Posterior heart field and epicardium in cardiac development : PDGFRα and EMT
Bax, N.A.M.
Citation
Bax, N. A. M. (2011, January 13). Posterior heart field and epicardium in cardiac development : PDGFRα and EMT. Retrieved from https://hdl.handle.net/1887/16330
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/16330
Note: To cite this publication please use the final published version (if applicable).
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In this thesis the role of platelet-derived growth factor receptor alpha (Pdgfrα) - signalling on cardiovascular development has been explored. Additionally, the role of EPDCs in epithelial-to-mesenchymal transformation and cardiomyocyte differentiation has been investigated.
Chapter 1 provides a general introduction of this thesis. Cardiac development is described with emphasis on the recruitment of heart progenitors from the primary or first and second heart field. Furthermore, we outline the addition of progenitors at the venous pole of the heart from the posterior heart field, where they contribute to a myocardial and mesenchymal component. All described processes of heart development are related to the role of Pdgfrα and epithelial-to-mesenchymal transformation.
Chapter 2 describes the expression patterns of PDGF-A, -C and PDGFR-α during avian heart development. We showed that their expression pattern was spatiotemporally associated to the development of the second heart field-derived myocardium at the arterial and venous pole of the heart and also to the development of the proepicardium and epicardium. Pharmacochemical distribution of Pdgfrα-signalling and mechanical inhibition of the outgrowth of the proepicardial organ resulted in absence of subepicardium covering the ventricular myocardium. Mechanical inhibition of epicardial outgrowth also showed that the expression of the PDGF-ligands and their receptor in ventricular myocardium was linked to epicardial and epicardium-derived cells (EPDCs) contribution. These data support a functional role of Pdgfrα-signalling in the development of proepicardial derivatives and in the remodelling of cardiac tissue during heart development.
In Chapter 3 the role of PDGFR-α in cardiac development is explored using Pdgfrα knockout and PDGFRαGFP knockin mouse embryos. GFP expression showed that PDGFR-α is present in both myocardium and mesenchyme at the venous pole of the heart which are derived from the posterior heart field. The mesenchymal population is the source of the dorsal mesenchymal protrusion (DMP) as well as the epicardium, the latter allowed us to study epicardial-myocardial interaction in the differentiation of the ventricular wall.
Pdgfrα deficient mice showed several cardiac malformations, such as hypoplasia of the atrial and sinus venosus myocardium, including venous valves and sinoatrial node (SAN).
The hypoplasia of the SAN was accompanied by increased expression of Nkx2.5.
Furthermore, the hypoplasia of the PEO and the DMP in the Pdgfrα mutants is most probably due to diminished addition of mesenchyme from the PHF. Epicardial dissociation or blebbing observed in Pdgfrα-/- embryos is caused by altered Integrin/VCAM interaction
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while disturbed EMT and diminished migration of EPDCs is due to increased expression of WT1. These data demonstrate a function for Pdgfrα-regulated PHF-derived myocardial differentiation and remodelling. Furthermore, our data provide novel insight in a possible role for Pdgfrα in transduction pathways that lead to repression of Nkx2.5 and WT1 during development of posterior heart field-derived cardiac structures
In Chapter 4, we used humans and model organisms to identify a susceptibility factor for the development of total anomalous pulmonary venous return (TAPVR). We reported genetic analysis in TAPVR-patients that implicate the PDGFRα gene in the development of TAPVR. Gene expression studies in mouse and chicken embryos for both the Pdgfrα receptor and its ligand Pdgf-a show temporal and spatial patterns consistent with a role in pulmonary vein (PV) development. Functional knockdown of PDGF-signalling in both chicken and mouse during the period of PV formation cause a spectrum of inflow tract abnormalities, including TAPVR. The role of the dorsal mesenchymal protrusion (DMP) in the development of TAPVR supports a function for PDGF-signalling in second heart field development. These defects occur with low penetrance (~7% for TAPVR and ~30%
for intermediate anomalies) suggesting the interaction of other genetic or environmental factors. We also show that the TAPVR seen in chicken en mouse is highly similar to that discovered in an abnormal early stage embryo from the Kyoto human embryo collection.
Taken together, these data from human genetics and animal models support a role for PDGF-signalling in normal PV development and provide important insight into the embryogenesis and molecular pathogenesis of TAPVR.
