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Increased regurgitant blood flow has been linked to endocardial cushion defects and resultant congenital heart diseases (CHDs). Prenatal alcohol exposure (PAE) has been shown to alter early blood flow resulting in abnormal endocardial cushions and CHDs. Compounds, including folic acid (FA), mitigate PAE effects and prevent CHDs, but few studies have assessed their effects on blood flow. We modeled binge drinking in quail embryos at gastrulation. Embryos were exposed to ethanol alone, FA (3.2 μg/egg) alone, and the two simultaneously. We quantified in cardiac looping stages (equivalent to 4 wk of human gestation), the regurgitant blood flow with Doppler optical coherence tomography (OCT) and endocardial cushion volumes using OCT imaging. Incidences of abnormal body curvature and heart rates were also measured. Embryos exposed to ethanol showed significantly increased regurgitant blood flow compared with controls, whereas embryos given FA with ethanol had significantly reduced regurgitant blood flow but without returning to control levels. Ethanol exposure led to significantly smaller, abnormal endocardial cushions, and the addition of FA improved their size, but they remained smaller than controls. Abnormal body curvatures after PAE were reduced in incidence but not fully prevented by FA. FA supplementation partially alleviated PAE-induced abnormal cardiovascular function and morphology. Normal blood flow and endocardial cushions are both required to produce a healthy four-chambered heart. These findings support that FA supplementation should begin early in pregnancy to prevent heart as well as neural tube defects. Investigations into the efficacy of combinations of compounds to prevent PAE-induced defects are warranted. NEW & NOTEWORTHY State-of-the-art biophotonic tools captured blood flow and endocardial cushion volumes in tiny beating quail embryo hearts, an accessible model for studying four-chambered heart development. Both hemodynamic flow and endocardial cushion volumes were altered with ethanol exposure but normalized when folic acid was introduced with ethanol. Folic acid supplementation preserved hemodynamic function that is intimately involved in sculpting the heart from the earliest stages of heart development.

BACKGROUND Fetal alcohol spectrum disorder (FASD) is caused by prenatal alcohol exposure (PAE), the intake of ethanol (C2 H5 OH) during pregnancy. Features of FASD cover a range of structural and functional defects including congenital heart defects (CHDs). Folic acid and choline, contributors of methyl groups to one-carbon metabolism (OCM), prevent CHDs in humans. Using our avian model of FASD, we have previously reported that betaine, another methyl donor downstream of choline, prevents CHDs. The CHD preventions are substantial but incomplete. Ethanol causes oxidative stress as well as depleting methyl groups for OCM to support DNA methylation and other epigenetic alterations. To identify more compounds that can safely and effectively prevent CHDs and other effects of PAE, we tested glutathione (GSH), a compound that regulates OCM and is known as a "master antioxidant." METHODS/RESULTS Quail embryos injected with a single dose of ethanol at gastrulation exhibited congenital defects including CHDs similar to those identified in FASD individuals. GSH injected simultaneously with ethanol not only prevented CHDs, but also improved survival and prevented other PAE-induced defects. Assays of hearts at 8 days (HH stage 34) of quail development, when the heart normally has developed 4-chambers, showed that this single dose of PAE reduced global DNA methylation. GSH supplementation concurrent with PAE normalized global DNA methylation levels. The same assays performed on quail hearts at 3 days (HH stage 19-20) of development, showed no difference in global DNA methylation between controls, ethanol-treated, GSH alone, and GSH plus ethanol-treated cohorts. CONCLUSIONS GSH supplementation shows promise to inhibit effects of PAE by improving survival, reducing the incidence of morphological defects including CHDs, and preventing global hypomethylation of DNA in heart tissues.

