Outflow Tract Defects Research Articles

Dysregulation of TBX1 dosage in the anterior heart field results in congenital heart disease resembling the 22q11.2 duplication syndrome.

Non-allelic homologous recombination events on chromosome 22q11.2 during meiosis can result in either the deletion (22q11.2DS) or duplication (22q11.2DupS) syndrome. Although the spectrum and frequency of congenital heart disease (CHD) are known for 22q11.2DS, there is less known for 22q11.2DupS. We now evaluated cardiac phenotypes in 235 subjects with 22q11.2DupS including 102 subjects we collected and 133 subjects that were previously reported as a confirmation and found 25% have CHD, mostly affecting the cardiac outflow tract (OFT). Previous studies have shown that global loss or gain of function (LOF; GOF) of mouse Tbx1, encoding a T-box transcription factor mapping to the region of synteny to 22q11.2, results in similar OFT defects. To further evaluate Tbx1 function in the progenitor cells forming the cardiac OFT, termed the anterior heart field, Tbx1 was overexpressed using the Mef2c-AHF-Cre driver (Tbx1 GOF). Here we found that all resulting conditional GOF embryos had a persistent truncus arteriosus (PTA), similar to what was previously reported for conditional Tbx1 LOF mutant embryos. To understand the basis for the PTA in the conditional GOF embryos, we found that proliferation in the Mef2c-AHF-Cre lineage cells before migrating to the heart, was reduced and critical genes were oppositely changed in this tissue in Tbx1 GOF embryos versus conditional LOF embryos. These results suggest that a major function of TBX1 in the AHF is to maintain the normal balance of expression of key cardiac developmental genes required to form the aorta and pulmonary trunk, which is disrupted in 22q11.2DS and 22q11.2DupS.

Fetal major cardiac defects and placental dysfunction at 11-13 weeks' gestation.

To investigate the relationship between fetal major cardiac defects and markers of placental perfusion and function. This was a prospective screening study in singleton pregnancies at 11-13 weeks' gestation. Uterine artery pulsatility index (UtA-PI), serum pregnancy-associated plasma protein-A (PAPP-A) and placental growth factor (PlGF) were measured and the values were converted into multiples of the normal median (MoM). Median MoM values in fetuses with isolated major cardiac defects were compared with those in fetuses without major defects. The 50 094 singleton pregnancies fulfilling the entry criteria included 49 898 pregnancies with normal cardiac anatomy and 196 (0.39%) with major congenital cardiac defects: 73 (37.2%) with conotruncal defects, 63 (32.1%) with left ventricular outflow tract defects and 60 (30.6%) with valvular defects. In the group with cardiac defects, compared with controls, there was lower median PAPP-A MoM (0.81 vs 1.00, P < 0.0001) and PlGF MoM (0.78 vs 1.00, P < 0.0001) but no significant difference in UtA-PI MoM (1.01 vs 1.00, P = 0.162). In pregnancies with isolated fetal major cardiac defects, there is evidence of placental dysfunction in the absence of impaired placental perfusion. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Preservation of conductive propagation after surgical repair of cardiac defects with a bio-engineered conductive patch

Both stable and biodegradable biomaterials have been used to surgically repair congenital cardiac defects. However, neither type of biomaterial can conduct electrical activity. We evaluated the conductivity and efficacy of a newly synthesized conductive polypyrrole-chitosan (Ppy+Chi) gelfoam patch to support cardiomyocyte (CM) viability and function in vitro and to surgically repair a cardiac defect in vivo. Ppy+Chi was incorporated into gelfoam (Gel) to form a 3-dimensional conductive patch. In vitro, patch characteristics were evaluated and biocompatibility and bioconductivity were investigated by culturing neonatal rat CMs on the patches. In vivo, a full-thickness right ventricular outflow tract defect was created in rats and the patches were implanted. Four weeks after patch repair, cardiac electrical activation and conduction velocity were evaluated using an optical mapping system. In vitro, the Ppy+Chi+Gel patch had a higher mean breaking stress than the Gel or Chi+Gel patches, and the highest conductivity. None of the patches altered cell growth. The Ca2+ transient velocity of CMs cultured on the Ppy+Chi+Gel patch was 2.5-fold higher than that of CMs cultured on the Gel or Chi+Gel patches. In vivo, optical mapping at 4 weeks post-implantation demonstrated that Ppy+Chi+Gel patch-implanted hearts had faster conduction velocities, as measured on the epicardial surface. Continuous electrocardiographic telemetry did not reveal any pathologic arrhythmias after patch implantation. Ex-vivo patch conductivity testing also revealed that the Ppy+Chi+Gel patch was more conductive than the Gel and Chi+Gel patches. The Ppy+Chi+Gel patch was biocompatible, safe and conductive, making it an attractive candidate for a new biomaterial platform for cardiac surgical repair to preserve synchronous ventricular contraction.

