Tetrahydrobiopterin Prevents Congenital Heart Defects Induced by Pregestational Diabetes

Congenital heart defects (CHDs) account for 1–5% of live births and are the leading cause of death in the first year life. Pregestational diabetes increases the risk for CHDs over five fold. We have recently shown that pregestational diabetes in mice induces CHDs in 58% of offspring. Exposure of the embryo to a hyperglycemic environment leads to oxidative stress. Reactive oxygen species (ROS) production results in oxidation of proteins vital for heart development, such as endothelial nitric oxide synthase (eNOS). Activity of eNOS is down‐regulated in diabetes as it becomes uncoupled, and its cofactors are oxidized, leading to decreased nitric oxide production and increased superoxide formation. Tetrahydrobiopterin (BH4) is a principal co‐factor required for eNOS dimer stabilization, and is an endogenous antioxidant. In states of oxidative stress, BH4 is itself oxidized leading to eNOS uncoupling. Treatment with BH4 has been shown to recouple eNOS and improve vascular endothelial function in diabetes. The aim of this study was to investigate the effects of BH4 on fetal heart development in mice with pregestational diabetes. Pregestational diabetes was induced by administering streptozotocin (STZ, 75 mg/kg, IP for 3 days) to adult female C57BL/6 mice. BH4 (Kuvan 10 mg/kg/day) was orally administered to the pregnant females via dissolution in peanut butter. Diabetic pregnant mice without BH4 treatment served as controls. Embryos were collected at E18.5 for histological analysis of cardiac morphology and coronary artery formation. Our data show that pregestational diabetes resulted in a spectrum of CHDs including atrial septal defect (ASD), ventricular septal defects (VSD), atrioventricular septal defect (AVSD), and double outlet right ventricle (DORV). Maternal diabetes also resulted in coronary artery malformations, including decreased left and right main coronary artery diameter, artery abundance, and smooth muscle content. Notably, BH4 treatment significantly decreased the incidence of CHDs from 59.4 to 26.7% and abrogated major CHDs such as VSD, AVSD and DORV. BH4 treatment also rescued coronary artery malformations. Furthermore, lineage tracing was performed with a global double fluorescent Mef2C‐Cre;mT/mG mouse, where anterior second heart field (SHF) progenitors are labeled with GFP. Fate mapping revealed significantly reduced numbers of GFP+ SHF progenitors contributing to the outflow tract cushions at E9.5, endocardial cushions at E12.5 and ventricular walls at E12.5, indicating defects in proliferation, migration and myocardialization induced by maternal diabetes. Finally, western blot analysis revealed low eNOS dimer to monomer ratio in the STZ‐induced diabetic E12.5 ventricles; this was reversed by BH4 treatment. These results suggest that eNOS uncoupling may be responsible for decreased SHF progenitor cell recruitment, thereby causing the CHDs observed. In conclusion, eNOS uncoupling contributes to the development of CHDs induced by pregestational diabetes in mice. BH4 treatment recouples eNOS and prevents CHDs, implicating it as a therapeutic target for the potential prevention of CHDs caused by pregestational diabetes in patients. Support or Funding Information This study was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to Q.F. (grant #MOP‐119600)