Background Despite decades of public education about dire consequences of prenatal alcohol exposure, drinking alcohol during pregnancy remains prevalent. As high as 40% of live-born infants exposed to alcohol during gestation and diagnosed with Fetal Alcohol Syndrome have congenital heart defects that can be life-threatening. In animal models, the methyl donor betaine, found in foods such as wheat bran, quinoa, beets and spinach, ameliorated neurobehavioral deficits associated with prenatal alcohol exposure (PAE) but effects on heart development are unknown. Methods Previously we modeled a binge drinking episode during the first trimester in avian embryos. Here we investigated whether betaine could prevent adverse effects of alcohol on heart development. Embryos exposed to ethanol with and without an optimal dose of betaine (5 μM) were analyzed at late developmental stages. Cardiac morphology parameters were rapidly analyzed and quantified using optical coherence tomography. DNA methylation at early stages was detected by immunofluorescent staining for 5-methylcytosine in sections of embryos treated with ethanol or co-treated with betaine. Results Compared to ethanol-exposed embryos, betaine-supplemented embryos had higher late-stage survival rates and fewer gross head and body defects than seen after alcohol exposure alone. Betaine also reduced the incidence of late-stage cardiac defects such as absent vessels, abnormal atrio-ventricular (AV) valves, and hypertrophic ventricles. Furthermore, betaine co-treatment brought measurements of great vessel diameters, interventricular septum (IVS) thickness, and AV leaflet volumes in betaine-supplemented embryos close to control values. Early-stage 5-methycytosine staining revealed that DNA methylation levels were reduced by ethanol exposure and normalized by co-administration with betaine. Conclusions This is the first study demonstrating efficacy of the methyl donor betaine in alleviating cardiac defects associated with PAE. These findings highlight the therapeutic potential of low-concentration betaine doses in mitigating PAE induced birth defects and has implications for prenatal nutrition policies, especially for women who may not be responsive to folate supplementation. This article is protected by copyright. All rights reserved.

Background The most commonly used method to analyze congenital heart defects involves serial sectioning and histology. However, this is often a time-consuming process where the quantification of cardiac defects can be difficult due to problems with accurate section registration. Here we demonstrate the advantages of using optical coherence tomography, a comparatively new and rising technology, to phenotype avian embryo hearts in a model of Fetal Alcohol Syndrome where a binge-like quantity of alcohol/ethanol was introduced at gastrulation.

Over 500,000 women per year in the United States drink during pregnancy, and 1 in 5 of this population also binge drink. Up to 40% of live-born children with prenatal alcohol exposure (PAE) present with congenital heart defects (CHDs) including life-threatening outflow and valvuloseptal anomalies. Previously we established a PAE model in the avian embryo and used optical coherence tomography (OCT) imaging to assay looping-stage (early) cardiac function/structure and septation-stage (late) cardiac defects. Early-stage ethanol-exposed embryos had smaller cardiac cushions (valve precursors) and increased retrograde flow, while late-stage embryos presented with gross head/body defects, and exhibited smaller atrio-ventricular (AV) valves, interventricular septae, and aortic vessels. However, supplementation with the methyl donor betaine reduced gross defects, prevented cardiac defects such as ventricular septal defects and abnormal AV valves, and normalized cardiac parameters. Immunofluorescent staining for 5-methylcytosine in transverse embryo sections also revealed that DNA methylation levels were reduced by ethanol but normalized by co-administration of betaine. Furthermore, supplementation with folate, another methyl donor, in the PAE model appeared to normalize retrograde flow levels which are typically elevated by ethanol exposure. Studies are underway to correlate retrograde flow numbers for folate with associated cushion volumes. Finally, preliminary findings have revealed that glutathione, a key endogenous antioxidant which also regulates methyl group donation, is particularly effective in improving alcohol-impacted survival and gross defect rates. Current investigations will determine whether glutathione has any positive effect on PAE-related CHDs. Our studies could have significant implications for public health, especially related to prenatal nutrition recommendations.

Alcohol-induced congenital heart defects are frequently among the most life threatening and require surgical correction in newborns. The etiology of these defects, collectively known as fetal alcohol syndrome, has been the focus of much study, particularly involving cellular and molecular mechanisms. Few studies have addressed the influential role of altered cardiac function in early embryogenesis because of a lack of tools with the capability to assay tiny beating hearts. To overcome this gap in our understanding, we used optical coherence tomography (OCT), a nondestructive imaging modality capable of micrometer-scale resolution imaging, to rapidly and accurately map cardiovascular structure and hemodynamics in real time under physiological conditions. In this study, we exposed avian embryos to a single dose of alcohol/ethanol at gastrulation when the embryo is sensitive to the induction of birth defects. Late-stage hearts were analyzed using standard histological analysis with a focus on the atrio-ventricular valves. Early cardiac function was assayed using Doppler OCT, and structural analysis of the cardiac cushions was performed using OCT imaging. Our results indicated that ethanol-exposed embryos developed late-stage valvuloseptal defects. At early stages, they exhibited increased regurgitant flow and developed smaller atrio-ventricular cardiac cushions, compared with controls (uninjected and saline-injected embryos). The embryos also exhibited abnormal flexion/torsion of the body. Our evidence suggests that ethanol-induced alterations in early cardiac function have the potential to contribute to late-stage valve and septal defects, thus demonstrating that functional parameters may serve as early and sensitive gauges of cardiac normalcy and abnormalities.

