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Research ArticleCardiologyDevelopment
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10.1172/jci.insight.183516
1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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1Center for Cardiovascular Research, Abigail Wexner Research Institute, and The Heart Center, Nationwide Children’s Hospital, Columbus, Ohio, USA.
2Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
3Center for Perinatal Research, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA.
4Department of Pediatrics and
5Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
Address correspondence to: Vidu Garg, Center for Cardiovascular Research and The Heart Center, Nationwide Children’s Hospital, Room WB4275 Columbus, Ohio 43205, USA. Phone: 614.355.5710; Email: vidu.garg@nationwidechildrens.org.
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Published October 22, 2024 - More info
Published in Volume 9, Issue 23 on December 6, 2024
AbstractCongenital heart disease (CHD) affects approximately 1% of live births. Although genetic and environmental etiologic contributors have been identified, the majority of CHD lacks a definitive cause, suggesting the role of gene-environment interactions (GxEs) in disease pathogenesis. Maternal diabetes mellitus (matDM) is among the most prevalent environmental risk factors for CHD. However, there is a substantial knowledge gap in understanding how matDM acts upon susceptible genetic backgrounds to increase disease expressivity. Previously, we reported a GxE between Notch1 haploinsufficiency and matDM leading to increased CHD penetrance. Here, we demonstrate a cell lineage–specific effect of Notch1 haploinsufficiency in matDM-exposed embryos, implicating endothelial/endocardial tissues in the developing heart. We report impaired atrioventricular cushion morphogenesis in matDM-exposed Notch1+/– animals and show a synergistic effect of NOTCH1 haploinsufficiency and oxidative stress in dysregulation of gene regulatory networks critical for endocardial cushion morphogenesis in vitro. Mitigation of matDM-associated oxidative stress via superoxide dismutase 1 overexpression did not rescue CHD in Notch1-haploinsufficient mice compared to wild-type littermates. Our results show the combinatorial interaction of matDM-associated oxidative stress and a genetic predisposition, Notch1 haploinsufficiency, on cardiac development, supporting a GxE model for CHD etiology and suggesting that antioxidant strategies alone may be ineffective in genetically susceptible individuals.
IntroductionCongenital heart disease (CHD) affects approximately 1% of live births and is the leading cause of birth defect–related infant mortality (1). Although high-throughput genome-sequencing technologies have made remarkable advances in uncovering genetic etiologies for CHD, the underlying cause for more than half of CHD cases is still unknown (2–5). A subset of these cases have long been proposed to be the combinatorial effect of genetic predisposition and environmental influences, resulting in complex inheritance patterns and variable CHD expressivity (6, 7). Among environmental contributors, maternal pregestational diabetes mellitus is a highly prevalent and well-established risk factor for CHD, increasing the risk of having an infant with CHD by 3- to 5-fold (8–13). The influence of maternal diabetes mellitus (matDM) on cardiac development has been studied in animal models, shedding light on key cellular and molecular pathways particularly vulnerable to this environmental milieu (14). Recently, matDM has been shown to have cell lineage–specific effects during cardiac morphogenesis, specifically affecting the second heart field and neural crest lineages to disrupt cardiomyocyte differentiation and anterior-posterior specification (15, 16). The incidence of CHD in a pregnancy complicated by pregestational diabetes mellitus has remained elevated even with substantial advancements in prenatal care, suggesting that matDM may cause CHD by acting on susceptible genetic backgrounds, i.e., gene-environment interaction (GxE) etiology. However, the cellular and molecular basis for GxE in CHD as postulated in the multifactorial hypothesis for CHD remain limited (6, 17, 18).
