Abstract 15665: Ablation of Nkx2-5 Beginning at Mid-Embryonic Stage Results in Premature Lethality and Cardiac Malformation
Background: Human congenital heart disease linked to mutations in NKX2-5 is characterized by cardiac anomalies (atrial and/or ventricular septal defects and tetralogy of Fallot), and conduction and contraction defects. In mice, homozygous germline deletion of Nkx2-5 gene results in lethality at E10.5. Whether Nkx2-5 is necessary for cardiac development beyond this embryonic stage is not well understood.
Methods and Results: We utilized tamoxifen-inducible Nkx2-5 knockout mice that carry homozygous floxed-Nkx2-5 alleles and a heterozygous Cre-ERTM transgene under the control of CMV enhancer and chicken-β-globin promoter. Tamoxifen injection into pregnant mice on gestational day 12.5 nearly completely deleted Nkx2-5 genes in the embryos demonstrated by quantitative RT-PCR and Western blotting. Nkx2-5-ablated embryos (n=13) demonstrated arrhythmias and contraction abnormalities at E16.5 by echocardiogram in utero and died by E17.5, which was not observed in control embryos after tamoxien-injection (flox/flox, n=8 or wild/wild/Cre, n=3). Nkx2-5 ablated hearts demonstrated markedly thinner outer compact layers and cardiac anomalies including atrial and/or ventricular septal defects. Quantitative serial section histology revealed growth retardation of the septum secundum from the atrial myocardium in Nkx2-5 ablated embryos [flox/flox 94 μm (n=3) vs. flox/flox/Cre 46 μm (n=5)], which partly explains the cardiac anomalies. Cardiac transcript levels of cardiac voltage-gated Na+ channel pore-forming α-subunit (Nav1.5-α), KcneI/minK and gap junction protein connexin40 were reduced greater than 70% within 4 days after tamoxifen-injection.
Conclusions: Because a previous study demonstrated no cardiac anomalies in mice with ventricular-restricted deletion of Nkx2-5, the observed embryonic lethality and septum anomalies are likely due to near complete deletion of Nkx2-5 in the entire heart. Reduction in target gene expression may contribute to the cardiac phenotype demonstrated in embryos with Nkx2-5 ablation beginning at E12.5. Our results provide evidence that Nkx2-5 is necessary for survival of embryos after the mid-embryonic stage for maintenance of cardiac function and formation by regulation of expression of its target genes.
- © 2010 by American Heart Association, Inc.