Anne-Marie Koop

6 231 were stained with hematoxylin and eosin (H&E) for routine histological analysis, Sirius Red for detection of fibrillar collagen and FITC-labelled wheat-germ-agglutinin antibody (WGA, Sigma, 1:100) to visualize and quantify the cell cross-sectional area. Isolectin B4 staining (GSI-biotin, Vector, 1:100) was performed to visualize the capillaries in cardiac tissue. Slides were visualized using a Zeiss Axioskop 2Plus with an AxioCamHRc. Modification of Isolectine B4 staining with additional fluorescence labeled-streptavidin (Dylight 595-conjugated streptavidin, Jackson Thermo, 1:100) and counterstaining with FITC-labeled WGA was performed to assess capillary to cardiomyocyte ratios. Collagen deposition, cell surface areas and capillary density were determined using ImageJ software. Slides were visualized using a Leica DM2000 and a Leica DM3000 microscope for bright field and fluorescence imaging, respectively. Statistical Analysis All data are presented as mean values ± standard error of mean (SEM), unless otherwise specified. The variables were analyzed using Student’s t-test and analysis of variance (ANOVA) to assess statistical significance between groups. The significant effects evaluation was conducted using Tukey’s multiple comparison tests, with an adjusted calculation of p -value. Probability values p<0.05 were considered statistically significant. The strength of relationship between cardiac output and miR- 199b expression as well as between RVEF and miR-199b expression was assessed by Pearson product correlation coefficient formula. All analyses were done using GraphPad Prism software V5.04 (GraphPad software, Inc, La Jolla, CA, USA). RESULTS Cardiac expression of miR-199b in RV remodelling induced by PAB To assess whether miR-199b is involved in RV failure, we subjected WT mice and TG mice with cardiac-specific overexpression of miR-199b (MHC-199b) 33 to sham or pulmonary artery banding (PAB) surgery for 6 weeks ( figure 1a ). Two weeks after PAB surgery, both WT and MHC-199b mice displayed a similar increase in PAB gradient ( figure 1b ) which slightly increases over time. Six-weeks after banding, both WT and MHC-199b showed similar PAB gradients indicating the same degree of pressure overload imposed on these groups. Real-time PCR revealed upregulation of miR- 199b in WT mice after PAB ( figure 1c ). Despite the elevated miR-199b expression levels in MHC-199b sham-operated mice, MHC-199b PAB animals revealed increased levels of miR-199b upon six weeks of RV pressure overload ( figure 1c ), indicating that RV stress further induces miR-199b expression.