Cardiac neural crest cell (CNCC) ablation creates congenital heart defects (CHDs) that resemble those observed in many syndromes with craniofacial and cardiac consequences. The loss of CNCCs causes a variety of great vessel defects, including persistent truncus arteriosus and double-outlet right ventricle. However, because of the lack of quantitative volumetric measurements, less severe defects, such as great vessel size changes and valve defects, have not been assessed. Also poorly understood is the role of abnormal cardiac function in the progression of CNCC-related CHDs. CNCC ablation was previously reported to cause abnormal cardiac function in early cardiogenesis, before the CNCCs arrive in the outflow region of the heart. However, the affected functional parameters and how they correlate with the structural abnormalities were not fully characterized. In this study, using a CNCC-ablated quail model, we contribute quantitative phenotyping of CNCC ablation-related CHDs and investigate abnormal early cardiac function, which potentially contributes to late-stage CHDs. Optical coherence tomography was used to assay early- and late-stage embryos and hearts. In CNCC-ablated embryos at four-chambered heart stages, great vessel diameter and left atrioventricular valve leaflet volumes are reduced. Earlier, at cardiac looping stages, CNCC-ablated embryos exhibit abnormally twisted bodies, abnormal blood flow waveforms, increased retrograde flow percentage, and abnormal cardiac cushions. The phenotypes observed in this CNCC-ablation model were also strikingly similar to those found in an established avian fetal alcohol syndrome model, supporting the contribution of CNCC dysfunction to the development of alcohol-induced CHDs.

Over 500,000 women per year in the United States drink during pregnancy, and 1 in 5 of this population also binge drink. As high as 20-50% of live-born children with prenatal alcohol exposure (PAE) present with congenital heart defects including outflow and valvuloseptal anomalies that can be life-threatening. Previously we established a model of PAE (modeling a single binge drinking episode) in the avian embryo and used optical coherence tomography (OCT) imaging to assay early-stage cardiac function/structure and late-stage cardiac defects. At early stages, alcohol/ethanol-exposed embryos had smaller cardiac cushions and increased retrograde flow. At late stages, they presented with gross morphological defects in the head and chest wall, and also exhibited smaller or abnormal atrio-ventricular (AV) valves, thinner interventricular septae (IVS), and smaller vessel diameters for the aortic trunk branches. In other animal models, the methyl donor betaine (found naturally in many foods such as wheat bran, quinoa, beets and spinach) ameliorates neurobehavioral deficits associated with PAE but the effects on heart structure are unknown. In our model of PAE, betaine supplementation led to a reduction in gross structural defects and appeared to protect against certain types of cardiac defects such as ventricular septal defects and abnormal AV valvular morphology. Furthermore, vessel diameters, IVS thicknesses and mural AV leaflet volumes were normalized while the septal AV leaflet volume was increased. These findings highlight the importance of betaine and potentially methylation levels in the prevention of PAE-related birth defects which could have significant implications for public health.

Fetal alcohol syndrome commonly results in neurological and craniofacial defects, additionally, as high as 54% of live-born children with this syndrome also possess cardiac abnormalities. We have previously shown that CNCC-ablated embryos exhibit similar structural and functional phenotypes as ethanol-exposed embryos. Here, we present progress on two fronts toward understanding the association between CNCC dysfunction and FAS-related CHDs. We have developed a technique for measuring the thickness of the cardiac cushions throughout the heart. These values were then mapped onto a surface mesh of the myocardial wall for 3-D visualization. The cushions were observed to be significantly reduced in the outflow tract of CNCC-ablated embryos. We also observed a correlation between abnormal pulsed Doppler waveforms and increased separation of the atrioventricular inferior and superior cushions. This correlation between function and structure will enable rapid phenotyping of perturbed embryos. Finally, we present our preliminary results using methyl donors to rescue ethanol-exposed embryonic CHDs. Betaine was administered along with the ethanol injection to embryos at 21 hours of development. The embryos were then analyzed at day 8 for survival and heart morphology. The administration of betaine resulted in a significant increase in survival and normalization of atrioventricular valve leaflet volume and interventricular septum thickness.