Chapter 5 describes enhanced proliferation, cellular maturation and alignment of cardiomyocytes in the presence of epicardium-derived cells. During cardiac development, the contribution of EPDCs is indispensible for the formation of the ventricular compact zone and myocardial maturation. In this study we used cocultures of embryonic quail EPDCs and neonatal mouse cardiomyocytes to investigate in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction. Expression studies for electrical and mechanical junctions (connexin 43, N-cadherin and focal adhesion kinase), sarcomeric proteins (Troponin-I, α-actinin) and extracellular matrix (collagen I and periostin) show elevated levels in the EPDCs-cardiomyocytes cocultures. We also show an enhanced cellular alignment of cardiomyocytes, which combined with the increased expression of sarcoplasmatic reticulum Ca2+-ATPase (SERCA2a) was related to increased contraction. Furthermore, we showed that the expression of electrical and mechanical junctions were downregulated in three reciprocal in vivo animal models for EPDC-depletion.
In conclusion, EPDCs provide the architectural clues needed for cardiomyocyte proliferation and maturation into an arrayed, electrically and mechanically coupled myocardium.
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These data also provide new insight into a role for EPDCs in cell-based therapies because they provide the prerequisites for correct integration of cardiomyocytes into a functional and mature cardiac syncytia.
Chapter 6 provides new insights in EMT of human adult epicardial cells. Human adult quiescent epicardium is re-activated after a myocardial injury. These re-activated cells undergo EMT and migrate into the injured myocardium where they contribute to cardiac repair. Gaining more insight into the factors driving EMT of adult epicardium is important to fully appreciate its regenerative capacity. We studied the process of EMT in an in vitro model of human adult epicardial cells. Stimulating with transforming growth factor beta (TGFβ) induced loss of the epithelial character of human adult epicardial cells. We also show that the process of EMT initiates the onset of mesenchymal differentiation. Loss of epithelial character was confirmed by immunofluorescent staining and Western blot and qPCR analysis for epithelial and EMT markers. The TGFβ-induced EMT was dependent on the ALK5 kinase activity but so far seems independent of endoglin. Interestingly, soluble VCAM-1 was able to inhibit the TGFβ-stimulated EMT. Furthermore, knockdown of the epicardial marker WT1 regulates EMT via Pdgfrα. Taken together, this study provides more insight in the role of the TGFβ/ALK5 pathway initiating EMT of human adult epicardial cells probably via transcriptional regulation of WT1 and Pdgfrα. These results might be beneficial for endogenous regulated cell-based cardiac repair.
Chapter 7 studied the electrophysiological properties of epicardial cells and whether epithelial-to-mesenchymal transformation (EMT) influenced electrical conductivity of these epicardial cells. Human adult epicardium-derived cells (EPDCs) in vitro undergo spontaneous EMT thereby changing the morphology from cobblestone-like (c) to spindle- shaped (s), as shown by a loss of β-catenin at their cell surface and increased expression of the myofibroblast marker vimentin. Furthermore, the expression of both gap junctions (connexin 40, Cx43 and Cx45) and ion channels (SCN5a, CACNA1C and Kir2.1) were downregulated after EMT. We used micro-electrode arrays to investigate the electrical conduction epicardial cells before and after EMT. Therefore, human adult epicardial cells were seeded in a channel between two neonatal rat cardiomyocyte (CMC) population.
We observed that the electrical conduction was influenced by EMT. While the presence of cEPDCs resulted in electrical coupling of the two CMC fields, within 24h, sEPDCs significantly reduced conductivity, associated with a conduction block. From this study, we concluded that electrical conduction across EPDCs is influenced by EMT and is associated with a decrease in gap junction and ion channel expression. This study may provide new insight in the relevance for the role of EPDCs in cardiac development and in EMT-related cardiac dysfunction.
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Chapter 8 provides a general discussion on the data presented in the thesis. We describe the role of Pdgfrα in cardiac development at the venous pole of the heart and related this to the development of human pathologies. Additionally, the role of Pdgfrα in epicardial EMT during embryonic development and in human adult epicardial cells is discussed.
Furthermore, we highlighted a possible role for EMT of human adult epicardial to orchestra endogenous cell-based cardiac therapies.