Cardiac development is a dynamic process with tight spatiotemporal regulation of several multipotent cardiac cell lineages (19, 20). Endothelial-mesenchymal transition (EndMT) is a crucial process that occurs early in heart development wherein endocardial cells lining the atrioventricular canal (AVC) and developing outflow tract (OFT) detach from the monolayer and migrate into the preformed cardiac jelly to form endocardial cushions (21–23). Subsequent remodeling of the endocardial cushions at the AVC leads to the formation of the atrioventricular valve leaflets, atrioventricular septum, and membranous portion of the ventricular septum (24, 25). At the OFT, endocardial cushions receive additional contributions from the migrating cardiac neural crest cells to form the semilunar valves and aorticopulmonary septum (26). Although matDM is associated with a spectrum of CHD phenotypes, the most commonly observed include septal and conotruncal (involving OFT and great vessels) heart defects, suggesting EndMT and endocardial cushion morphogenesis are particularly susceptible to disruption by the abnormal diabetic environment (10, 11). This is supported by several published studies in animal models showing dysregulation of key EndMT signaling pathways in embryonic hearts exposed to matDM, including BMP, TGF-β, and Notch signaling (27–31). Additionally, oxidative stress is a hallmark of diabetic embryopathy, and impaired redox signaling has been implicated in dysregulation of several cardiac developmental pathways, suggesting that matDM-associated oxidative stress may be culpable for the elevated risk of CHD (31, 32).
Previously, we found matDM interacts with Notch1 haploinsufficiency in mice to increase the incidence of membranous ventricular septal defects (VSDs), supporting a GxE between Notch1 and matDM (27). Pathogenic NOTCH1 variants are associated with a spectrum of CHD in humans, and Notch signaling is known to interact with other signaling pathways such as BMP and TGF-β to facilitate EndMT in the developing heart (33–38). To better characterize the Notch1-matDM GxE and place it in the context of cardiac development, we hypothesized that matDM and Notch1 haploinsufficiency functionally converge to disrupt endocardial cushion morphogenesis and EndMT to increase the risk of CHD.
In this study, we demonstrate that matDM and Notch1 haploinsufficiency interact within the developing endothelial/endocardial and endocardially derived mesenchyme to increase the incidence of membranous VSD. We report abnormal atrioventricular (AV) cushion morphogenesis in matDM-exposed Notch1-haploinsufficient (Notch1+/–) embryonic hearts compared with nondiabetic controls. In a human induced pluripotent stem cell–based (iPSC-based) in vitro model, we show that NOTCH1 haploinsufficiency sensitizes endothelial cells to the effects of oxidative stress and disrupts a network of genes and biological processes underlying EndMT and endocardial cushion morphogenesis. Consistent with this, we find matDM-exposed Notch1+/– embryos are insensitive to antioxidant-based therapeutic strategy, despite rescue of VSD observed in matDM-exposed WT littermates. Overall, the results from this study elucidate mechanisms by which matDM interacts with a genetic susceptibility, i.e., Notch1 haploinsufficiency, to bring about cell lineage–specific effects and increase penetrance of CHD. This work serves as experimental proof supporting a multifactorial etiology for CHD, underscoring the need to identify novel genetic modifiers that act in conjunction with environmental teratogens to increase the incidence of CHD, and shows the variability in phenotypic rescue with antioxidant therapies in a genetically susceptible background.
ResultsGxE between endothelial Notch1 haploinsufficiency and matDM causes CHDs. Previously, we identified a novel GxE between Notch1 haploinsufficiency and matDM in mice, wherein Notch1+/– embryos exposed to matDM had significantly higher incidence of VSD compared with WT littermates at embryonic day (E) 13.5 (27). We first sought to determine if this GxE is evident at a later developmental stage in mice. For this, we generated streptozotocin-induced (STZ-induced) diabetic WT females for timed mating with nondiabetic Notch1+/– males. Analysis of matDM-exposed embryos at E14.5 (n = 3 litters) verified a previously reported GxE resulting in a significantly higher incidence of membranous VSD in Notch1+/– (10/14, 71%) embryos compared with WT littermates (1/10, 10%, Fisher’s exact test P value = 0.005) (Supplemental Figure 1, A–C; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.183516DS1).