The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just prior to septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear-localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox-1-positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not become Prox-1-positive even in the adult heart.

The great arteries develop from symmetrical aortic arch arteries which are extensively remodeled. These events are vulnerable to perturbations. Hemodynamic forces have a significant role in this remodeling. In this study, optical coherence tomography (OCT) visualized live avian embryos for staging and measuring pharyngeal arch morphology. Measurements acquired with our orientation-independent, dual-angle Doppler OCT technique revealed that ethanol exposure leads to higher absolute blood flow, shear stress, and retrograde flow. Ethanol-exposed embryos had smaller cardiac neural crest (CNC) derived pharyngeal arch mesenchyme and fewer migrating CNC-derived cells. These differences in forces and CNC cell numbers could explain the abnormal aortic arch remodeling.

The most commonly used method to analyze congenital heart defects involves serial sectioning and histology. However, this is often a time-consuming process where the quantification of cardiac defects can be difficult due to problems with accurate section registration. Here we demonstrate the advantages of using optical coherence tomography, a comparatively new and rising technology, to phenotype avian embryo hearts in a model of fetal alcohol syndrome where a binge-like quantity of alcohol/ethanol was introduced at gastrulation.The rapid, consistent imaging protocols allowed for the immediate identification of cardiac anomalies, including ventricular septal defects and misaligned/missing vessels. Interventricular septum thicknesses and vessel diameters for three of the five outflow arteries were also significantly reduced. Outflow and atrioventricular valves were segmented using image processing software and had significantly reduced volumes compared to controls. This is the first study to our knowledge that has 3D reconstructed the late-stage cardiac valves in precise detail to examine their morphology and dimensions.We believe, therefore, that optical coherence tomography, with its ability to rapidly image and quantify tiny embryonic structures in high resolution, will serve as an excellent and cost-effective preliminary screening tool for developmental biologists working with a variety of experimental/disease models.

Differential tissue hypoxia drives normal cardiogenic events including coronary vessel development. This requirement renders cardiogenic processes potentially susceptible to teratogens that activate a transcriptional pathway that intersects with the hypoxia-inducible factor (HIF-1) pathway. The potent toxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause cardiovascular defects by way of reduced myocardial hypoxia, inhibition of angiogenic stimuli, and alterations in responsiveness of endothelial cells to those stimuli. Our working hypothesis is that HIF-1 levels and thus HIF-1 signaling in the developing myocardium will be reduced by TCDD treatment in vivo during a critical stage and in particularly sensitive sites during heart morphogenesis. This inadequate HIF-1 signaling will subsequently result in outflow tract (OFT) and coronary vasculature defects. Our current data using the chicken embryo model showed a marked decrease in the intensity of immunostaining for HIF-1α nuclear expression in the OFT myocardium of TCDD-treated embryos. This area at the base of the OFT is particularly hypoxic during normal development; where endothelial cells initially form a concentrated anastomosing network known as the peritruncal ring; and where the left and right coronary arteries eventually connect to the aortic lumen. Consistent with this finding, anomalies of the proximal coronaries were detected after TCDD treatment and HIF-1α protein levels decreased in a TCDD dose-dependent manner.

Coronary vessels are of particular clinical interest to the general public and adult cardiologists because of their propensity in populations of developed countries to get clogged leading to chest pain (angina) and heart attacks (myocardial infarctions; MIs). Coronary artery disease (CAD) is the leading type of heart disease and the leading cause of deaths in the USA in both men and women (representing 51% of all cardiovascular diseases in 2006; American Heart Association Heart Disease and Stroke Statistics 2010 Update-at-a-glance; http://www.americanheart.org/downloadable/heart/1265665152970DS3241%20HeartStrokeUpdate_2010.pdf; http://www.nlm.nih.gov/medlineplus/coronaryartery disease.html). While the developmental biology of cardiac vessels seems removed from this concern for the adult population, findings from this field may be beneficial and relevant for all age groups. Interest in the development of coronary vessels has increased due to findings that suggest the embryonic epicardium provides components of the coronary vessels and factors that positively influence the myocardium. The possibility has been raised that the epicardium might be sufficiently activated to repair cardiac tissue in the adult in part by promoting coronary vascularization. The aim of this Chapter is to call attention to questions regarding coronary vessels that still require answers and to introduce hypotheses that may lead to answers and strategies for cardiotherapy.

