NFAT signalling and the differentiation of coronary smooth muscle cells.

Development of the heart is complex and cells with different embryonic origins participate in cardiac organogenesis. The first and second heart fields deliver cells to generate the atrial and ventricular chambers and also participate in venous inflow and arterial outflow tract formation. There are also important cellular contributions from two extra-cardiac cell populations, i.e. cardiac neural crest cells and the pro-epicardium. The neural crest cells populate the outflow tract, are involved in septation of the arterial pole into the aorta and pulmonary trunk, and contribute to the smooth muscle layer of these large vessels. The pro-epicardium forms at the venous pole of the heart as a cauliflowerlike cluster, which establishes contact to the myocardial surface and through proliferationand migration rapidly invest the myocardium to establish the epicardium. The embryonic epicardium gives rise to epicardium-derived cells (EPDCs) that undergo epithelial mesenchymal transformation (EMT) and migrate into the myocardial wall to participate in coronary artery formation and to differentiate into interstitial and perivascular fibroblasts (Figure 1). In the article by Yang et al., the role of calcineurin nuclear factor of activated T cell (NFAT) signalling for smooth muscle differentiation and coronary artery wall development has been thoroughly investigated. NFATs constitute a family of transcription factors that are essential for cardiac development and their importance for cardiac valve formation and cardiac hypertrophy has been established. The NFAT family consists of five genes Nfatc1–Nfatc4 and Nfat5 of which Nfatc1, Nfatc3, and Nfatc4 are expressed in the embryonic heart. When phosphorylated the NFAT proteins are kept in the cytoplasm. Signalling pathways that raise intracellular calcium causes an activation of the phosphatase calcineurin, which dephosphorylates NFATs causing nuclear import resulting in a change in gene expression (Figure 1). The group of Ching-Pin Chang has recently demonstrated that calcineurin signalling is essential forpropercoronary vessel formation. In the current study, Yang et al. employed the GATA5-Cre mouse line, in order to ablate calcineurin in the epicardium. In contrast to global and endothelial, cell-specific ablation of the calcineurin gene epicardiumspecific inactivation is fully compatible with embryonic and postnatal life. However, when these animals were subjected to an exhaustive treadmill exercise themutants displayeda reduction in exercise capacity, accompanied by a reduction in left ventricular fractional shortening and an increase in cardiac stress marker gene expression. The coronary arteries displayed specific alterations including an increased level of MMP9, collagen, and elastin expression. Moreover, the smooth muscle layer of the coronary arteries showed a specific mutant phenotype. While thickness of the media as well as cell number appeared normal, the expression levels of smooth muscle cell-specific differentiation marker genes were strongly reduced in the mutants. Cyclosporin A (CsA) treatment of embryos to test when calcineurin signalling was essential for coronary smooth muscle differentiation defined a time window between embryonic day 12.5 and 13.0. To rule out the possibility that ablation of calcineurin in differentiated smooth muscle or cardiac muscle would be responsible for the phenotype, the authors also ablated calcineurin with the Sm22a-Cre line. These mice, however, revealed no defect in coronary smooth muscle cell differentiation, which corroborates the results of the CsA experiments, suggesting that there is a specific requirement for calcineurin in the epicardium or in EPDCs. The question, which of the Nfatc genes was involved in the signalling pathway, was addressed by ablating Nfatc1 in the epicardium using GATA5-Cre. These mice did not reveal any defects in coronary smooth muscle differentiation and were fully viable ruling out that Nfatc1 is part of the pathway. The authors assume that Nfactc3 and Nfatc4 are likely to be involved; however, this was not further investigated in this study. To identify which target gene is downstream of NFAT, different signalling pathway intermediates were tested and nuclear Smad2, which mediates TGF-b signalling, was found to be reduced in coronary smooth muscle cells of calcineurin-ablated mice. Analysis of the Smad2 promoter revealed a number of putative NFAT-binding site, which based on reporter gene assays and chromatin immunoprecipitation analysis were found to be functional (Figure 1). This study adds to the already large list of functions of the calcineurin-NFAT signalling pathway in the heart. Intriguingly, NFAT signalling in the embryo, therefore, is first required to establish the