Impact of genetic factors on antioxidant rescue of maternal diabetes–associated congenital heart disease

Figures

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  Figure 1. Endothelial/endocardial haploinsufficiency of Notch1 is sufficient for GxE with matDM. (A) Breeding scheme to generate diabetic and nondiabetic E14.5 Notch1fl/wt and Notch1fl/wt Tie2-Cre+ mice for histological analysis. (B) Table showing incidence of VSD in E14.5 embryos. (C) Representative images of matDM-exposed and nondiabetic Notch1fl/wt and Notch1fl/wt Tie2-Cre+ embryonic hearts. Asterisk denotes VSD. P value obtained from Fisher’s exact test; scale bar = 200 μm. ND, nondiabetic; DM, diabetic; RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; IVS, interventricular septum; VSD, ventricular septal defect.

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  Figure 2. matDM impairs AV cushion size in Notch1-haploinsufficient embryonic hearts. (A) Three-dimensional (3D) projection of AV cushions in E11.5 control and matDM-exposed WT and Notch1+/– embryonic hearts. Top panel shows overlay of 3D projection on corresponding 2D transverse section of the heart; scale bar = 100 μm. Bottom panel shows AV cushion 3D projections rotated 90° to show AV cushion span right to left across AVC. (B) Quantification of AV cushion volume from 3D projections. (C) Control and matDM-exposed WT and Notch1+/– embryonic hearts showing Tie2-Cre–driven RosamT;mG-based GFP expression. GFP+ cells in the heart represent all endocardially and endocardially derived cells. Top panel shows GFP expression throughout the embryonic heart; scale bar = 200 μm. Bottom panel shows corresponding 240 × 240 µm zoomed-in images of AV cushions for each genotype. (D) Quantification of GFP+ cell nuclei within AV cushion based on DAPI staining. Each point represents a single animal of corresponding genotype/condition. P values obtained from t tests for the estimated marginal means for all pairwise comparisons using Tukey’s correction to adjust for multiple testing. AV, atrioventricular; dor,…

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  Figure 3. matDM impairs proteoglycan distribution within AV cushion of Notch1-haploinsufficient embryonic hearts. (A) Alcian blue staining of control and matDM-exposed E11.5 WT and Notch1+/– AV cushion. (B) Immunostaining of cleaved versican (DPEAAE) in control and matDM-exposed WT and Notch1+/– AV cushion. (C) Quantification of Alcian blue staining from A. (D) Quantification of DPEAAE staining from B. Each point represents a single animal of corresponding genotype/condition. P values obtained from t tests for the estimated marginal means for all pairwise comparisons using Tukey’s correction to adjust for multiple testing. Scale bar = 100 μm.

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  Figure 4. NOTCH1 haploinsufficiency and oxidative stress act synergistically to dysregulate processes involved in endocardial cushion morphogenesis. (A) Principal component analysis shows the grouping of biological replicates and high variance between genotype and response to oxidative stress treatment. (B) Volcano plot showing upregulated and downregulated DEGs in NOTCH1+/– versus NOTCH1WT iECs in control condition. (C) Volcano plot showing upregulated and downregulated DEGs in NOTCH1+/– and NOTCH1WT iECs in oxidative stress. (D) Venn diagram showing number of common and unique DEGs in NOTCH1+/– iECs versus NOTCH1WT in control and oxidative stress condition followed by heatmap showing top 50 significant DEGs in NOTCH1+/– versus NOTCH1WT in control and oxidative stress condition. (E) Top significantly enriched GO BP terms in NOTCH1+/– iECs versus NOTCH1WT in control. (F) Top significantly enriched GO BP terms in NOTCH1+/– iECs versus NOTCH1WT in oxidative stress. (G) Heatmap of dysregulated pathways in NOTCH1+/– versus NOTCH1WT in control and oxidative stress. (H) DEGs in NOTCH1+/– iECs versus NOTCH1WT in oxidative stress that affect GO term extracellular matrix organization. Asterisk…

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  Figure 5. SOD1 overexpression does not rescue matDM-associated VSD in the setting of Notch1 haploinsufficiency. (A) Breeding scheme to generate E14.5 DM WT, SOD1+, Notch1+/–, and Notch1+/– SOD1+ embryos. (B and C) Graph and table showing incidence of VSD in E14.5 embryos by genotype. (D) Representative images of matDM-exposed WT, SOD1+, Notch1+/–, and Notch1+/– SOD1+ embryonic hearts. Asterisk denotes VSD. P values obtained from Fisher’s exact test; scale bar = 200 μm. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; IVS, interventricular septum; VSD, ventricular septal defect; DM, diabetic.