Epithelial-to-mesenchymal transformation alters electrical conductivity of human epicardial cells

Abstract

The myocardium of the developing heart tube is covered by epicardium. These epicardial cells undergo a process of epithelial-to-mesenchymal transformation (EMT) and develop into epicardium-derived cells (EPDCs). The ingrowing EPDCs differentiate into several celltypes of which the cardiac fibroblasts form the main group. Disturbance of EMT of the epicardium leads to serious hypoplasia of the myocardium, abnormal coronary artery differentiation and Purkinje fibre paucity. Interestingly, the electrophysiological properties of epicardial cells and whether EMT influences electrical conductivity of epicardial cells is not yet known. We studied the electrophysiological aspects of epicardial cells before and after EMT in a dedicated in vitro model, using micro-electrode arrays to investigate electrical conduction across epicardial cells. Therefore, human adult epicardial cells were placed between two neonatal rat cardiomyocyte populations. Before EMT the epicardial cells have a cobblestone (epithelium-like) phenotype that was confirmed by staining for the cell-adhesion molecule β-catenin. After spontaneous EMT in vitro the EPDCs acquired a spindle-shaped morphology confirmed by vimentin staining. When comparing both types we observed that the electrical conduction is influenced by EMT, resulting in significantly reduced conductivity of spindle-shaped EPDCs, associated with a conduction block. Furthermore, the expression of both gap junction (connexins 40, Cx43 and Cx45) and ion channel proteins (SCN5a, CACNA1C and Kir2.1) was down-regulated after EMT. This study shows for the first time the conduction differences between epicardial cells before and after EMT. These differences may be of relevance for the role of EPDCs in cardiac development, and in EMT-related cardiac dysfunction.

Fig 1

Epithelium-to-mesenchymal transformation is accompanied by a decrease in β-catenin and connexins expression levels. Immunofluorescence microscopy of cEPDCs (before EMT) and sEPDCs (after EMT) labelled with antibodies directed against β-catenin (A), Cx40 (B), Cx43 (C) and Cx45 (D). The expression of β-catenin is strongly expressed at the cell borders of cEPDCs. Expression of β-catenin is downregulated in the cytoplasm of sEPDCs. Scale bar, 20 μm.

Fig 2

Immunofluorescent staining of ion channels in epicardial cells before EMT (cEPDCs) and after EMT (sEPDCs). Immunofluorescence analysis of Kir2.1 (B), SCN5a (C) and CACNA1C (D) in EPDCs before and after EMT. Expression of ion channels was reduced by EMT. Vimentin was used to determine cell morphology of sEPDCs (A). Scale bar, 20 μm.

Fig 3

Semiquantitiative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of connexins and ion channels in EPDCs before and after EMT. mRNA levels of Cx40 (A, F), Cx43 (B, F), Cx45 (C, F) and ionchannels Kir2.1 (F), SCN5a (D, F) and CACNA1C (E, F) were quantified. Equal amounts of input cDNA were used as indicated by β-actin (F). C: cEPDCs; S: sEPDCs.

Fig 4

Overview of conduction velocities, and corresponding conduction delays, measured in EPDCs in-between two adjacent fields of CMCs. Conduction velocity was significantly decreased in sEPDCs. In fact, with increasing distance (360 μm) sEPDCs were no longer able to conduct the electrical impulse across the channel, resulting in asynchronized beating of the two CMC fields. No significant differences were found during follow-up at 48 hrs (A). Extracellular electrograms derived from cocultures of labelled EPDCs and neonatal rat cardiomyocytes, 24 hrs (light grey) and 48 hrs (dark grey) after plating (B). These electrograms clearly show the decremental nature of conduction across EPDCs, regardless of EMT. However, as conduction across these EPDCs depends on electrotonic interaction, the decrease in connexin levels that occurs in these cells during EMT is expected to result in conduction block over a certain distance (B). ^#P < 0.05 versus sEPDCs.