The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5-positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1alpha), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. This led to our hypothesis that there is a "template" of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study, we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF-1alpha protein expression, and its downstream target genes without high mortality. We also altered HIF-1alpha gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF-1alpha (AdCA5). We assayed for coronary anomalies using anti-alpha-smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression.

Quantification of microvascular remodeling as a meaningful discovery tool requires mapping and measurement of site-specific changes within vascular trees and networks. Vessel density and other critical vascular parameters are often modulated by molecular regulators as determined by local vascular architecture. For example, enlargement of vessel diameter by vascular endothelial growth factor (VEGF) is restricted to specific generations of vessel branching (Parsons-Wingerter et al., Microvascular Research72: 91, 2006). The averaging of vessel diameter over many successively smaller generations is therefore not particularly useful. The newly automated, user-interactive software VESsel GENeration Analysis (VESGEN) quantifies major vessel parameters within two-dimensional (2D) vascular trees, networks, and tree-network composites. This report reviews application of VESGEN 2D to angiogenic and lymphangiogenic tissues that includes the human and murine retina, embryonic coronary vessels, and avian chorioallantoic membrane. Software output includes colorized image maps with quantification of local vessel diameter, fractal dimension, tortuosity, and avascular spacing. The density of parameters such as vessel area, length, number, and branch point are quantified according to site-specific generational branching within vascular trees. The sole user input requirement is a binary (black/white) vascular image. Future applications of VESGEN will include analysis of 3D vascular architecture and bioinformatic dimensions such as blood flow and receptor localization. Branching analysis by VESGEN has demonstrated that numerous regulators including VEGF(165), basic fibroblast growth factor, transforming growth factor beta-1, angiostatin and the clinical steroid triamcinolone acetonide induce 'fingerprint' or 'signature' changes in vascular patterning that provide unique readouts of dominant molecular signaling.

Notch1 is an important regulator of intercellular interactions in cardiovascular development. We show that the nuclear-localized, cleaved and active form of Notch1, the Notch1 intracellular domain (N1ICD), appeared in mesothelial cells of the pro-epicardium during epicardial formation at looped heart stages. N1ICD was also present in mesothelial cells and mesenchymal cells specifically within the epicardium at sulcus regions. N1ICD-positive endothelial cells were detected within the nascent vessel plexus at the atrioventricular junction and within the compact myocardium (Hamburger and Hamilton stage [HH] 25-HH30). The endothelial cells expressing N1ICD were surrounded by N1ICD-positive smooth muscle cells after coronary orifice formation (HH32-HH35), while N1ICD expression was absent in the mesenchymal and mesothelial cells surrounding mature coronary vessels. We propose that differential activation of the hypoxia/HIF1-VEGF-Notch pathway may play a role in epicardial cell interactions that promote epicardial epithelial/mesenchymal transition and coronary progenitor cell differentiation during epicardial development and coronary vasculogenesis in particularly hypoxic sulcus regions.

Apoptosis occurs at high frequency in the myocardium of the developing avian cardiac outflow tract (OFT). Up- or down-regulating apoptosis results in defects resembling human conotruncal heart anomalies. This finding suggested that regulated levels of apoptosis are critical for normal morphogenesis of the four-chambered heart. Recent evidence supports an important role for hypoxia of the OFT myocardium in regulating cell death and vasculogenesis. The purpose of this study was to determine whether apoptosis in the outflow tract myocardium occurs in the mouse heart during developmental stages comparable to the avian heart and to determine whether differential hypoxia is also present at this site in the murine heart. Apoptosis was detected using a fluorescent vital dye, Lysotracker Red (LTR), in the OFT myocardium of the mouse starting at embryonic day (E) 12.5, peaking at E13.5-14.5, and declining thereafter to low or background levels by E18.5. In addition, high levels of apoptosis were detected in other cardiac regions, including the apices of the ventricles and along the interventricular sulcus. Apoptosis in the myocardium was detected by double-labeling with LTR and cardiomyocyte markers. Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) and immunostaining for cleaved Caspase-3 were used to confirm the LTR results. At the peak of OFT apoptosis in the mouse, the OFT myocardium was relatively hypoxic, as indicated by specific and intense EF5 staining and HIF1alpha nuclear localization, and was surrounded by the developing vasculature as in the chicken embryo. These findings suggest that cardiomyocyte apoptosis is an evolutionarily conserved mechanism for normal morphogenesis of the outflow tract myocardium in avian and mammalian species.