Analysis of the Endocardial-to-Mesenchymal Transformation of Heart Valve Development by Collagen Gel Culture Assay
Department of Medicine, Division of Cardiovascular Medicine, Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA. Methods in molecular biology (Clifton, N.J.)
(Impact Factor: 1.29).
01/2012; 843:101-9. DOI: 10.1007/978-1-61779-523-7_10
Malformations of heart valves are one of the most common serious congenital defects. Heart valves are developed from endocardial cushions of the heart. The endocardial cushion in early heart development consists of two cell layers: an outer myocardial cell layer and an inner endocardial cell layer with abundant extracellular matrix (cardiac jelly) in between. Endocardial cells of the cushion, triggered by signals from myocardial cells, delaminate from the surface of the endocardial cushion and undergo transdifferentiation into mesenchymal cells. This process of endocardial-to-mesenchymal transformation (EMT) begins in the atrioventricular canal at embryonic day 9 (E9) and in the cardiac outflow tract at E10 of mouse development. Once formed by the EMT, the mesenchymal cells invade the cardiac jelly, proliferate, and populate the endocardial cushion. The cellularized endocardial cushion then undergoes morphological remodeling; it lengthens and matures into a thin elongated valve leaflet. Here we describe a method to culture endocardial cushions and measure EMT ex vivo. EMT can thus be analyzed independent of other concurrent developmental defects in mice. This culture method also enables ex vivo manipulations of signaling or gene function during EMT to delineate molecular pathways essential for heart valve development.
Available from: Dagmar Wachten
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ABSTRACT: Calcineurin is a heteromeric Ca(2+)-dependent serine/threonine phosphatase. It dephosphorylates the transcription factor nuclear factor of activated T cells (NFAT) in the cytoplasm, which subsequently undergoes nuclear translocation. NFAT regulates numerous biological processes, including inflammatory T cell responses and cardiac development. Our study identifies the Cysteine-Rich with EGF-Like Domains 1 (Creld1) gene as a regulator of calcineurin/NFATc1 signaling. We show that Creld1 is sufficient to promote NFATc1 dephosphorylation and translocation to the nucleus. Creld1 is contained in a joint protein complex with the regulatory subunit of calcineurin, CnB, thereby controlling calcineurin function. Localization of Creld1 at the endoplasmic reticulum (ER) is important to exert its action on calcineurin. By using Creld1KO mice, we demonstrate that Creld1 is essential for heart development. Creld1 function is required for the VEGF-dependent proliferation of endocardial cells by promoting the expression of NFATc1 target-genes. Collectively, our study identifies Creld1 as an important regulator of calcineurin/NFATc1 signaling.
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