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

Bax NA, Pijnappels DA, van Oorschot AA, Winter EM, de Vries AA, van Tuyn J, Braun J, Maas S, Schalij MJ, Atsma DE, Goumans MJ, Gittenberger-de Groot AC - J. Cell. Mol. Med. (2011)

Bottom Line: 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.These differences may be of relevance for the role of EPDCs in cardiac development, and in EMT-related cardiac dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.

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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.
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fig02: 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.

Mentions: To evaluate whether EMT may have an effect on their conductivity of epicardial cells, we first analysed their capacity to couple. Therefore, the two types of EPDCs were analysed by immunofluorescence microscopy using antibodies recognizing specific connexins (Fig. 1) and ion channels (Fig. 2) antibodies. EMT induced changes in cellular morphology were confirmed using immunofluorescent staining for β-catenin (Fig. 1A1 and A2) and vimentin (Fig. 2A1 and A2). cEPDCs displayed a marked membrane expression of β-catenin, especially at sites of cell–cell contact, confirming their epithelial nature (Fig. 1A1). After EMT, the level of β-catenin was reduced and in the sEPDCs the protein was down-regulated in the cytoplasm in sEPDCs (Fig. 1A2). Cx40 (Fig. 1B1 and B2) and Cx45 (Fig. 1D1 and D2) were weakly expressed in the cytoplasm of both cEPDCs and sEPDCs, whereas some of some Cx45 staining was also present in the nucleus (Fig. 1D1 and D2). Analysis of mRNA expression confirmed that both Cx40 and Cx45 were present before and after EMT (Fig. 3). The quantification of Cx40 mRNA expression showed a 3.1 times higher expression in sEPDCs as in cEPDcs (P < 0.05) (Fig. 3A). The mRNA expression of Cx45 was not significantly affected by EMT as the Cx45/β-actin ratio was almost equal in cEPDCs and sEPDCs (Fig. 3C and F). Cx43 was present in the cytoplasm of cEPDCs and between adjacent cEPDCs. The punctuated pattern of the staining reflected the presence of gap junctions (Fig. 1C1). Cx43 levels were reduced in the cytoplasm of sEPDCs and between adjacent sEPDCs (Fig. 1C2) compared to cEPDCs. Quantification of PCR analysis revealed that the expression of Cx43 mRNA was 53% higher in cEPDC (P < 0.05) (Fig. 3B).


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

Bax NA, Pijnappels DA, van Oorschot AA, Winter EM, de Vries AA, van Tuyn J, Braun J, Maas S, Schalij MJ, Atsma DE, Goumans MJ, Gittenberger-de Groot AC - J. Cell. Mol. Med. (2011)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4373436&req=5

fig02: 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.
Mentions: To evaluate whether EMT may have an effect on their conductivity of epicardial cells, we first analysed their capacity to couple. Therefore, the two types of EPDCs were analysed by immunofluorescence microscopy using antibodies recognizing specific connexins (Fig. 1) and ion channels (Fig. 2) antibodies. EMT induced changes in cellular morphology were confirmed using immunofluorescent staining for β-catenin (Fig. 1A1 and A2) and vimentin (Fig. 2A1 and A2). cEPDCs displayed a marked membrane expression of β-catenin, especially at sites of cell–cell contact, confirming their epithelial nature (Fig. 1A1). After EMT, the level of β-catenin was reduced and in the sEPDCs the protein was down-regulated in the cytoplasm in sEPDCs (Fig. 1A2). Cx40 (Fig. 1B1 and B2) and Cx45 (Fig. 1D1 and D2) were weakly expressed in the cytoplasm of both cEPDCs and sEPDCs, whereas some of some Cx45 staining was also present in the nucleus (Fig. 1D1 and D2). Analysis of mRNA expression confirmed that both Cx40 and Cx45 were present before and after EMT (Fig. 3). The quantification of Cx40 mRNA expression showed a 3.1 times higher expression in sEPDCs as in cEPDcs (P < 0.05) (Fig. 3A). The mRNA expression of Cx45 was not significantly affected by EMT as the Cx45/β-actin ratio was almost equal in cEPDCs and sEPDCs (Fig. 3C and F). Cx43 was present in the cytoplasm of cEPDCs and between adjacent cEPDCs. The punctuated pattern of the staining reflected the presence of gap junctions (Fig. 1C1). Cx43 levels were reduced in the cytoplasm of sEPDCs and between adjacent sEPDCs (Fig. 1C2) compared to cEPDCs. Quantification of PCR analysis revealed that the expression of Cx43 mRNA was 53% higher in cEPDC (P < 0.05) (Fig. 3B).

Bottom Line: 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.These differences may be of relevance for the role of EPDCs in cardiac development, and in EMT-related cardiac dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.

Show MeSH
Related in: MedlinePlus