Clinical and molecular effects of CHD7 in the heart.

Heart defects caused by loss-of-function mutations in CHD7 are a frequent cause of morbidity and mortality in CHARGE syndrome. Here we review the clinical and molecular aspects of CHD7 that are related to the cardiovascular manifestations of the syndrome. The types of heart defects found in patients with CHD7 mutations are variable, with an overrepresentation of atrioventricular septal defect and outflow tract defect including aortic arch anomalies compared to nonsyndromic heart defects. Chd7 haploinsufficiency in mouse is a good model for studying the heart effects seen in CHARGE syndrome, and mouse models reveal a role for Chd7 in multiple lineages during heart development. Formation of the great vessels requires Chd7 expression in the pharyngeal surface ectoderm, and this expression likely has an non-autonomous effect on neural crest cells. In the cardiogenic mesoderm, Chd7 is required for atrioventricular cushion development and septation of the outflow tract. Emerging knowledge about the function of CHD7 in the heart indicates that it may act in concert with transcription factors such as TBX1 and SMADs to regulate genes such as p53 and the cardiac transcription factor NKX2.5.

Dose-dependent effect of Hyperglycemia during Cardiac Development

Congenital heart disease (CHD) constitutes a major proportion of birth defects among infants. It has a complex etiology, involving both genetic and environmental determinants. Pre-existing maternal diabetes mellitus (DM) is one environmental risk factor associated with increased risk of CHDs. Previous studies have reported a direct correlation between maternal glucose level and the onset of specific cardiac malformations. Hyperglycemia (HG) is the primary teratogen in DM mediated complications and results in increased levels of reactive oxygen species (ROS) and reduced nitric oxide (NO) bioavailability. However, the exact molecular mechanism underlying the influence of altered redox homeostasis on the development of CHD is unclear. In this study, we hypothesized that in response to various concentrations of glycemic stress in utero, there is dose-dependent change in ROS regulating enzymes. We utilized a Streptozotocin induced type1 DM mouse model as well as atrioventricular cushion mesenchymal cells (AVM), an embryonic cardiac cell line for our study. AVM cells were cultured for 6, 24 and 48-hours in normal glucose (NG, 5.5mM) and three different concentrations of D-glucose (HG; 10mM, 20mM and 30mM). Our preliminary data indicated that mitochondrial ROS generation constituted more than 70% of total ROS across HG concentrations compared to NG after 24hr exposure. ROS regulating genes did not show a linear trend in expression across different time points: Sod1 (p=0.0004) and Sod2 (p=0.0108) gene expression was found to be significantly increased after 24hr and 48hr exposure with HG (30mM)while Nox1 (p=0.0004) showed an opposite trend after 24hr exposure (30mM)). In addition, E13.5 embryos from chemically induced maternal type 1 model revealed a spectrum of CHD’s, including septal and outflow tract defects, and neural tube malformations with increased glucose levels. Further histologic and molecular characterization in vivo will provide a better understanding of the mechanism of how maternal hyperglycemic dose influences cardiac development.

