Analysis of the GPCR-induced RhoA signaling in healthy and diseased adult cardiomyocytes

Background: The aim of the presented thesis was to analyze the activation of the monomeric G protein RhoA in adult cardiomyocytes and further to unravel the role of the guanine nucleotide exchange factor p63RhoGEF in this context. Previous data demonstrated that RhoA is activated in neonatal cardiomyocytes by GPCR ligands like endothelin-1 (ET-1), phenylephrine (PE) and angiotensin II (AngII). So far, a contribution of the Gq/11 protein-regulated p63RhoGEF was demonstrated in the ET-1-mediated RhoA activation in neonatal cardiomyocytes. Moreover, RhoA was described to be permanently activated in pressure-overloaded hearts and p63RhoGEF to be higher expressed in the same system. This led to the hypothesis that p63RhoGEF is involved in the GPCR-dependent RhoA regulation in adult cardiomyocytes. Methods: The obtained data result from combined analysis of isolated wildtype (WT), healthy (sham) and diseased (transverse aortic constriction, TAC) adult mouse cardiomyocytes (AMCM) as well as of cholesterol-depleted AMCM (MβCD-AMCM) and of heterozygous (HET) and homozygous (KO) p63RhoGEF-knockout AMCM. p63RhoGEF-constructs were adenovirally overexpressed in WT-AMCM. The activity of RhoA was studied with the help of an antibody which specifically recognizes the conformation of active RhoA (RhoA-GTP) by immunofluorescence stainings and confocal imaging. Changes in RhoA activity were exemplarily confirmed by a biochemical binding assay. Protein distribution in AMCM was also analyzed by immunofluorescence stainings and confocal imaging, and protein expression was detected by immunoblot analysis. Quantitative analyses of protein co-localization and distribution were performed with ImageJ. Results: Independent of the investigated condition, active RhoA was predominately localized at the sarcolemma in AMCM. Application of ET-1, PE and AngII to WT- or sham-AMCM resulted in an increased level of active RhoA and a redistribution towards the costameric regions of the sarcolemma. Neither in TAC-, MβCD-, HET- nor in KO-AMCM an activation occurred in response to the GPCR ligands. In contrast, in all four types of AMCM, ET-1 and PE reduced the RhoA activity compared to control. Interestingly, in TAC-, MβCD- and KO-AMCM the basal RhoA activity was increased indicating that the tight regulation of RhoA in its signal context is disturbed when i) the sarcolemma is disorganized as after TAC, ii) cholesterol is depleted after MβCD treatment, or iii) p63RhoGEF is fully absent. This prompted the idea that p63RhoGEF is not only involved in the activation of RhoA in response to GPCRs, but also important for the membrane homeostasis in AMCM. By overexpression of p63RhoGEF, we could confirm its formerly described localization at the sarcolemma and found in addition a perinuclear distribution. This led amongst others to an increase in the number of cis-Golgi apparatus particles similar as observed in TAC-AMCM. In contrast, the loss of p63RhoGEF reduced particle number arguing that p63RhoGEF is involved in Golgi apparatus regulation. Interestingly, p63RhoGEF also affected the size of the AMCM nuclei. Overexpression of a dominant negative p63RhoGEF construct (p63ΔN) reduced the area of the nuclei, comparable as seen in isolated AMCM and intact myocardium after p63RhoGEF depletion. Opposing to this, in TAC-AMCM the mean nucleus area was increased. This was in accordance with the observed changes in cell size. Although there were differences in the type and height of the affected parameters, p63RhoGEF-overexpression and TAC increased cell size, whereas p63RhoGEF depletion reduced it. Summary: The presented data show for the first time that RhoA is activated in healthy adult cardiomyocytes in response to important cardiovascular GPCR ligands. Disturbances of the sarcolemmal organization ultimately lead to uncoupling of RhoA from its physiological signaling context. p63RhoGEF, verified as a direct mediator of the Gq/11 protein-dependent RhoA activation, plays an additional role in the regulation of RhoA and of membranous compartments in adult cardiomyocytes. Most importantly, its depletion leads to an uncoupling of RhoA from its signaling context.