| dc.description.abstracteng | The guanine exchange factor p63RhoGEF was identified as a specific activator of the RhoGTPase RhoA and its transcript was found to be abundant in brain and heart tissue. Amongst cardiac cells, the highest expression of p63RhoGEF was detected in smooth muscle cells and cardiac fibroblasts. Within this thesis the relevance of p63RhoGEF in the healthy myocardium and under pathological conditions was under investigation with a special focus on its function in cardiac fibroblasts in the context of auto- and paracrine signaling.
In a model of cardiac fibrosis, in which wild type mice were subjected to transverse aortic constriction (TAC), p63RhoGEF expression was found to be up-regulated in the myocardium. Furthermore, its expression correlated positively with the fibrosis marker CTGF (connective tissue growth factor) as well as with the level of hypertrophy, and negatively with the contractility of the heart. Due to the lack of specific mouse models targeting cardiac fibroblasts, the role of p63RhoGEF was first assessed with the help of 2D and 3D cultures of neonatal rat cardiac fibroblasts (NRCF). In 2D culture it was demonstrated that p63RhoGEF regulates the expression and secretion of CTGF involving the activation of the serum response factor (SRF) and its actin-dependent co-factor myocardin-related transcription factor (MRTF). Moreover, confocal microscopy studies revealed a partial co-localization of p63RhoGEF and CTGF at the trans-Golgi network. In 3D engineered connective tissue cultures (ECT), p63RhoGEF was demonstrated to regulate viscoelastic properties. While overexpression of p63RhoGEF increased the stiffness, the dominant negative truncated p63ΔN decreased the rigidity of the tissue. An influence of p63RhoGEF on CTGF expression in ECT could be detected. CTGF itself was however not sufficient to regulate viscoelastic properties of ECT. Next the heterogeneous engineered heart muscle model (EHM) was used to demonstrate that p63RhoGEF overexpression in cardiac fibroblasts enhanced resting and twitch tension, whereas p63ΔN reduced both. In contrast, overexpression of p63RhoGEF in cardiomyocytes (CM) failed to improve the contractile function validating the importance of p63RhoGEF in cardiac fibroblasts for cell-cell communication via paracrine signaling.
In the following, a global p63RhoGEF knockout mouse line was used to study the effect of a complete (KO) and partial (HET) deletion of p63RhoGEF. In HET mice already under basal condition an impaired cardiac phenotype was present, while the KO mice showed no major difference compared to the WT phenotype. Mice subjected to TAC developed all heart failure within 5 weeks after intervention. While the HET mice showed the most detrimental cardiac dysfunction accompanied by a faster dilation of the left ventricle, the KO animals resembled the WT phenotype. Interestingly, mortality was increased in KO male mice leading to a survival rate of less than 60% within the first 2 weeks after TAC. So far, excessive hypertrophy and fibrosis as a cause could be excluded. In the surviving animals the degree of hypertrophy was identical after 5 weeks independent of the genotype. However, in the HET mice a decrease in fibrosis could be detected.
To further study the role p63RhoGEF in adult mouse cardiac fibroblasts (AMCF), cells were isolated from the generated mouse line and displayed a prominent myofibroblast character in culture regardless of the genotype. The genetic deletion of p63RhoGEF in these cells exhibited a paradox signaling. While the total RhoA and cytoskeletal protein expression was unchanged, the genetic deletion resulted in a higher phosphorylation of direct ROCK targets cofilin and ERM proteins, increased CTGF secretion and up-regulation of fibrosis-associated factors TGF- and collagen in a dose-dependent manner showing the most pronounced response detected in the KO AMCF. Moreover, the knockout of p63RhoGEF led to activation of cell survival and growth pathways and improved proliferation as well as prevented cell death. | de |