The expression pattern of activated NOTCH1 in the developing heart has been well characterized, being highly expressed within endocardial and endocardially derived mesenchymal cells, which form the endocardial cushions at the AVC and OFT (39, 40). Subsequently, we sought to determine if endothelium-specific Notch1 haploinsufficiency is sufficient to sensitize the effects of matDM on cardiac development. For this experiment, STZ-induced diabetic and nondiabetic Notch1fl/fl females were bred with nondiabetic Tie2-Cre+ males to generate control and matDM-exposed Notch1fl/wt Tie2-Cre+ embryos, which are conditionally heterozygous for Notch1 in all endothelial cells, including endocardial cells and their derived tissues in the heart (Figure 1A). While no VSD was found in nondiabetic Notch1fl/wt Tie2-Cre+ or Notch1fl/wt littermates, we found a significantly higher incidence of membranous VSD in E14.5 matDM-exposed Notch1fl/wt Tie2-Cre+ embryos (7/17, 41%) compared with non-Cre+ littermates (3/31, 9.7%, Fisher’s exact test P value = 0.022) (Figure 1, B and C). To account for variability across multiple matDM-exposed litters (n = 6), we used a general linear mixed model and included litter as the random effect, and the probability of VSD for Notch1fl/wt Tie2-Cre+ was found to be higher compared with Notch1fl/wt (P value = 0.06, see Supporting Data Values) (41). While this is slightly higher than the traditional 0.05 cut point, this still small P value provides evidence that the data are not compatible with the null hypothesis (42). We also noted that the incidence of VSD in matDM-exposed Notch1fl/wt Tie2-Cre+ (41%) was found to be lower than the incidence observed in matDM-exposed Notch1+/– (71%). We attribute this difference to potential roles of Notch1 haploinsufficiency in non-endothelium-derived cells of the heart in the GxE with matDM, or this variability may be due to incomplete deletion of Notch1 in all endothelial cells. Overall, these results suggest that the observed GxE between Notch1 haploinsufficiency and matDM can act within the developing endothelium, including the endocardium- and endocardium-derived cells to impair cardiac morphogenesis, particularly ventricular septation.
Figure 1Endothelial/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.
Notch1 haploinsufficiency exacerbates the effects of matDM on AV cushion development. During cardiac development, endocardial cells undergo EndMT to form endocardial cushions at the AVC and OFT. Subsequent remodeling of the proximal OFT and AV cushions gives rise to the AV septal complex, which includes the mitral and tricuspid valve leaflets and the membranous ventricular septum (24, 25, 43). Here, we sought to determine if interaction between Notch1 haploinsufficiency and matDM impairs endocardial cushion morphogenesis at the OFT and AVC. As Notch signaling is known to regulate EndMT during endocardial cushion development, we utilized the Rosa26mT/mG locus to trace endothelial cells that have undergone EndMT in endocardial cushions of matDM-exposed Notch1+/– embryos compared with nondiabetic controls. We analyzed histological sections of OFT and AV cushions at E11.5; volumetric analysis using 3D reconstruction of serial histological sections revealed matDM-exposed Notch1+/– hearts had significantly smaller AV cushion size compared with nondiabetic Notch1+/– and nondiabetic WT hearts (Figure 2, A and B). On the other hand, matDM-exposed WT hearts did not show a statistically significant difference in AV cushion size compared to nondiabetic controls. We found no significant difference in OFT cushion size across the 4 groups, suggesting that this GxE affects AV cushion morphogenesis to contribute to membranous VSD (Supplemental Figure 2, A and B). Upon examination of GFP+ EndMT-derived cells within the AV cushions, GFP+ cells appeared more compacted within the AV cushion of matDM-exposed Notch1+/– hearts, which was likely due to the smaller AV cushion size (Figure 2C). This was quantified by counting the number of GFP+ cell nuclei within the AV cushion, and a statistically significant increase in cell density was noted between matDM-exposed Notch1+/– embryos compared with nondiabetic Notch1+/– control (Figure 2D). Again, this effect was not observed in the matDM-exposed WT littermates, and we noted a high degree of variability in this group, possibly due to the low penetrance of disease in matDM-exposed WT embryos. These results show that the overall size of the AV cushion is significantly smaller in the matDM-exposed Notch1+/– compared with nondiabetic controls, causing EndMT-derived cells to become more densely packed within the AV cushion.
Figure 2matDM 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, dorsal; R, right; L, left; ven, ventral.
