Anne-Marie Koop

2 53 at dual wavelengths of 450/540 nm. Data are presented as quantities of cGK activity expressed in units per ug protein, as measured with the BioRad DC Protein Assay. PKA activity was measured using the MESACUP Protein Kinase Assay (MBL CO., Ltd, Nagoya, Japan). Sample preparation was done similarly to PKG sample preparation with the same extraction buffers. 100 uL of each prepared sample was added to each well (incubated for 10 min, 25°C) followed by stop solution. After washing 100 uL biotinylated antibody 2B9 was added (incubated for 60 min, 25°C) and again the plate was washed. The addition of POD-conjugated streptavidin (incubated for 60 minutes at 25°C), substrate solution (incubated for 3 min at 25°C ) and stop solution was interspersed by washing steps. The absorbance was read at a wavelength of 492 nm. Data are presented as relative PKA activity. qRT-PCR To characterize the hypertrophy response and study the regulation fibrosis and capillary growth, expression of the fetal gene program (myosin heavy chain isoforms, natriuretic pro peptides type A and B) and markers of hypertrophy (ACTA, ACTC, RCAN1), fibrosis (TGF β β -1, OPN-1, Col1A2, Col3A1), capillary growth (VEGF-A, VEGF-R1, VEGF-R2), and oxidative stress (HO-1, NOX-4) were measured. We also specifically measured mRNA expression of genes involved in the regulation of systolic and diastolic function (SERCA-ATPase, phospholamban, sodium-calcium exchanger (NCX) and titin isoforms N2B and N2Ba. RV (free wall) tissue was snap-frozen in liquid nitrogen. 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), before conversion to cDNA by QuantiTect Reverse Transcription (Qiagen, Venlo, the Netherlands). Gene expression was measured with Absolute QPCR SYBR Green ROX mix (Abgene, Epsom, UK) in the presence of 7.5ng cDNA and 200nM forward and reverse primers. qRT-PCR was carried out on the Biorad CFX384 (Bio-Rad, Veenendaal, the Netherlands) using a standard protocol. Primer sequences are available upon request. mRNA levels are expressed in relative units based on a standard curve obtained by a calibrator cDNAmixture. All measured mRNA expression levels were corrected for 36B4 reference gene expression. Transcriptome-wide expression profiling Total RNA was isolated from the right ventricular free wall using TRI reagent (Sigma, St. Louis, MO) according to the manufacturer’s protocol. RNA was purified for individual rats (n=7/4/5 CON/PAB-/PAB+CF) using the Qiagen RNeasy mini kit (Venlo, The Netherlands); RNA quality was verified (RIN >9) (Agilent, Amstelveen, the Netherlands). Biotin-labeling, hybridization, washing and scanning of GeneChip Rat