Abstract

Cardiac malformations are very prevalent and can be caused both by defective genes and environmental teratogens. Among the latter, caffeine causes malformations when exposed during early cardiac development, whereas its later effects are still unclear. We exposed three-day incubated (D3) chick embryos to 2 mg caffeine and analyzed them at D5, D7 and D9. The embryos were serially sectioned and analyzed two-dimensionally. Alternatively, the sections of D9 embryos were reconstructed three-dimensionally using Amira® software and analyzed volumetrically. The expression of genes involved in endothelial-mesenchymal transformation (EMT) was studied by real-time PCR. Interestingly, caffeine treatment at D3 embryos did not induce cardiac malformations, but did delay growth, in particular that of the ventricles and ventricular trabeculae. Furthermore, it affected EMT in the endocardial cushion and atrioventricular valves. Gene-expression analysis revealed that caffeine had a progressively deleterious effect on the expressions of GATA4, MMP2, SNAIL1, TWIST1, and VIMENTIN. The effect of late caffeine administration on the chicken embryos would provide suggestive evident towards a possible heart developmental defect in humans, particularly heavy caffeine consumers during pregnancy.