Tissue specific requirements for WNT11 in developing outflow tract and dorsal mesenchymal protrusion

Correct cardiac development is essential for fetal and adult life. Disruptions in a variety of signaling pathways result in congenital heart defects, including outflow and inflow tract defects. We previously found that WNT11 regulates outflow tract development. However, tissue specific requirements for WNT11 in this process remain unknown and whether WNT11 is required for inflow tract development has not been addressed. Here we find that germline Wnt11 null mice also show hypoplasia of the dorsal mesenchymal protrusion (DMP), which is required for atrioventricular septation. Ablation of Wnt11 with myocardial cTnTCre recapitulated outflow tract defects observed in germline Wnt11 null mice, but DMP development was unaffected. In contrast, ablation of Wnt11 with Isl1Cre fully recapitulated both outflow tract and DMP defects of Wnt11 germline nulls. DMP hypoplasia in Wnt11 mutants was associated with reduced proliferation within the DMP, but no evident defects in myocardial differentiation of the DMP. Examination of Pitx2-, Axin2-, or Patched-lacZ reporter mice revealed no alterations in reporter expression, suggesting that WNT11 was required downstream of, or in parallel to, these signaling pathways to regulate DMP formation. These studies revealed a previously unappreciated role for WNT11 for DMP formation and distinct tissue-specific requirements for WNT11 in outflow tract and DMP development.

Loss of function in ROBO1 is associated with tetralogy of Fallot and septal defects

BackgroundCongenital heart disease (CHD) is a common birth defect affecting approximately 1% of newborns. Great progress has been made in elucidating the genetic aetiology of CHD with advances in genomic...

Neonatal outcomes in fetuses with cardiac anomalies and the impact of delivery route

Congenital fetal cardiac anomalies compromise the most common group of fetal structural anomalies. Several previous reports analyzed all types of fetal cardiac anomalies together without individualized neonatal morbidity outcomes based on cardiac defect. Mode of delivery in cases of fetal cardiac anomalies varies greatly as optimal mode of delivery in these complex cases is unknown. We sought to determine rates of neonatal outcomes for fetal cardiac anomalies and examine the role of attempted route of delivery on neonatal morbidity. Gravidas with fetal cardiac anomalies and delivery >34 weeks, excluding stillbirths and aneuploidies (n= 2166 neonates, n= 2701 cardiac anomalies), were analyzed from the Consortium on Safe Labor, a retrospective cohort study of electronic medical records. Cardiac anomalies were determined using International Classification of Diseases, Ninth Revision codes and organized based on morphology. Neonates were assigned to each cardiac anomaly classification based on the most severe cardiac defect present. Neonatal outcomes were determined for each fetal cardiac anomaly. Composite neonatal morbidity (serious respiratory morbidity, sepsis, birth trauma, hypoxic ischemic encephalopathy, and neonatal death) was compared between attempted vaginal delivery and planned cesarean delivery for prenatal and postnatal diagnosis. We used multivariate logistic regression to calculate adjusted odds ratio for composite neonatal morbidity controlling for race, parity, body mass index, insurance, gestational age,maternal disease, single or multiple anomalies, and maternal drug use. Most cardiac anomalies were diagnosed postnatally except hypoplastic left heart syndrome, which had a higher prenatal than postnatal detection rate. Neonatal death occurred in 8.4% of 107 neonates with conotruncal defects. Serious respiratory morbidity occurred in 54.2% of 83 neonates with left ventricular outflow tract defects. Overall, 76.3% of pregnancies with fetal cardiac anomalies underwent attempted vaginal delivery. Among patients who underwent attempted vaginal delivery, 66.1% had a successful vaginal delivery. Women with a fetal cardiac anomaly diagnosed prenatally were more likely to have a planned cesarean delivery than women with a postnatal diagnosis (31.7 vs 22.8%; P < .001). Planned cesarean delivery compared to attempted vaginal delivery was not associated with decreased composite neonatal morbidity for all prenatally diagnosed (adjusted odds ratio, 1.67; 95% confidence interval, 0.85-3.30) or postnatally diagnosed (adjusted odds ratio, 0.99; 95% confidence interval, 0.77-1.27) cardiac anomalies. Most fetal cardiac anomalies were diagnosed postnatally and associated with increased rates of neonatal morbidity. Planned cesarean delivery for prenatally diagnosed cardiac anomalies was not associated with less neonatal morbidity.

