Pregestational diabetes is a major risk factor for CHDs in humans. However, the molecular mechanisms that lead to the development of CHDs and possible therapeutic approaches to prevent those defects are still not fully understood. It is generally believed that oxidative stress plays a major role in the induction of birth defects in diabetic fetus [12, 16, 30]. Here, we employed a mouse model of pregestational diabetes induced by STZ and studied the effects of NAC treatment on CHDs in the offspring of diabetic mice. Our data showed that pregestational diabetes resulted in a high incidence of CHDs and decreased cell proliferation associated with altered expression levels of Gata4, Gata5 and Vegf-a. Importantly, GSH levels were decreased while ROS levels were increased in the fetal heart of pregestational diabetes. Notably, these abnormalities in the fetal heart were rescued by maternal treatment with NAC. Our study provides new evidence on the critical role of glutathione in embryonic heart development and suggests that NAC may have therapeutic potential in preventing CHDs in patients with pregestational diabetes.
To simulate congenital malformations induced by maternal diabetes without genetic modifications, several experimental approaches have been used, which include STZ- or alloxan-induced diabetes, and infusion of glucose to induce hyperglycemia. When diabetes or hyperglycemia is induced at the time of mating or within a few days after gestation, congenital defects in the central nervous system and skeletal malformation are observed in the offspring [31, 32]. While congenital heart malformations have been observed in animal studies of diabetic pregnancy, a spectrum of defects that can arise has been less well characterized [33–35]. In the present study, diabetes was induced by STZ in female mice for at least one week before gestation. Our results show that pregestational diabetes induces embryopathy with a wide range of cardiovascular malformations including ASD, VSD, AVSD, TGA, DORV and TOF. These malformations of the cardiovascular system mirror congenital defects of neonates born to females with pregestational diabetes . Thus, our model represents an appropriate animal model to study CHDs induced by pregestational diabetes.
Diabetes increases ROS production through increased activity of ROS generating enzymes and decreased activity of antioxidant enzymes [36, 37]. Extensive evidence have shown the involvement of oxidative stress in diabetic embryopathy  and the importance of glutathione in regulating ROS levels and redox signaling . In the present study, we demonstrated that ROS levels were significantly increased in the fetal heart of diabetic offspring. Furthermore, total glutathione, GSH and GSSG levels were decreased in the embryonic heart of diabetic offspring. To replenish GSH levels in the diabetic fetal heart, female mice with pregestational diabetes were treated with NAC, a precursor of cysteine essential for the production of GSH [18, 39, 40]. Notably, treatment with NAC increased GSH levels and decreased ROS levels in the diabetic fetal heart. Importantly, NAC treatment also significantly decreased CHDs induced by pregestational diabetes. These data suggest an important role of GSH depletion and excessive ROS production in the development of CHDs. Previous studies have shown that treatment with NAC in vitro or GSH ethyl ester in vivo reduces gross embryonic malformation induced by high glucose or maternal diabetes [10, 16]. However, the beneficial effect of glutathione on cardiac development was limited to outflow tract defects induced by high glucose [20, 34]. The present study further demonstrated the beneficial effects of NAC on a wide spectrum of cardiovascular malformations induced by pregestational diabetes in vivo. It should be noted that NAC treatment did not alter total glutathione levels in the fetal hearts of control mice. This is not surprising because intracellular GSH levels are regulated by a feedback inhibition to glutamate-cysteine ligase (GCL), a rate limiting enzyme in the production of GSH . As such, the exogenous NAC participates in GSH synthesis only during oxidative stress conditions . In addition, NAC also protects GSH from oxidation through its antioxidant properties independent of GSH synthesis , leading to an increased GSH/total glutathione ratio in both diabetic or control mice in the present study.
It is well documented that ROS regulates gene expression, cell proliferation and apoptosis . In the present study, transcription factors that are critical to embryonic heart development including Gata4, Gata5[44, 45] were downregulated in diabetic fetal hearts at E11.5. However similar to previous studies, Vegf-a mRNA levels were increased in diabetic fetal hearts . Elevated expression levels of VEGF-A are associated with congenital heart defects [46, 47]. High VEGF-A levels in fetal hearts inhibit epithelial-to-mesenchymal transition (EMT) in the endocardial cushion, which contributes to formation of atrioventricular septum [48, 49]. Although a causal relationship between altered expression of Gata4, Gata5, Vegf-a and the development of CHDs in our study cannot be established, the fact that treatment with NAC restored their expression, improved cell proliferation via restoring cyclin D1 expression and prevented CHDs in diabetic offspring suggests that these transcription factors are sensitive to redox regulation and their alteration may contribute at least in part to cardiac malformation in pregestational diabetes.
Apoptosis is a highly regulated process and aberrant apoptosis may result in cardiovascular defects [50, 51]. The present study showed that apoptosis in the endocardial cushion was increased in diabetic embryos. Additionally, treatment with NAC increased apoptosis and induced 10% septal defects in control embryos. These data are consistent with a role of apoptosis in cardiac malformation [51, 52]. Cell proliferation was assessed using pHH3 staining, which marks cells undergoing mitosis. Notably, the number of pHH3+ cells in the endocardial cushion and myocardium was decreased in diabetic embryos, which was rescued by NAC treatment. Cell proliferation in endocardial cushion was also increased by NAC treatment in the control embryos. Since NAC treatment did not inhibit apoptosis in diabetic embryos in our study, increases in cell proliferation may represent a major effect of NAC in preventing CHDs in diabetes.
In conclusion, the present study demonstrated that pregestational diabetes induces a wide spectrum of CHDs similar to humans. Treatment with NAC increases GSH levels, decreases ROS levels in the fetal heart and prevents the development of CHDs in the offspring of pregestational diabetes. In women with pregestational diabetes, insulin is the primary treatment to achieve good glycemic control . However, insulin treatment is not sufficient to decrease the risk of CHDs in the diabetic offspring to normal levels [38, 54]. Even with optimal care and planning of diabetic pregnancies, the risk of CHDs in the offspring of diabetic mothers is not as low as in the offspring of nondiabetic mothers. Further studies are required to investigate whether NAC, an FDA approved drug either alone or in combination with insulin prevents CHDs in infants of women with pregestational diabetes.