ERp44 is Required for Endocardial Cushion Development by Regulating VEGFA Secretion in Myocardium

Rationale: Endocardial cushions are precursors of the valvoseptal complex that separates the four heart chambers and control blood flow through the heart. Abnormalities in endocardial cushion development lead to atrioventricular septal defects (AVSDs), which affect 1 in 2,100 live births. Several genes have been implicated in the development of endocardial cushions. Specifically, endoplasmic reticulum-resident protein 44 (ERp44) has been found to play a role in the early secretory pathway, but its function in heart development has not been well studied. Objective: The goal of this study was to investigate the role of ERp44 in heart development in mice. Approach and Results: Using conventional and tissue-specific knockout mouse models, we demonstrated that ERp44 plays a specific role in heart development. ERp44 knockout (KO) mice were smaller in size, and most mice died during early postnatal life. KO hearts exhibited the typical phenotypes of congenital heart diseases, such as abnormal heart shapes as well as severe septal and valvular defects. Similar phenotypes were found in cTnt-cre+/-; Erp44fl/fl mice, which indicated that myocardial ERp44 principally controls endocardial cushion formation. Further studies demonstrated that the deletion of ERp44 significantly decreased the proliferation of cushion cells and impaired the endocardial-mesenchymal transition (EndMT), which was followed by endocardial cushion dysplasia. Finally, we found that ERp44 directly bound to VEGFA and controlled its release. Conclusions: ERp44 contributes to the development of the endocardial cushion by affecting the EndMT of cushion cells by regulating VEGFA release in myocardial cells.

Download Full-text

From cushions to leaflets: morphogenesis of cardiac atrioventricular valves

10.1093/med/9780198757269.003.0017 ◽

2018 ◽

Author(s):

Donal MacGrogan ◽

José Maria Pérez-Pomares ◽

Bill Chaudhry ◽

José Luis de la Pompa ◽

Deborah J. Henderson

Keyword(s):

Heart Development ◽

Cellular Proliferation ◽

Valve Disease ◽

Signalling Pathways ◽

Valve Leaflet ◽

Endocardial Cushions ◽

Developmental Defects ◽

Mesenchymal Transition ◽

Valve Development ◽

Developmental Signalling

At the looping stage of heart development, tissue patterning of myocardium and endocardium at the atrioventricular (AV) junction defines a morphogenic field competent to form valves that initially appear as protrusions of proteoglycan-rich extracellular matrix (ECM) called endocardial cushions (ECs) which are cellularized by an endocardial-mesenchymal transition (EMT). Cellular proliferation results in fusion of the major AV mesenchymal cushions and AV septation, whereas smaller cushions receive a supply from epicardially derived cells. These various sources of mesenchyme precursors give rise to most of the valve structures, leaflets, annuli, and supporting tension apparatus. During valve leaflet maturation, the ECM matrix accumulates collagen and elastin and assembles into a thin flexible fibrous structure, which is remarkably tough. Valve development is regulated by the cross-talk between developmental signalling pathways. Pathogenic mutations in a subset of developmentally important genes have been linked to valve disease, suggesting that developmental defects may underlie valve disease in adulthood.

Download Full-text

A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects

Development ◽

10.1242/dev.128.9.1531 ◽

2001 ◽

Vol 128 (9) ◽

pp. 1531-1538 ◽

Cited By ~ 5

Author(s):

Y. Dor ◽

T.D. Camenisch ◽

A. Itin ◽

G.I. Fishman ◽

J.A. McDonald ◽

...

Keyword(s):

Growth Factor ◽

Heart Development ◽

Heart Defects ◽

Negative Regulator ◽

Congenital Defects ◽

Endocardial Cushion ◽

Potential Contribution ◽

Vegf Receptor ◽

Endocardial Cushions ◽

Mesenchymal Transformation

Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation.