The CapZ interacting protein Rcsd1 is required for cardiogenesis downstream of Wnt11a in Xenopus laevis

Wnt proteins are critical for embryonic cardiogenesis and cardiomyogenesis by regulating different intracellular signalling pathways. Whereas canonical Wnt/β-catenin signalling is required for mesoderm induction and proliferation of cardiac progenitor cells, β-catenin independent, non-canonical Wnt signalling regulates cardiac specification and terminal differentiation. Although the diverse cardiac malformations associated with the loss of non-canonical Wnt11 in mice such as outflow tract (OFT) defects, reduced ventricular trabeculation, myofibrillar disorganization and reduced cardiac marker gene expression are well described, the underlying molecular mechanisms are still not completely understood. Here we aimed to further characterize Wnt11 mediated signal transduction during vertebrate cardiogenesis. Using Xenopus as a model system, we show by loss of function and corresponding rescue experiments that the non-canonical Wnt signalling mediator Rcsd1 is required downstream of Wnt11 for ventricular trabeculation, terminal differentiation of cardiomyocytes and cardiac morphogenesis. We here place Rcsd1 downstream of Wnt11 during cardiac development thereby providing a novel mechanism for how non-canonical Wnt signalling regulates vertebrate cardiogenesis.

Cyp26 Enzymes Facilitate Second Heart Field Progenitor Addition and Maintenance of Ventricular Integrity.

Although retinoic acid (RA) teratogenicity has been investigated for decades, the mechanisms underlying RA-induced outflow tract (OFT) malformations are not understood. Here, we show zebrafish embryos deficient for Cyp26a1 and Cyp26c1 enzymes, which promote RA degradation, have OFT defects resulting from two mechanisms: first, a failure of second heart field (SHF) progenitors to join the OFT, instead contributing to the pharyngeal arch arteries (PAAs), and second, a loss of first heart field (FHF) ventricular cardiomyocytes due to disrupted cell polarity and extrusion from the heart tube. Molecularly, excess RA signaling negatively regulates fibroblast growth factor 8a (fgf8a) expression and positively regulates matrix metalloproteinase 9 (mmp9) expression. Although restoring Fibroblast growth factor (FGF) signaling can partially rescue SHF addition in Cyp26 deficient embryos, attenuating matrix metalloproteinase (MMP) function can rescue both ventricular SHF addition and FHF integrity. These novel findings indicate a primary effect of RA-induced OFT defects is disruption of the extracellular environment, which compromises both SHF recruitment and FHF ventricular integrity.

Abstract 11921: Risk Stratification for Ventricular Arrhythmia in Patients With Repaired Tetralogy of Fallot (TOF) via Image-Based Computational Simulations: A Pilot Study

Introduction: Patients with repaired TOF have increased arrhythmia risk due to myocardial fibrosis, scar and right ventricular dilation. Current risk stratification lacks consistent predictive value and clinical practicality. We surmised that personalized models developed from cardiac late gadolinium enhancement MRI (LGE-MRI) could correctly assess risk of arrhythmia in patients with repaired TOF using our previously-validated Virtual Arrhythmia Risk Prediction (VARP) protocol. Methods: As a proof-of-concept retrospective study, we developed personalized 3D computational models of TOF hearts from LGE-MRI scans. Three patients with clinically disparate arrhythmia status were included: patient 1 (P1) had clinical episodes of sustained ventricular tachycardia (VT) and inducibility in electrophysiologic study (EPS); patient 2 (P2) had negative EPS despite documented runs of non-sustained VT; patient 3 (P3) had no clinical evidence of VT. Then, for each heart, we applied our VARP protocol to generate a computational model, including personalized representations of scar and fibrosis. We then assessed the inducibility of reentrant arrhythmia from 26 pacing sites. Results: For all three cases, VARP outcomes correlated with clinical observations. In the P1 model sustained VT was induced with rapid ventricular pacing, while only non-sustained VT was induced in the P2 model (Fig 1). No arrhythmia was induced in the P3 model. Reentrant wavefronts in P1 and P2 occurred near the right ventricular outflow tract (RVOT) or ventricular septal defect patch, common sites of reentry observed in EPS in patients with TOF (Fig 1). Conclusion: Application of the VARP approach in repaired TOF patients is feasible and has the potential to correctly identify patients with a high risk of developing arrhythmia. With additional validation this non-invasive technique could serve as an effective tool for longitudinal risk assessment and help guide clinical decision making.

