Anne-Marie Koop

52 occlusion caused immediate fatal deterioration of cardiac function in the PAB+CF rats precluding this method to determine elastance. Organ weights, staining After heart catheterization, the rats were euthanized by removing the heart from the thorax. Heart, lungs and liver were dissected. RV, interventricular septum, LV and both atria were separated and weighed. The liver lobe and lung lobe were weighed, dried overnight at 65°C and weighed again to determine wet weight/dry weight ratio. Midventricular RV sections were fixated (formalin) and stained to assess cardiomyocyte cross-sectional area (wheat germ agglutinin), fibrosis (Masson Tri-chrome), capillary density (lectin) and macrophages (CD68) as described previously. 3,4,7,8 Microscopy-imaging was performed at the UMCG Imaging Center (UMIC), which is supported by the Netherlands Organization for Health Research and Development (ZonMW grant 40-00506-98-9021). Western blot Protein was extracted using RIPA buffer; protein concentration was measured using Protein Assay (Bio-Rad Laboratories B.V., Veenendaal). Protein was put on a gel and after electrophoresis semi-dry blotting was performed. Protein transfer to the blot was confirmed with Ponceau S staining. After blotting, the membrane was blocked using 5% Elk in TBS/0,1% Tween for at least 30 min and incubated with antibodies specific to SERCA, PLN and phosphorylated PLN and standard secondary antibodies. Tubulin was used as a loading control. Protein detection and quantification was done with ImageQuant LAS 4000 (GE Healthcare Life Sciences). Protein kinase activity assays PKG activity was measured in RV (free wall) tissue using the cyclex cyclic GMP dependent protein kinase (cGK) assay kit (CycLex Co., Nagano, Japan). Samples were prepared in extraction buffer (50 mM potassium phosphate buffer, 1 mM EDTA, 1 mM EGTA, 5 mM DTT, 4 ul/mL phosphate inhibitor), potassium phosphate buffer and DE-buffer (20 mM Tris-HCl, 60 mM NaCl, 0,5 mM EDTA, 1 mM EGTA, 4 uL/mL phosphatase inhibitors) according to the assay protocol. 100uL of each sample was then added to the plate, which was precoated with recombinant G-kinase substrate. After incubation (30 min, 30°C) and a washing step, incubation with 100uL of HRP conjugated anti-phopho-specific antibody for an hour at room temperature was performed. After another washing step, substrate reagent was added to incubate for 15 minutes; with stop solution the reaction was terminated. Absorbance was read in