Project 6: Analysis of differential ROCK activities in cardiac fibroblasts
PI Göttingen: S. Lutz; PI London: A. Ridley; PhD student: G.L. Santos - finished PhD
Scientific background and preliminary results
Rho-associated kinases ROCK1 and ROCK2 are serine/threonine kinases that are downstream targets of the small GTPases RhoA, RhoB, and RhoC. ROCKs are involved in diverse cellular activities including actin cytoskeleton organization, cell adhesion and motility, proliferation and apoptosis, remodelling of the extracellular matrix and cell contraction. The functions of ROCK1 and ROCK2 have long been considered to be similar; however, it is now clear that while having mostly common functions, there are in addition isoform-specific targets like Rnd3, which was shown to be phosphorylated by ROCK1 but not ROCK2. Moreover, depending on their ubcellular localization, activation, and other environmental factors, ROCK signalling can have different effects on cellular function. With respect to the heart, findings in isoform-specific knockout mice argue for a role of ROCK1 and ROCK2 in the pathogenesis of cardiac fibrosis and cardiac hypertrophy, respectively. However, the distinct roles of both kinases in the two main cell populations, cardiomyocytes and cardiac fibroblasts, are not understood in detail. Especially, due to a lack of specific genetic models allowing the regulation of genes in cardiac fibroblasts, the knowledge about ROCK’s function in these cells is sparse. The available data on the function of ROCK in cardiac fibroblasts are mainly obtained by purified 2D cultures of these cells and by usage of ROCK inhibitors. It was shown that ROCKs plays a role in the regulation of cardiac fibroblast behavior in response to metabolic factors like leptin and glucose, to force and to G protein-coupled receptor agonist including sphingosine 1 phosphate, prostaglandins and angiotensin II. In neither of these studies an isoform-specific analysis was performed.
In contrast to these studies, we recently obtained data from 2D cardiac fibroblasts cultures showing that both ROCKs, in part differentially, influence cardiac fibroblast gene expression, morphology, proliferation and migration. For example, downregulation of ROCK1 but not of ROCK2 led to a moderated reduction in proliferation, however, downregulation of both kinases or general ROCK inhibition was more effective which argues for a cooperative function of both kinases in cell proliferation with a more dominant role of ROCK1. In accordance, inhibition of ROCK2 by Slx-2119 was without effect. In contrast, for migration we found no differences between the role of ROCK1 and ROCK2 and down-regulation of each isoform was sufficient to impair migration. As cardiac fibroblasts are, however, influenced by other cells in the myocardium and their culture on stiff plastic surfaces immediately lead to their transdifferentiation into myofibroblasts, we performed experiments with engineered tissues. These 3D cultures were either composed of total cardiac cells (EHM) or only cardiac fibroblasts (engineered connective tissue, ECT), suggested that ROCKs are also involved in the regulation of extracellular matrix (ECM) composition and thus influenced the tissues’ contractile function and viscoelastic properties. RNA sequencing of ECT treated with the ROCK inhibitor H1152p and qPCR studies on cultured cardiac fibroblasts with lowered expression of ROCK isoforms showed that both kinases are involved in the regulation of less recognized cardiac ECM proteins like the collagens 4a2, 6a and 8a1. Moreover, a switch in the collagen organizers biglycan and decorin was detected. Elastin was strongly downregulated and its degrading enzyme elastase was up-regulated independent on the kinase isoform. Interestingly, the hyaluronan synthase 2 was down-regulated only after knockdown of ROCK1, but not of ROCK2. So far neither the underlying signal mechanisms nor the impact of the change in single ECM protein expression on tissue properties is clear.
Hypotheses of the PhD project
Based on our data on general and distinct ROCK functions in cardiac fibroblasts, we hypothesize that both kinases share similar and differential downstream signalling cascades influencing cardiac fibroblasts gene expression and function.
In the project the doctoral researcher will test the hypotheses that
1. ROCK1 and ROCK2 share similar and differential targets in cardiac fibroblasts.
2. ROCK1 and ROCK2 influence gene transcription in a common and distinct manner in cardiac fibroblasts.
3. ROCK1 and ROCK2 regulate biomechanical properties of 3D engineered tissues differentially.
Speaker IRTG 1816, Gender equality coordinator
Research interests: Monomeric G protein signalling in cardiovascular cells
Research interests: Rho GTPase signalling and endothelial activation
PhD student 2nd IRTG 1816 cohort
Current Position: Postdoctoral Researcher, University Medical Center Göttingen, Germany