0535: A novel approach to exploration of complex congenital heart diseases through 3D CT scans

One of the key success factors of the surgical treatment of complex congenital heart defects is a detailed preoperative exploration. The main issue is the lack of detailed visibility of the intracardiac anatomical structures. 2D echocardiography provides a partial view and doesn’t provide fine anatomical details. Novel imagery technology with 3D cardiac tomography (CT) may represent a valuable tool to explore complex congenital heart defects. To determine the quality and limitations of this technique to study the intracardiac anatomy in complex outflow tract defects. The secondary objective is to generate the CT scan views in preoperative assessment fitting precisely to the surgical views. We performed CT scans with high resolution in 15 heart specimens with double outlet right ventricle. All 3D images were produced with a 3D reconstruction platform from Paris Descartes University. The 3D view from the right ventricle showed the anatomic details for all hearts. We described the VSD, its localization, borders and surface. We described also the relationship with the aorta and the pulmonary trunk and the conal septum (length and orientation). The 3D view from the right atrium was more difficult then expected and needs to be improved. This study summarizes the role of 3D scan reconstruction in congenital heart diseases as an imaging modality to increase the precision of preoperative assessment.

Abstract 17616: Recurrent Copy Number Variants are Enriched in Bicuspid Aortic Valve and Affect Cardiac Developmental Genes

Introduction: Bicuspid Aortic Valve (BAV) is the most common adult congenital heart defect. The spectrum of BAV ranges from chromosomal disorders such as Turner syndrome to sporadic isolated disease of unknown cause. We hypothesized that genomic copy number variants (CNVs) contribute causally to BAV. Methods: We conducted genome-wide SNP array analyses of BAV cases in six European American cohorts (3533 total subjects, 31% BAV, 45% female, ages 1-94 years) with sporadic or familial thoracic aortic aneurysms (37%), congenital left ventricular outflow tract obstructive defects (47%) or Turner syndrome (10%). Genotyping was performed on Illumina Omni microarrays, using PennCNV, Nexus and CNVPartition for CNV detection. Thresholds were set at a minimum of six consecutive SNPs for CNVs that were identified by at least two algorithms and intersect with known genes. A total of 1380 BAV cases were compared to 1065 cases with tricuspid aortic valves and 7013 dbGAP controls without a history of cardiovascular disease using association tests for rare CNVs as implemented in PLINK. Pathway and functional analyses of rare CNVs were performed using the ToppGene analysis suite. Genes within rare CNVs were prioritized by their connectivity to genes in the Notch pathway or established roles in human or animal cardiac phenotypes. Results: We identified 12 rare CNV regions that were recurrent in at least three different BAV groups and were absent or extremely rare (frequency &lt;0.1%) in controls. Four of these regions involved genes that cause aortic valve or outflow tract defects when mutated. The largest and most prevalent of the recurrent CNVs were at Xq28 (four duplications and two deletions), at 22q11.21, telomeric to the DiGeorge syndrome critical region (four deletions, three duplications), and at 7p15.2 (four deletions). The percentage of individuals harboring rare CNVs was significantly greater in BAV cases than in tricuspid or dbGAP controls. Conclusions: We identified multiple loci affected by rare CNVs in BAV subjects, demonstrating the genetic heterogeneity of BAV and implicating alterations of candidate genes at these loci in the pathogenesis of BAV.

Heparan Sulfate Biosynthesis Enzyme, Ext1, Contributes to Outflow Tract Development of Mouse Heart via Modulation of FGF Signaling.

Glycosaminoglycans are important regulators of multiple signaling pathways. As a major constituent of the heart extracellular matrix, glycosaminoglycans are implicated in cardiac morphogenesis through interactions with different signaling morphogens. Ext1 is a glycosyltransferase responsible for heparan sulfate synthesis. Here, we evaluate the function of Ext1 in heart development by analyzing Ext1 hypomorphic mutant and conditional knockout mice. Outflow tract alignment is sensitive to the dosage of Ext1. Deletion of Ext1 in the mesoderm induces a cardiac phenotype similar to that of a mutant with conditional deletion of UDP-glucose dehydrogenase, a key enzyme responsible for synthesis of all glycosaminoglycans. The outflow tract defect in conditional Ext1 knockout(Ext1 f/f:Mesp1Cre) mice is attributable to the reduced contribution of second heart field and neural crest cells. Ext1 deletion leads to downregulation of FGF signaling in the pharyngeal mesoderm. Exogenous FGF8 ameliorates the defects in the outflow tract and pharyngeal explants. In addition, Ext1 expression in second heart field and neural crest cells is required for outflow tract remodeling. Our results collectively indicate that Ext1 is crucial for outflow tract formation in distinct progenitor cells, and heparan sulfate modulates FGF signaling during early heart development.

