Abstract Body (Do not enter title and authors here): Background: Pregestational diabetes is an established risk factor for congenital heart disease (CHD), including heterotaxy syndrome, which results from abnormal left-right (L-R) axis. However, the specific mechanism by which hyperglycemia disturbs the L-R axis still remains unsolved. In addition, it is unknown how a variety of heterotaxy is provoked by pregestational diabetes.
Methods: We conducted whole-mount in situ hybridization, reporter analysis and RNA-seq in mouse embryos of streptozotocin-induced diabetic dams and Wnt3a^–/– embryos. Additionally, the effect of high-glucose conditions on intracellular pH (pH_i) was investigated. We also examined the influence of maternal vitamin A intake on L-R axis formation in embryos of diabetic dams.
Results: We observed various patterns of Pitx2 expression in embryos of diabetic dams, indicating disrupted L-R axis. The initial step of L-R axis formation was impaired, with Nodal expression, which induces Nodal-Pitx2 cascade in the lateral plate mesoderm, being regressed in the node. Consistent with the resemblance to the phenotype of Wnt3a^–/– embryos, canonical Wnt signaling was downregulated in embryos of diabetic dams. As the RNA-seq analysis suggested dysregulation of glycolysis, we assessed pH_i to find that hyperglycemia lowers pH_i in the primitive streak, leading to the regression of Nodal expression in the node. Furthermore, maternal vitamin A intake was found to increase the incidence and diversity of L-R axis defects, likely as a result of dysregulation of retinoic acid metabolism apparent in embryos of diabetic dams and Wnt3a^–/– embryos.
Conclusions: Hyperglycemia downregulates canonical Wnt signaling by lowering pH_i, which not only impairs L-R axis formation but also dysregulates retinoid metabolism. Consequently, embryos of diabetic dams become vulnerable to fluctuation of maternal vitamin A intake, resulting in diverse L-R axis defects. These findings provide insights into the etiology of CHD and suggest the importance of diet for prevention of heterotaxy during diabetic pregnancy.