Anne-Marie Koop

230 parasternal long axis and four-chamber views respectively. MRI was performed at six weeks in a 9.4T MRI scanner (Bruker BioSpin, Ellingen, Germany) equipped with 1,500 mT/m gradient set. Respiratory and heart rate were derived using pressure pad placed under the chest of the mouse. The longitudinal axis of the right ventricle was determinedwith a two and four chamber scout scans, where after axes were adjusted to actual axes. Slices of longitudinal axis, four chamber views, and ten or eleven slices of the short-axis of onemillimeter and no slice gap, were obtained. Sliceswere 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 parameters: TR = 6.8 ms, TE = 1.3 ms, number of movie frames = 15, slice thickness = 1 mm, matrix = 256 x 256, field- of-view = 30 x 30 mm 2 . 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). Semi- automatic 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 100%(EDV - ESV)/EDV. Cardiac output (CO) was calculated manually as SV x mean observed heart rate. Septal flattening is expressed by 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 mid-papillary level. RNA isolation, cDNA conversion and Real-time RT-PCR Total RNA was isolated from mouse heart tissue using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. Then RNA (1 mg) was reverse-transcribed with either M-MLV reverse transcriptase (Promega, Madison, WI, USA) or for miRNA transcript detection with miScript Reverse Transcription Kit (Qiagen). Real-time PCR was performed on a BioRad iCycler (Biorad) using SYBR Green (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 U6 (miScript Primer Assays) for miRNAs) and relative to the control sample is given by 2–ΔCt. Primer sequences for mRNA detection are depicted in table 1 . Histology, Immunohistochemistry and immunofluorescence microscopy For histological analysis, hearts were arrested in diastole, perfusion-fixed with 4% paraformaldehyde, embedded in paraffin and cut into 4-μmsections. Paraffin sections