The deposition and distribution of extracellular matrix (ECM) components, including sulfated proteoglycans such as hyaluronic acid and versican, are crucial for endocardial cushion formation and remodeling into the AV valvuloseptal complex, a process highly regulated by both endocardial and myocardial signaling adjacent to the AV cushion (44–46). Alcian blue staining of WT and Notch1+/– embryos of diabetic and nondiabetic dams at E11.5 showed reduced proteoglycan deposition in matDM-exposed Notch1+/– AV cushion compared with nondiabetic control while matDM-exposed WT embryos did not show a difference compared to nondiabetic controls (Figure 3, A and C). During AV cushion development, full-length versican undergoes proteolytic cleavage by the action of matrix metalloproteinases MMP2 and ADAMTS1 (47). Similarly, expression of cleaved versican, labeled by an antibody against neo-epitope DPEAAE, was significantly decreased in the matDM-exposed Notch1+/– AV cushion compared with nondiabetic Notch1+/– control (Figure 3, B and D). Similar to the AV cushion size and cellular density data, we found a high degree of variability in the matDM-exposed WT littermates, and as a result no statistical significance was reached between matDM-exposed WT and nondiabetic controls in either the Alcian blue staining or DPEAAE expression. Overall, these results indicate that GxE between Notch1 haploinsufficiency and matDM impairs AV cushion morphogenesis potentially via disruption of ECM organization.
Figure 3matDM 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.
NOTCH1 haploinsufficiency acts synergistically with oxidative stress to dysregulate genes involved in EndMT and endocardial cushion morphogenesis. Previously published research from us and others have proposed matDM-associated oxidative stress as the key driver of cardiac maldevelopment, leading to increased incidence of CHD. To determine if there is elevated oxidative stress in matDM-exposed Notch1+/– embryos compared with nondiabetic controls, we probed E11.5 matDM-exposed and nondiabetic WT and Notch1+/– embryonic hearts with an antibody against 4-hydroxynoneal (4-HNE), a stable by-product of lipid peroxidation and a robust biomarker for cellular oxidative stress (48). We detected increased 4-HNE labeling in matDM-exposed Notch1+/– and WT littermates compared with nondiabetic controls while no difference was detected between Notch1+/– and WT matDM-exposed hearts, including in the AV cushions, suggesting there are comparable levels of oxidative stress in matDM-exposed WT and Notch1+/– littermates at this time point (Supplemental Figure 3, A and C). Increased oxidative stress can induce cellular apoptosis in diabetic embryos, particularly during the neurulation stage at E8.75 (49, 50). To probe for increased apoptosis in matDM-exposed WT and Notch1+/– embryonic hearts, we performed TUNEL assay on E11.5 matDM-exposed and nondiabetic Notch1+/– and WT embryonic hearts. However, no significant differences were observed across groups, suggesting there is no increase in apoptosis in matDM-exposed cardiac tissues in either Notch1+/– or WT embryos at this time point (Supplemental Figure 3, B and D).
To determine the molecular mechanisms underlying GxE between endothelial Notch1 and matDM-associated oxidative stress, we utilized NOTCH1WT and isogenic NOTCH1+/– iPSC lines, generated as previously described (51, 52). We differentiated NOTCH1WT and NOTCH1+/– iPSCs to induced endothelial cells (iECs) using a previously published differentiation protocol (53). Following differentiation, subsets of NOTCH1WT and NOTCH1+/– iECs were either exposed to oxidative stress (50 μM H2O2) or left untreated as control, to mimic matDM-associated oxidative stress in vitro. After 4 days, total RNA was isolated from each iEC subset and bulk RNA sequencing performed for differential gene expression analysis. Principal component analysis of the sequenced samples revealed distinct clustering of biological replicates (n = 3 per genotype/condition), with the largest variance observed between NOTCH1+/– and NOTCH1WT in oxidative stress (Figure 4A). As expected, we observed high variance between