Hoxb1 regulates proliferation and differentiation of second heart field progenitors in pharyngeal mesoderm and genetically interacts with Hoxa1 during cardiac outflow tract development

Outflow tract (OFT) anomalies are among the most common congenital heart defects found at birth. The embryonic OFT grows by the progressive addition of cardiac progenitors, termed the second heart field (SHF), which originate from splanchnic pharyngeal mesoderm. Development of the SHF is controlled by multiple intercellular signals and transcription factors; however the relationship between different SHF regulators remains unclear. We have recently shown that Hoxa1 and Hoxb1 are expressed in a sub-population of the SHF contributing to the OFT. Here, we report that Hoxb1 deficiency results in a shorter OFT and ventricular septal defects (VSD). Mechanistically, we show that both FGF/ERK and BMP/SMAD signaling, which regulate proliferation and differentiation of cardiac progenitor cells and OFT morphogenesis, are enhanced in the pharyngeal region in Hoxb1 mutants. Absence of Hoxb1 also perturbed SHF development through premature myocardial differentiation. Hence, the positioning and remodeling of the mutant OFT is disrupted. Hoxa1−/− embryos, in contrast, have low percentage of VSD and normal SHF development. However, compound Hoxa1−/−; Hoxb1+/− embryos display OFT defects associated with premature SHF differentiation, demonstrating redundant roles of these factors during OFT development. Our findings provide new insights into the gene regulatory network controlling SHF and OFT formation.

Selective serotonin reuptake inhibitors and venlafaxine in early pregnancy and risk of birth defects: population based cohort study and sibling design

Objective To assess whether use of specific selective serotonin reuptake inhibitors (SSRIs) or venlafaxine in early pregnancy is associated with an increased risk of birth defects, with emphasis on cardiovascular...

MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus.

Cardiac left ventricular outflow tract (LVOT) defects represent a common but heterogeneous subset of congenital heart disease for which gene identification has been difficult. We describe a 46,XY,t(1;5)(p36.11;q31.2)dn translocation carrier with pervasive developmental delay who also exhibited LVOT defects, including bicuspid aortic valve (BAV), coarctation of the aorta (CoA) and patent ductus arteriosus (PDA). The 1p breakpoint disrupts the 5′ UTR of AHDC1, which encodes AT-hook DNA-binding motif containing-1 protein, and AHDC1-truncating mutations have recently been described in a syndrome that includes developmental delay, but not congenital heart disease [Xia, F., Bainbridge, M.N., Tan, T.Y., Wangler, M.F., Scheuerle, A.E., Zackai, E.H., Harr, M.H., Sutton, V.R., Nalam, R.L., Zhu, W. et al. (2014) De Novo truncating mutations in AHDC1 in individuals with syndromic expressive language delay, hypotonia, and sleep apnea. Am. J. Hum. Genet., 94, 784–789]. On the other hand, the 5q translocation breakpoint disrupts the 3′ UTR of MATR3, which encodes the nuclear matrix protein Matrin 3, and mouse Matr3 is strongly expressed in neural crest, developing heart and great vessels, whereas Ahdc1 is not. To further establish MATR3 3′ UTR disruption as the cause of the proband's LVOT defects, we prepared a mouse Matr3Gt-ex13 gene trap allele that disrupted the 3′ portion of the gene. Matr3Gt-ex13 homozygotes are early embryo lethal, but Matr3Gt-ex13 heterozygotes exhibit incompletely penetrant BAV, CoA and PDA phenotypes similar to those in the human proband, as well as ventricular septal defect (VSD) and double-outlet right ventricle (DORV). Both the human MATR3 translocation breakpoint and the mouse Matr3Gt-ex13 gene trap insertion disturb the polyadenylation of MATR3 transcripts and alter Matrin 3 protein expression, quantitatively or qualitatively. Thus, subtle perturbations in Matrin 3 expression appear to cause similar LVOT defects in human and mouse.

