Enhanced cardiogenesis in embryonic stem cells overexpressing the GATA-4 transcription factor

Abstract

GATA-4 is a cardiac-specific member of the GATA family of zinc finger transcription factors. During embryogenesis, GATA-4 expression is detected very early in the cardiogenic area and persists later in the developing heart. Studies have shown that GATA-4 is a potent transcriptional activator of several cardiac muscle-specific genes and a key regulator of the cardiomyocyte gene program. Consistent with a role for GATA-4 in cardiomyocyte formation, inhibition of GATA-4 expression by antisense transcripts interferes with expression of cardiac muscle genes and blocks development of beating cardiomyocytes in P19 embryonic stem cells. In order to better define the function of GATA-4 in cardiogenesis, we have carried out molecular analysis of early stages of cardiomyocyte differentiation in GATA-4-deficient P19 cell lines and in P19 cells stably overexpressing GATA-4. The results indicate that GATA-4 is not required for either endodermal or mesodermal commitment or for initiation of the cardiac pathway. However, in the absence of GATA-4, differentiation is blocked at the precardiac (cardioblasts) stage and cells are lost through extensive apoptosis. In contrast, ectopic expression of GATA-4 in P19 cells accelerates cardiogenesis and markedly increases (over 10-fold) the number of terminally differentiated beating cardiomyocytes following cell aggregation. Together, these findings suggest that, in addition to its role in activation of the cardiac genetic program, GATA-4 may be the nuclear target of inductive and/or survival factors for precardiac cells.

Discovery of Small Molecules Targeting the Synergy of Cardiac Transcription Factors GATA4 and NKX2-5

“…The primary screening using a reporter gene assay and the in vitro cell culture studies revealed several biologically active compounds, i.e., 3, 4, 5, 6, and 7, with distinct profiles. The structure−activity relationship studies of inhibitory compounds 3, 4, and 5 demonstrated the indispensable role of the two heavy atom linker together with a hydrogen acceptor feature next to the five-member ring, while a completely opposite agonistic effect was achieved by introducing a substituted fivemember ring into the amide bond (7). In the cardiomyocyte cultures, the compounds either augmented or inhibited the hypertrophic agonist-induced ANP and BNP expression.…”

“…The precipitated product was filtered, washed with n-heptane (2 × 50 mL), and dried under vacuum at 45 °C overnight to yield 8.52 g (62%) of compound 3 as an offwhite powder. NMR: 10.12 ppm (s, 1H, NH), 7 4-[(4-Propoxybenzylidene)amino]-5-(pyridin-4-yl)-4H-1,2,4triazole-3-thiol (4). A mixture of 4-propoxybenzaldehyde (10.00 g, 60.9 mmol) and 4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol (11.8 g, 60.9 mmol) in glacial acetic acid (100 mL) was heated to reflux for 2.5 h, and the solution was filtered hot.…”

“…GATA4 is one of the earliest-expressed TFs during cardiac development, and its up-regulation is sufficient to induce cardiogenesis in embryonic stem cells and promote a cardiac cell fate in noncardiogenic cells. 7 In the adult heart, GATA4 is a critical regulator of cardiac regeneration 8 and acts as a key transcriptional controller of numerous cardiac genes, including those encoding atrial natriuretic peptide (ANP), 9 Btype natriuretic peptide (BNP), 10 α-myosin heavy chain (α-MHC), 11 and β-MHC. 12 GATA4 binds to specific DNA response elements with or without cofactors, e.g., NKX2-5, TBX5, myocyte-specific enhancer factor 2 (MEF2), and serum response factor (SRF).…”

“…Subsequently, during the small molecule optimization of the inhibitory compound 3, surprisingly, we detected a compound that strongly augmented the GATA4− NKX2-5 synergy in the reporter gene assay (Figure 1A). The structure of this activator compound, N-(4-chlorophenyl)-5methyl-N-(4-methyl-4,5-dihydrothiazol-2-yl)-3-phenylisoxazole-4-carboxamide (7), partially overlaps the pharmacophore model, which was developed based on inhibitory compounds with an additional five-member ring moiety attached. Due to the complexity of the compound structure, the synthesis of its derivatives was not pursued in this study, and only one related compound was tested to confirm this finding.…”

Cyclosporine A Induces Cardiac Developmental Toxicity in Zebrafish by Up-Regulation of Wnt Signaling and Oxidative Stress

“…To study the underlying mechanism of cardiac developmental toxicity induced by CsA, expression levels of genes correlated with cardiac development, and apoptosis were detected by qPCR. GATA4 is one of the earliest developing transcription factors during heart development, and its up-regulated expression can promote the differentiation of embryonic stem cells into heart ( Grepin et al, 1997 ). Nkx2.5 regulates cardiac tube elongation and has different effects on the number of ventricular and atrial cells ( Targoff et al, 2008 ).…”