NOTCH1+/– and NOTCH1WT control samples, suggesting there are intrinsic transcriptomic differences between NOTCH1+/– and NOTCH1WT iECs at baseline untreated condition. Differential gene expression analysis revealed there were 965 genes significantly differentially expressed (FDR-adjusted P value < 0.05, absolute log2 fold-change > 0.75) between NOTCH1+/– and NOTCH1WT iECs in control condition (437 upregulated and 528 downregulated) (Figure 4B and Supplemental Table 1). Next, we turned our attention to how these genotypes differed from each other during exposure to oxidative stress. Differential gene expression analysis revealed there were 2,890 genes significantly differentially expressed (FDR-adjusted P value < 0.05, absolute log2 fold-change > 0.75) between NOTCH1+/– and NOTCH1WT iECs under oxidative stress (1,508 upregulated and 1,382 downregulated) (Figure 4C and Supplemental Table 2). We verified approximately 40% downregulation of Notch1 mRNA in NOTCH1+/– iECs under both conditions (log2 fold-change = –0.64, FDR-adjusted P value < 0.05 in control; log2 fold-change = –0.61, FDR-adjusted P value < 0.05 in oxidative stress); however, it was not assigned as a DEG because of our stringent log2 fold-change cutoff of 0.75. Comparison of DEGs from control and oxidative stress revealed 638 genes were dysregulated in NOTCH1+/– iECs in both control and oxidative stress while 2,252 genes were dysregulated in NOTCH1+/– iECs only under oxidative stress (Figure 4D). Overrepresentation analysis of DEGs in NOTCH1+/– iECs under each condition was performed. Top significant biological processes in NOTCH1+/– iECs in control condition included embryonic organ development, skeletal system morphogenesis, and extracellular structure organization (Figure 4E and Supplemental Table 3). In contrast, top dysregulated biological processes in NOTCH1+/– in oxidative stress included terms related to cell division (MKI67, MYBL2, CDC20, PLK1, CCNB1), mesenchyme development (APLNR, EFNB1), and extracellular matrix organization (COL1A1, COL11A1, MMP2) (Figure 4F and Supplemental Table 4). While several biological pathways were commonly dysregulated in NOTCH1+/– iECs in both control and oxidative stress, including GO terms mesenchyme development, connective tissue development, collagen metabolic processes, and proteoglycan metabolic processes, the number of DEGs in each process was higher in NOTCH1+/– iECs in oxidative stress compared with control condition (Figure 4G). Additionally, many biological processes were found to be dysregulated in NOTCH1+/– iECs in oxidative stress, including the terms nuclear division, mitotic sister chromatid segregation, extracellular matrix organization, heart morphogenesis, Notch signaling, and nitric oxide signaling pathway among others (Figure 4G). Considering that extracellular matrix organization and proteoglycan metabolic processes are highly relevant processes in the context of endocardial cushion morphogenesis, we investigated the effect of oxidative stress in NOTCH1+/– iECs on genes within these terms, reporting several interconnected genes to be dysregulated across both processes (Figure 4, H and I). Among DEGs affecting ECM organization, the majority were upregulated, and many of these genes are known to be negative regulators of ECM organization (DPP4, FAP, ANTXR1, CST3, EMILIN1), while the majority of genes in proteoglycan metabolic processes were downregulated (BMPR1B, IGF1, COL11A1). Taken together, these results suggest Notch1 haploinsufficiency acts synergistically with oxidative stress to dysregulate gene regulatory networks crucial for endocardial cushion morphogenesis.
Figure 4NOTCH1 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 indicates genes that were also dysregulated in NOTCH1+/– iECs versus NOTCH1WT in control. (I) DEGs in NOTCH1+/– iECs versus NOTCH1WT in oxidative stress that affect GO term proteoglycan metabolic processes. Asterisk indicates genes that are also significantly differentially expressed in NOTCH1+/– iECs versus NOTCH1WT in control. DEGs, differentially expressed genes; FC, fold-change; GO, gene ontology; BP, biological processes; Padj, FDR-adjusted P value.
