Anne-Marie Koop

6 233 indicative of a stronger cardiac stress-induced MHC-isotype switch these animals ( figure 3d-e ). Taken together, these results indicate that cardiac miR-199b overexpression sensitizes the RV to pressure overload by inducing stress marker gene expressi Downstream effectors of miR-199b in RV remodelling Next, we investigated the mechanisms by which miR-199b may induce exaggerated RV remodelling and dysfunction following chronic pressure overload. Our group has previously established that miR-199b exerts its pro-hypertrophic function in the pressure-overloaded LV by directly regulating the calcineurin/nuclear factor of activated T-cell (CnA/NFAT) pathway. As calcineurin activation also may contribute to RV remodelling induced by pulmonary artery banding 32 we assessed Rcan1-4 transcript expression levels, a sensitive marker of cardiac NFAT activity, in PAB hearts. As expected, a significant upregulation of calcineurin/NFAT signaling was induced by RVpressure overload. However, no differenceswere observed betweenWTandMHC- 199b hearts ( figure 3f ). Although we could observe lower levels of dual-specificity tyrosine-phosphorylation regulated kinase 1a ( Dyrk1a ), a previously validated target gene of miR-199b, these differences were not statistically significant ( figure 3g ). We further assessed expression levels of transforming growth factor beta ( Tgfb ) as a growth factor involved in fibrosis and cardiomyocyte hypertrophy, 35 and as a potential target gene of the miR-199 family. Although we observed a clear increase in Tgfb mRNA in banded-hearts, no differences were observed between WT and MHC- 199b mice ( figure 3h ). Similar results were obtained for Endoglin , a coreceptor for Tgf β - signaling ( figure 3i ). As we did not detect major differences regarding collagen deposition at the histological level between the WT and TG animals after PAB, we also assessed the expression levels of collagen type I alpha 1 chain ( Col1a1 ) as a fibrosis-related gene. At the molecular level, we observed a clear upregulation of Col1a1 in MHC-199b hearts compared to WT hearts when subjected to RV pressure overload ( figure 3j ).