Project 6: Redox-sensitive interplay of RhoGTPases in cardiac cells
PI Göttingen: S. Lutz; PI London: E. Ehler, P. Eaton; PhD student: Alisa N. DeGrave
Scientific background and preliminary results
The RhoGTPases RhoA and Rac1 are expressed in all cardiac cells and were found to be involved in detrimental remodeling processes of the heart such as hypertrophy and fibrosis (Jatho et al, 2015; Vettel et al, 2012; Ongherth et al, 2015). Both GTPases are activated downstream of disease-relevant G protein-coupled receptors (GPCR), including the endothelin-1 and angiotensin II-type 1 receptors. These receptors are in addition known to induce the production of reactive oxygen species (ROS) via NADPH oxidases (NOX) in cardiac cells (Tanaka et al, 2001; Zhang et al, 2015). There are several lines of evidence that all three pathways are complexly interconnected. Rac1 was identified as an essential co-factor of the cardiac-relevant NOX2. In addition, Rac1 activity was shown to be enhanced by ROS in a cardiomyocyte-like cell type suggesting a positive feedforward mechanism (Nagase et al, 2012). This activity enhancement could be indirect, but also direct as Rac1 possesses a redox-sensitive cysteine-containing motif (GXXXXGK[S/T]C) adjacent to the phosphoryl-binding loop. Oxidation of this motif led in vitro to an acceleration of the release of GDP and thus mimicked the action of a guanine nucleotide exchange factor (Heo et al, 2005). RhoA possesses a similar redox motif (GXXXCGK(S/T)C) as Rac1. This motif contains, however, a second cysteine residue (Cys 16) located N-terminally to the Rac1-homolog cysteine residue (Cys 20). In vitro oxidation of RhoA led to the same enhanced release of GDP from the binding pocket as demonstrated for Rac1, but in addition a disulfide bond was formed between both cysteines in the empty GTPase (Heo et al, 2006). This disulfide bond sterically hindered the GTP to enter the binding pocket and rendered the GTPase inactive. Addition of ascorbate prevented the disulfide bond formation (Heo et al, 2005). Based on this data it was hypothesized that under physiological conditions both GTPases are activated in response to ROS, whereas under conditions of high oxidative stress enhanced Rac1 activation might lead to the inactivation of RhoA. So far there is no study, which tested this hypothesis. Moreover, there is only one study which demonstrated that the cysteines in the redox-sensitive motif of RhoA are important for its activation in response to exogenously added ROS. This study showed that after treatment of fibroblasts with 100 nM H2O2, RhoA became activated within few minutes and more importantly the exchange of the redox-sensitive cysteines to alanines fully prevented the activation. These point mutations did not interfere with other signal pathways leading to RhoA activation (Aghajanian et al, 2009.
Besides this direct oxidation and activation of RhoA, indirect redox mechanisms have been proposed leading to a change in the activity status of RhoA. In Hela cells, it was demonstrated that high Rac1 activity and thus high ROS production led to the oxidation and inhibition of the low-molecular weight protein tyrosine phosphatase. The consequence was an increased phosphorylation and activity of p190A-RhoGAP which silenced RhoA activity (Nimnual et al, 2003). In contrast, for cardiac cells it was suggested that the activation of the transient receptor potential channel 3 in conjunction with NOX2 results in the activation of RhoA via a microtubule-dependent mechanism (Numaga-Tomita et al, 2016).
Based on the current data situation, we want to answer the following questions in our study:
- How do different ROS levels and sources influence the activities of Rac1 and RhoA in human embryonic stem-cell derived cardiomyocytes (hCM) and human cardiac fibroblasts (hCF)?
- Is there a redox-sensitive interplay between Rac1 and RhoA in hCM and hCF?
- Which impact has the redox regulation of Rac1 and RhoA on the biomechanical properties of engineered connective and muscle tissues (hECT and hEHM)?
Ad1: To study the regulation of Rac1 and RhoA by ROS, we will generate bicistronic lentiviruses which encode for specific shRNAs in tandem with knockdown-inert variants of the GTPases. This will include the respective wildtype proteins and the following redox-insensitive and pseudo-oxidized mutants: Rac1-C18S, Rac1-C18D, RhoA-C16S, RhoA-C20S, RhoA-C16SC20S, RhoA-C16D, RhoA-C20D, RhoA-C16DC20D. We will express these mutants in hCM and hCF and analyse their activation in response to GPCR ligands (Ang II and ET-1 for hCM, Ang II of hCF) and exogenously added H2O2 (10, 100, 1000 nM) by a biochemical G-Lisa assay. We will further determine the redox status of wildtype Rac1 and RhoA, the localization of all Rac1 and RhoA variants, the Rac1-dependent ROS production, the activation of divers downstream mediators such as p21-and Rho-associated kinases (PAK, ROCK), changes in the phosphorylation of cytoskeletal proteins, and the arrangement of the actin cytoskeleton (Jaho et al, 2015; Vettel et al, 2012). We will perform confocal imaging, life cell imaging of the biosensor HyPer, the biotin switch assay, and immunoblot analysis. Based on the observed changes, hCM hypertrophy and apoptosis as well as the phenotypic status of the hCF will be investigated in transduced 2D cultures.
Ad2: Most studies suggest that in the hierarchy of the redox-sensitive interplay between both GTPases, Rac1 is superordinate. Therefore, we will implement different strategies to block Rac1 activity. This will include the expression of the dominant negative mutant Rac1-T17N, the use of the RacGEF and Rac inhibitors NSC 23766 and EHT 1864, and the inhibition of the NADPH oxidase activation via gp91 ds-tat. RhoA oxidation and activity will be studied via the biotin-switch and G-Lisa assays, respectively. Vice versa, the influence of the dominant negative RhoA-T19N and of the RhoA-C inhibitor C3 transferase on Rac1 activity and ROS production will be investigated by the G-Lisa assay and the biosensor HyPer. We will further consider indirect pathways which e.g. includes the activation of p190B-RhoGAP. This negative regulator of RhoA was shown to be activated by Rac1 binding and is based on our RNA sequencing data the most abundant RhoGAP in hCM and hCF (Bustos et al, 2008).
Ad3: Depending on the results obtained in 2D cultures, we will generate engineered human connective and muscle tissues (ECT and EHM) with transduced cells and will study the impact of the redox-sensitive interplay of Rac1 and RhoA on tissue properties. This will include tissue contraction and stiffness analyses. To assess changes in cell characteristics, the cells will be re-isolated from the tissues and subjected to a detailed FACS analysis. By this cell viability and the phenotypic state of both cell types will be assessed.
Speaker IRTG 1816, Gender equality coordinator
Research interests: Monomeric G protein signalling in cardiovascular cells
Research interests: Cytoarchitecture during cardiomyocyte development and disease: myofibrillogenesis, dilated cardiomyopathy, sarcomere, cytoskeleton, intercalated disc, M-band, formin ( FHOD3, FHOD1)
Research interests: Molecular mechanisms of redox signalling and heart faillure
PhD student 3rd cohort
RP 6.3: Redox-sensitive interplay of RhoGTPases in cardiac cells