P6: The Role of the RAS-MAPK Pathway in myocardial (dys)function
Current state of relevant research
RASopathies, including Noonan syndrome (NS), are a clearly defined class of developmental disorders [1]. They are caused by germline gain-of-function mutations in genes encoding proteins of the Ras/mitogen-activated protein kinase (MAPK) pathway, including KRAS [2], NRAS [3], and RAF1 [4]. This pathway is critical in the regulation of essential cellular processes in mammalian development and dysregulation causes a variety of systemic abnormalities. The majority of the patients who have NS develop congenital cardiac disease, including hypertrophic cardiomyopathy (HCM) [1]. Remarkably, gain-of-function mutations of RAF1 in NS patients promote HCM [4-6].
The function and downstream targets of RAF1 in cardiomyocytes remains poorly understood. Cells harboring RAF1 mutations are sensitive to calcineurin-mediated inhibition of nuclear factor of activated T-cells (NFAT), which is an important regulator of cardiac hypertrophy [7]. Very recently, Matthew Wolf and colleagues have shown that activated RAF induces cardiac hypertrophy in flies, recapitulating aspects of NS, and these phenotypes are dependent on Yorkie (Yki), a transcriptional coactivator in the Hippo pathway regulating organ size [8]. This and other works suggest that RAF1 activity is not restricted to ERK activation [9]. Although several alternative RAF1 substrates have been described, none has been unequivocally validated to date. Activation of the Hippo pathway by activated RAF is an interesting novel pathway leading to hypertrophy. The Hippo pathway is conserved among species, controls organ size, and has been implicated in a variety of human diseases. Cell-cell contacts, the actin cytoskeleton as well as a wide range of signals, including cellular energy status, mechanical forces, and hormonal signals that act through G-protein-coupled receptors have been implicated as regulators of Hippo signaling. Thus, we hypothesize that mutated forms of RAF1 mimic altered intra-organ communication leading to cardiac hypertrophy via aberrant activation of the Hippo pathway. The goal of our project is to move beyond the paradigm that activation of the MAP kinase cascade constitutes the major function of RAF1 by investigating novel molecules that are responsible for RAF1-mediated signalling in cardiovascular diseases.
Preliminary work
Induced pluripotent stem cell (iPSC)-derived human embryoid bodies (EBs)
differentiated to contractile cardiac muscle cells (called cardiac bodies or CBs) and bioartificial cardiac tissue (BCT; usually cells organoids) were very recently established in the Ahmadian lab in collaboration with Dr. George Kensah, Department of Cardiothoracic Surgery, University Hospital Magdeburg [10-13]. The iPSCs originated from dermal fibroblasts of NS patients with RAF1 mutations (e.g., S257L), respectively. CRISPR/Cas9 gene-edited and wild-type iPSCs will serve as controls. Preliminary biochemical data clearly show that these cells differ in their signalling activities (e.g., AKT, ERK2, p38, cTnT and YAP).
The fruit fly Drosophila melanogaster is a versatile model organism and has been established in the Wolf lab to model human heart diseases and better understand cardiomyopathies [14]. The wealth of mutants, rapid breeding times, and robust resources make Drosophila an ideal genetic model to dissect and analyse signalling pathways in an in vivo context. Moreover, the conservation of genes and signalling molecules between flies and mammals provides a robust strategy to translate findings form flies to cell culture and mammalian models. Recently, a crosstalk between Raf and Yki has been identified using the fly genetics and heart phenotyping employing Optical Coherence Tomography (OCT) to measure cardiac function and histology to assess morphology. Additionally, crosstalk between RAF and Hippo pathways was validated in mammalian cells using luciferase-based assays. These investigations identified that the Hippo pathway can influence Raf-mediated cardiac hypertrophy [6].
Research objectives of the joint program
This collaborative project focuses on cardiac-specific functions of RAF1 using RAF1 knockout (RAF1-/-) and NS-associated S257L and L613V mutations. However, the planned work will be distributed between both labs to capitalize on each laboratory’s expertise. A large part of the planned biochemical analysis will be performed in the Ahmadian lab by the German and American doctoral researchers. In vivo functional analysis will be performed at the Wolf lab using the Drosophila model as a rapid screening tool. OCT will be used to analyse cardiac function in flies, similar to echocardiography in mice. To identify, evaluate and characterize novel RAF1 substrates, their downstream pathways, and alteration of signal transduction, the German doctoral researcher will knockout the RAF1 gene in mouse HL1 and human IPSC lines by CRISPR/Cas9. RAF1-/-, RAF1+/+ and RAF1S257L cells, including HL1 and iPSC-differentiated myocardial cells, will be used to perform quantitative SILAC phosphoproteomics of myocardial cells to decipher regulatory networks which are disturbed by the RAF1 mutations. Moreover, RAF1 binding partners of WT and mutated RAF1 will be identified by fractionation, 2D blue native PAGE and LC/MS. The American doctoral researcher will perform phosphopeptide mapping of substrates of WT and constitutively active Raf1 (Raf1L613V mutation). Moreover, the doctoral researcher will employ sophisticated Drosophila genetics to knockout RAF1 substrates and binding proteins identified in iPSC-derived cardiac myocytes and examine the in vivo effects on cardiac hypertrophy using OCT and histology. This analysis will be performed also for the S257L mutant in cooperation with the German doctoral researcher. The role of the Hippo/YAP pathway in myocardial cell growth and differentiation will be analysed in detail. The German doctoral researcher will characterize the Hippo pathway, especially RASSF-regulated LATS/YAP activity (RAS/RASSF/MST, YAP/pYAP, CTGF, NOTCH2) in myocardial cells differentiated from RASopathy patient-derived iPSCs (WT and NS-associated mutations). To further translate findings from iPCS and HL1 cells to a mammalian heart, the American graduate student will use the Yki-Sd luciferase-based activity in fly. Additionally, the Wolf lab has transgenic Raf1L613V/+ knock-in and floxed YAP1 knockout mice that will be used to translate findings from flies to mice using echocardiography, cardiac MRI, histology, and biochemical analyses in the context of transverse-aortic constriction (TAC)-induced pressure overload mediated cardiac hypertrophy. Using these mouse models, specific phosphorylation events – guided by cell based experiments- will be examined in vivo. The German doctoral researcher will differentially analyse patient-derived iPSC-based myocardial cells (2d) as well as 3D cardiac bodies and bioartificial cardiac tissue (organoids) regarding cell size, MHC-isoforms, sarcomere length in collaboration with Dr. George Kensah, Department of Cardiothoracic Surgery, University Hospital Magdeburg.
The synergy of the two different complementing model systems involves the biochemical expertise of the Ahmadian group and the Drosophila and mouse model expertise of the Wolf group. The project provides an ideal opportunity for both understanding the molecular link between RAF1 signalling and the pathogenesis of HCM, and guiding future work directed to a mechanism-based identification of novel targets for therapeutic interventions.