Vangl2-Regulated Polarisation of Second Heart Field-Derived Cells Is Required for Outflow Tract Lengthening during Cardiac Development

Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.

Anatomy of the ventricular septal defect in outflow tract defects: Similarities and differences

ObjectiveThe study objective was to analyze the anatomy of the ventricular septal defect found in various phenotypes of outflow tract defects. MethodsWe reviewed 277 heart specimens with isolated outlet ventricular septal defect without subpulmonary stenosis (isolated outlet ventricular septal defect, 19); tetralogy of Fallot (71); tetralogy of Fallot with pulmonary atresia (51); common arterial trunk (54); double outlet right ventricle (65) with subaortic, doubly committed, or subpulmonary ventricular septal defect; and interrupted aortic arch type B (17). Special attention was paid to the rims of the ventricular septal defect viewed from the right ventricular side and the relationships between the tricuspid and aortic valves. ResultsThe ventricular septal defect was always located in the outlet of the right ventricle, between the 2 limbs of the septal band. There was a fibrous continuity between the tricuspid and aortic valves in 74% of specimens with isolated outlet ventricular septal defect, 66% of specimens with tetralogy of Fallot, 39% of specimens with tetralogy of Fallot with pulmonary atresia, 4.6% of specimens with double outlet right ventricle, 1.8% of specimens with common arterial trunk, and zero of specimens with interrupted aortic arch type B (P < .005). When present, this continuity always involved the anterior tricuspid leaflet. ConclusionsThe ventricular septal defect in outflow tract defects is always an outlet ventricular septal defect, cradled between the 2 limbs of the septal band. However, there are some differences regarding the posteroinferior and superior rims of the ventricular septal defect. These differences suggest an anatomic continuum from the isolated outlet ventricular septal defect to the interrupted aortic arch type B rather than distinct physiologic phenotypes, related to various degrees of abnormal rotation of the outflow tract during heart development: minimal in isolated outlet ventricular septal defect; incomplete in tetralogy of Fallot, tetralogy of Fallot with pulmonary atresia, and double outlet right ventricle; absent in common arterial trunk; and excessive in interrupted aortic arch type B.

Abstract 19285: Notch1 Haploinsufficiency Increases Risk of Congenital Heart Defects in the Setting of Maternal Diabetes by an Epigenetic Mechanism

Congenital heart disease (CHD) is the most common type of birth defect. Epidemiologic studies have shown the importance of genetic and environmental factors in the multifactorial etiology of CHD. Maternal diabetes mellitus (DM) is one of the non-genetic risk factors that predisposes to CHD predominantly cardiac septation and cardiac outflow tract defects. DM is known to be associated with endothelial cell dysfunction and we recently demonstrated a genetic interaction between endothelial nitric oxide synthase and Notch1, which encodes a receptor that functions in an important cardiac developmental signaling pathway. We hypothesized that maternal DM in the setting of Notch1 heterozygosity of the developing embryo will predispose to CHD. Notch1 +/- embryos (E13.5) exposed to maternal DM demonstrated an increased incidence (86%) of ventricular septal defects compared to wildtype littermates (22%) (Table). Gene expression studies in non-diabetic wildtype, diabetic wildtype and Notch1 +/- embryos showed DM was associated with decreased Notch1 mRNA levels and upregulation in Jarid2, a histone H3K9 demethylase known to regulate Notch1. In H9C2 and endocardial-derived cells and chick embryos, we showed that hyperglycemia led to decreased expression of Notch1 and its downstream targets in a dose-dependent manner. Similarly, Jarid2 mRNA levels increased with high glucose. Furthermore, we found reduced luciferase reporter activity in cells transfected with a constitutively active Notch1 intracellular domain with hyperglycemia. Studies demonstrating the relative enrichment of Jarid2 on Notch1 locus with hyperglycemia by ChIP-qPCR will also be presented. Our findings reveal that maternal DM disrupts cardiac development by deregulating the Notch1 signaling pathway and suggest that this gene-environment interaction is mediated by an epigenetic mechanism involving Jarid2 providing the first mechanistic insights for this association.