO) according to manufacturer’s instructions. RNA (1 ug) was then reverse-transcribed with either M-MLV reverse transcriptase (Promega, Madison, WI, USA). Quantitative real-time polymerase chain reaction (qPCR) was performed on a BioRad iCycler (Biorad) using SYBR Green reagent (VWR). Transcript quantities were compared using the relative Ct method, where the amount of target normalized to the amount of endogenous control (L7 for mRNAs) and relative to the control sample is given by 2–ΔCt. Primer sequences for mRNA detection are depicted in supplemental table 1 . Histology, Immunohistochemistry and immunofluorescence For histological analysis, hearts were perfusion-fixed with 4% paraformaldehyde, embedded in paraffin and cut into 4-μm sections. Paraffin sections were stained