Pregestational Diabetes and Congenital Heart Defects: Role of Reactive Oxygen Species

Women with pregestational diabetes are at a high risk of having babies with congenital heart defects (CHDs). The mechanisms of CHD induction in the offspring of women with pregestational diabetes remain elusive. With an ever-increasing rate of diabetes in young adults, there is a pressing need to understand the underlying mechanisms and initiate effective preventative strategies. The aim of this thesis is to study the role of reactive oxygen species (ROS) signalling in heart morphogenesis, unravel molecular mechanisms of CHDs induced by pregestational diabetes and provide new insights on potential therapeutic strategies. To this end, a mouse model of pregestational diabetes induced by streptozotocin was employed. A spectrum of septal, conotruncal and coronary artery malformation were identified in the offspring of mice with pregestational diabetes. Reactive oxygen species (ROS) levels were elevated and glutathione levels were diminished in the fetal hearts of diabetic female mice. Oral treatment with an antioxidant, N-Acetylcysteine (NAC), significantly diminished the incidence of CHDs and prevented coronary artery malformation in the offspring of pregestational diabetic mice. Furthermore, pregestational diabetes reduced cell proliferation, altered transcript levels, and disrupted epithelial to mesenchymal transition (EMT) in the fetal heart of pregestational diabetic mice, which were all prevented by NAC treatment. To further study the role of basal ROS production in embryonic heart development, a NADPH oxidase Nox2 knockout mouse was utilized. We demonstrated that loss of Nox2 expression decreased ROS production, and impaired TGF-β/BMP signalling and endocardial EMT in embryonic hearts. This ultimately resulted in cardiac septum and valve defects. Thus, under normal physiological conditions ROS production promotes heart development whereas excess ROS levels during pregestational diabetes induce CHDs. These studies show maintaining a balance of ROS levels is essential for normal embryonic heart development in mice. Furthermore, NAC may have a therapeutic potential in preventing the development of CHDs during pregestational diabetes

A regulatory subunit o f Protein Phosphatase 2A, PP2A-B\u27, is dispensable for activation of Sex Comb Reduced activity in Drosophila melanogaster

The Drosophila HOX transcription factor, Sex Combs Reduced (SCR), is required for determining labial and the first thoracic segmental identity. A Protein Phosphatase 2A holoenzyme assembled with the PP2A-B\u27 regulatory subunit has been proposed to specifically interact with and dephosphorylate SCR homeodomain activating SCR protein activity. To test this hypothesis, a null mutation was created in the PP2A-B\u27 gene, PP2A- B\u27a, using Flip mediated site-specific recombination. The number of sex comb bristles, salivary gland nuclei and pseudotracheal rows are SCR-dependent and were counted as a measure of SCR activity in vivo. Adults and larvae homozygous for PP2A-B\u27a showed no decrease in SCR activity. In addition no evidence of functional redundancy of PP2A-B\u27 with other regulatory subunits, Twins (TWS) and Widerborst (WDB) for dephosphorylation and activation of SCR activity was observed suggesting that PP2A has no role in activation of SCR activity

N-Acetylcysteine prevents congenital heart defects induced by pregestational diabetes

Background: Pregestational diabetes is a major risk factor of congenital heart defects (CHDs). Glutathione is depleted and reactive oxygen species (ROS) production is elevated in diabetes. In the present study, we aimed to examine whether treatment with N-acetylcysteine (NAC), which increases glutathione synthesis and inhibits ROS production, prevents CHDs induced by pregestational diabetes.Methods: Female mice were treated with streptozotocin (STZ) to induce pregestational diabetes prior to breeding with normal males to produce offspring. Some diabetic mice were treated with N-acetylcysteine (NAC) in drinking water from E0.5 to the end of gestation or harvesting of the embryos. CHDs were identified by histology. ROS levels, cell proliferation and gene expression in the fetal heart were analyzed.Results: Our data show that pregestational diabetes resulted in CHDs in 58% of the offspring, including ventricular septal defect (VSD), atrial septal defect (ASD), atrioventricular septal defects (AVSD), transposition of great arteries (TGA), double outlet right ventricle (DORV) and tetralogy of Fallot (TOF). Treatment with NAC in drinking water in pregestational diabetic mice completely eliminated the incidence of AVSD, TGA, TOF and significantly diminished the incidence of ASD and VSD. Furthermore, pregestational diabetes increased ROS, impaired cell proliferation, and altered Gata4, Gata5 and Vegf-a expression in the fetal heart of diabetic offspring, which were all prevented by NAC treatment.Conclusions: 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. Our study suggests that NAC may have therapeutic potential in the prevention of CHDs induced by pregestational diabetes. © 2014 Moazzen et al.; licensee BioMed Central Ltd

NOX2 Is Critical to Endocardial to Mesenchymal Transition and Heart Development

NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of Nox2 disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of Nox2-/- neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, Nox2-/- embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, Nox2 deficiency disrupted EndMT of atrioventricular cushion explants ex vivo. Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of Nox2 was associated with reduced expression of Gata4, Tgfβ2, Bmp2, Bmp4, and Snail1, which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development