Van Neck, R.; Lu, X.; Wisse, L. J.; DeRuiter, M. C.; Feng, Q.

Background: Pregestational diabetes (PGD) increases congenital heart defect (CHD) risk over five-fold. Maternal exercise enhances endothelial nitric oxide synthase (eNOS) activity, benefiting embryos, though its causal role remains unclear. This study investigated eNOS\'s role in maternal exercise-mediated protection of fetal heart development in a PGD mouse model. Methods: PGD was induced in eNOS+/- or wild-type (WT) female mice via streptozotocin before breeding with WT or eNOS+/- males. Pregnant females had access to a running wheel for voluntary exercise or remained sedentary. Fetuses were collected at embryonic day (E) 18.5 for genotyping and CHD assessment. E12.5 hearts were analyzed for proliferation, apoptosis, oxidative stress, and eNOS protein levels. Results: Maternal exercise normalized litter size and mortality rates in offspring of diabetic eNOS+/- females but did not reduce CHD incidence in offspring of WT or eNOS+/- females with PGD. CHDs included septal defects, double outlet right ventricle, and valve defects. Exercise increased coronary artery density but not capillary density. Proliferation deficits at E12.5 were restored by exercise, yet oxidative stress remained elevated. Maternal exercise in eNOS+/- dams during PGD did not significantly change eNOS protein levels in both eNOS+/+ and eNOS+/- fetal hearts. Offspring genotype did not impact CHD incidence, cell proliferation, apoptosis or oxidative stress. Conclusions: Maternal exercise does not prevent CHDs in PGD offspring of eNOS+/- mice. Its ability to mitigate PGD-induced oxidative stress is eNOS-dependent and essential for improving heart morphology.