Abstract
Krüppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress, such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death by mouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental role for KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF2−/− embryos die earlier, by E11.5. E9.5 FVB/N KLF2−/− hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normal single layer. By E10.5, traditional and endothelial-specific FVB/N KLF2−/− AV cushions are hypocellular, suggesting that the cells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF2−/− hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number of mesenchymal cells migrating from FVB/N KLF2−/− AV explants into a collagen matrix is reduced considerably compared to wild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/N KLF2−/− embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF2−/− hearts have largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF2−/− embryos have a delay in the formation of the atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucose dehydrogenase (Ugdh), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and Ugdh promoters in chromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. In conclusion, KLF2−/− heart phenotypes are genetic background-dependent. KLF2 plays a role in EMT through its regulation of important cardiovascular genes.
Highlights
Congenital heart defects (CHDs) are a leading cause of infant morbidity and mortality
We show that FVB/N KLF22/2 hearts are hyperplastic with respect to cells lining the AV canal, and hypoplastic with respect to endocardial cushion mesenchymal cells
FVB/N KLF22/2 embryos die by E11.5, sooner than KLF22/2embryos in a mixed genetic background, which die by E14.5 [1,11,13]
Summary
Congenital heart defects (CHDs) are a leading cause of infant morbidity and mortality (reviewed in [1]). Valve and septal defects account for the majority of CHDs. Mutations in transcription factor genes, including Nkx2-5 [2] and the cardiac T-box gene TBX5 [3], are important for normal valve development in humans. Mutations or variants in other transcription factor genes are likely to be involved in valve defects. The endocardial cushions are formed by endothelial to mesenchymal transformation (EMT). During EMT, AV endocardial cushion cells undergo hypertrophy, loss of cell-cell contacts, lateral mobility, formation of mesenchymal-like cell processes (filopodia), and migration into the cardiac jelly (Reviewed in [4]). Endothelial cells fail to transform and to migrate, resulting in hypoplastic endocardial cushions [5]. Extensive remodeling and proliferation of the endocardial cushions occurs to form the adult heart valves
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