Overexpression of antioxidant gene, SOD1, does not reduce incidence of matDM-associated CHD in the setting of Notch1 haploinsufficiency. Oxidative stress is characterized by an imbalance of reactive oxygen species (ROS) production and antioxidant defense response. Superoxide dismutases (SODs) are crucial for neutralizing superoxide radicals, converting them to H2O2, which can be degraded to H2O and O2 by endogenous catalase and glutathione peroxidase. To determine if a genetic antioxidant strategy via overexpression of SOD1 is effective in rescuing CHD in a Notch1+/– background, we bred SOD1-overexpressing transgenic male mice (SOD1+) to diabetic Notch1+/– females to generate WT, Notch1+/–, SOD1+, and SOD1+ Notch1+/– compound-mutation embryos (n = 11 litters) (Figure 5A). We performed histological analysis of E14.5 embryonic hearts and found a significantly decreased incidence of VSD in SOD1+ (1/25) compared with WT (6/23, Fisher’s exact test P value = 0.044) (Figure 5, B–D). This is consistent with prior publications showing overexpression of SOD1 in embryonic hearts can reduce the incidence of matDM-associated CHD in a WT setting (31, 32). However, no significant reduction in the incidence of VSD was noted when comparing Notch1+/– (12/19) and SOD1+ Notch1+/– embryos (10/17, Fisher’s exact test P value = 0.99). To account for litter variability, we used binomial regression with litter as a random effect to further analyze these results, and while statistical significance was not achieved, a similar trend was noted (see Supporting Data Values). We compared the level of oxidative stress via 4-HNE labeling between the above genotypes and found decreased immunostaining of 4-HNE in SOD1+ compared with WT while no difference was observed between the Notch1+/– and SOD1+ Notch1+/– (Supplemental Figure 4, A and B), suggesting SOD1 overexpression cannot reduce oxidative stress in a Notch1+/– background. The failure of SOD1 overexpression to reduce the incidence of VSD resulting from the Notch1-matDM interaction is consistent with our in vitro results showing an exacerbated effect on cardiac developmental process in NOTCH1+/– iECs exposed to H2O2. Overall, our results solidify the role of Notch1 haploinsufficiency as a genetic modifier in matDM-associated CHD, highlighting the presence of GxEs and their mechanisms in CHD pathogenesis.
Figure 5SOD1 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.
DiscussionmatDM is an established risk factor for CHD; however, its effect on genetic susceptibilities has not been well described. In this study, we show matDM interacts with Notch1 haploinsufficiency in vivo to increase the disease penetrance of a membranous VSD phenotype. We find that this GxE acts within the developing endothelial/endocardial and endocardially derived cells and that Notch1 haploinsufficiency sensitizes the effects of matDM on EndMT and AV cushion morphogenesis, affecting deposition of ECM components including versican. Using a human iPSC-based model, we demonstrate that oxidative stress–exposed NOTCH1+/– endothelial cells have exacerbated effect on cardiac developmental processes compared with oxidative stress–exposed NOTCH1WT iECs. We show effects of oxidative stress and NOTCH1 haploinsufficiency converge to dysregulate networks underlying endocardial cushion morphogenesis. Finally, we find that targeting matDM-associated oxidative stress via overexpression of SOD1 is unable to rescue VSD in a Notch1-haploinsufficient background as compared with successful rescue in WT littermates. Taken together, these results reveal that susceptible genetic variation acts in combination with effects of matDM to disrupt cardiac development and increase the incidence of CHD.
Complex inheritance patterns and incomplete penetrance of CHD are often explained by genetic heterogeneity among individuals, with strong speculation as to the requirement of specific GxE or gene-gene interaction for disease manifestation (6, 54). In humans, pathogenic NOTCH1 variants are associated with a spectrum of CHD with variable expressivity (33–35). The Notch signaling pathway is highly conserved, and Notch1 heterozygosity in mice has been reported to interact with both environmental factors (e.g., gestational hypoxia, maternal diabetes) as well as genetic factors (e.g., deletion of Nos3) to increase the incidence of heart defects (27, 55, 56). Hence, NOTCH1 variation may play a crucial role in multifactorial cases of CHD, and results from this study corroborate the role of NOTCH1 as both a genetic modifier and driver in CHD pathogenesis.
During cardiac development, activated NOTCH1 (N1CD) is expressed in the developing endocardium lining the AVC and OFT; in synchrony with myocardial BMP signaling, N1CD coordinates initiation of EndMT and mesenchyme development at these regions to form the endocardial cushions (37, 57). EndMT-derived mesenchymal cells communicate with adjacent endocardial and myocardial layers to dynamically regulate organization and stratification of the ECM to form the valvuloseptal complex (24, 25). Based on our in vivo results, matDM-exposed Notch1+/– end
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