Right ventricular remodeling refers to changes in size, shape and function of the right heart after cardiac injury. It is a progressive disorder connected to several conditions, including pulmonary hypertension. It is also a leading predictor for the development of right heart failure and death. Nonetheless, until recently the importance of right ventricular function in health and disease has been often underestimated. Previous studies showed that Wnt signaling pathway plays an important role in left ventricular remodeling in response to hypertrophic stimuli. While all of the available literature describes the impact of this signaling cascade in maladaptive growth of the left ventricle, its role in the right heart hypertrophy is unknown. Given that, we hypothesized that canonical Wnt signaling could also play a role in the development of right heart hypertrophy. As the Wnt pathway is crucial for the development of right ventricle we speculated its contribution to RVH could be more prominent.In this study, we employed and characterized the PAB and MCT animal models of right ventricular hypertrophy. Both models presented with substantial right heart hypertrophy as judged by RV/LV+S ratios as well as mRNA expression of known hypertrophic markers. Additionally, the right ventricles of PAB and MCT animals showed advanced fibrotic changes.We were able to show an upregulation of several Wnt signaling molecules, including beta-catenin, GSK3ß and Frizzled receptors on mRNA level in hypertrophied right ventricles of PAB animals. Most importantly, beta-catenin and GSK3beta protein levels were also elevated in those animals. Similarly, we found increased mRNA levels of GSK3beta and Fz2 receptor in the right ventricles of MCT rats. beta-catenin and GSK3beta protein levels showed a tendency to be elevated in those animals but did not reach significance level. Furthermore we found a significant upregulation in beta-catenin expression in cardiac fibroblasts derived from right ventricles of PAB animals as compared to Sham-RVs implying that CFs could be at least in part responsible for observed changes. Based on BrdU incorporation experiments, we alsofound that right ventricular fibroblasts exhibit increased proliferation rates. To further elucidate if this increased proliferation could be dependent on activated canonical Wnt signaling we performed several experiments employing Wnt3a stimulation and siRNA mediated beta-catenin knockdown.The hallmark of Wnt canonical pathway is the accumulation and nuclear translocation of beta-catenin. We could show both, accumulation of beta-catenin in the cytoplasm as well as increased beta-catenin nuclear levels in RCFs stimulated with Wnt3a. Wnta3a-mediated activation of Wnt signaling resulted further in the upregulated expression of several genes, most prominently Axin2 and Cyclin B1. We could further link Wnt/beta-catenin signaling with elevated collagen synthesis as well as increased cardiac fibroblast proliferation, two fundamental processes of myocardial fibrosis. We showed that Wnt3a stimulation leads to elevated mRNA levels of Collagens 1, 3 and 4 as well as total collagen synthesis in RCFs. Additionally we could observe increased collagen secretion. In accordance, siRNA-mediated beta-catenin knockdown resulted in decreased collagen expression. We further showed that Wnt3a stimulation was sufficient to increase the levels of expression of two Wnt/beta-catenin downstream genes - Cyclin D1 and c-Myc, both of which positively regulate cell proliferation. Likewise, Wnt3a stimulation and LiCl treatment resulted in significantly elevated rates of BrdU incorporation as compared to control. Finally, beta-catenin knockdown resulted in reduction in DNA synthesis rates as compared to scrambled siRNA control.In order to confirm our findings in vivo we recruited BAT-Gal reporter mice and subjected them to pulmonary artery banding procedure. Activation of Wnt-canonical pathway was assessed by beta-Galactosidase nuclear staining of cryosections derived from those animals. Although beta-Galactosidase positive cells were present in all included groups, we observed a substantially greater amount of those cells in the right ventricles of rats subjected to PAB. Thus, activation of Wnt/beta-catenin signaling pathway occurs in vivo and we can conclude that this signaling is important for the development of right ventricular remodeling and could eventually offer new treatment strategies.
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