Download Full-text

Transcriptional atlas of cardiogenesis maps congenital heart disease interactome

Physiological Genomics ◽

10.1152/physiolgenomics.00015.2014 ◽

2014 ◽

Vol 46 (13) ◽

pp. 482-495 ◽

Cited By ~ 29

Author(s):

Xing Li ◽

Almudena Martinez-Fernandez ◽

Katherine A. Hartjes ◽

Jean-Pierre A. Kocher ◽

Timothy M. Olson ◽

...

Keyword(s):

Heart Development ◽

Congenital Heart ◽

Time Course ◽

Molecular Mechanisms ◽

Heart Diseases ◽

Expression Profiles ◽

Embryonic Stem ◽

Specific Gene ◽

Mesenchymal Transition ◽

Transcriptional Landscape

Mammalian heart development is built on highly conserved molecular mechanisms with polygenetic perturbations resulting in a spectrum of congenital heart diseases (CHD). However, knowledge of cardiogenic ontogeny that regulates proper cardiogenesis remains largely based on candidate-gene approaches. Mapping the dynamic transcriptional landscape of cardiogenesis from a genomic perspective is essential to integrate the knowledge of heart development into translational applications that accelerate disease discovery efforts toward mechanistic-based treatment strategies. Herein, we designed a time-course transcriptome analysis to investigate the genome-wide dynamic expression landscape of innate murine cardiogenesis ranging from embryonic stem cells to adult cardiac structures. This comprehensive analysis generated temporal and spatial expression profiles, revealed stage-specific gene functions, and mapped the dynamic transcriptome of cardiogenesis to curated pathways. Reconciling known genetic underpinnings of CHD, we deconstructed a disease-centric dynamic interactome encoded within this cardiogenic atlas to identify stage-specific developmental disturbances clustered on regulation of epithelial-to-mesenchymal transition (EMT), BMP signaling, NF-AT signaling, TGFb-dependent EMT, and Notch signaling. Collectively, this cardiogenic transcriptional landscape defines the time-dependent expression of cardiac ontogeny and prioritizes regulatory networks at the interface between health and disease.

Download Full-text

Folic acid prevents functional and structural heart defects induced by prenatal ethanol exposure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00817.2020 ◽

2021 ◽

Author(s):

Stephanie M Ford ◽

Cameron J Pedersen ◽

Matthew R Ford ◽

Jun W Kim ◽

Ganga H Karunamuni ◽

...

Keyword(s):

Blood Flow ◽

Folic Acid ◽

Heart Diseases ◽

Prenatal Alcohol Exposure ◽

Heart Defects ◽

Alcohol Exposure ◽

Ethanol Exposure ◽

Endocardial Cushion ◽

Prenatal Ethanol Exposure ◽

Endocardial Cushions

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 weeks of human gestation) 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 to controls, while embryos given FA with ethanol had significantly reduced regurgitant blood flow but did not return 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 is warranted.

Download Full-text

NOX2 Is Critical to Endocardial to Mesenchymal Transition and Heart Development

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/1679045 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Hoda Moazzen ◽

Yan Wu ◽

Anish Engineer ◽

Xiangru Lu ◽

Simran Aulakh ◽

...

Keyword(s):

Cell Proliferation ◽

Heart Development ◽

Ex Vivo ◽

Heart Defects ◽

Embryonic Heart ◽

Abnormal Development ◽

Endocardial Cushion ◽

Atrioventricular Canal ◽

Mesenchymal Transition ◽

Septal Defects

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

Download Full-text

Abstract 12937: Single-cell Transcriptomic Analysis Reveals Developmentally Impaired Endocardial Population in Hypoplastic Left Heart Syndrome

Circulation ◽

10.1161/circ.142.suppl_3.12937 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Yifei Miao ◽

Lei Tian ◽

Marcy Martin ◽

Sharon Paige ◽

Francisco X Galdos ◽

...

Keyword(s):

Single Cell ◽

Hypoplastic Left Heart Syndrome ◽

Heart Development ◽

Heart Diseases ◽

Critical Role ◽

Abnormal Development ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Mesenchymal Transition ◽

Left Heart Syndrome

Hypoplastic left heart syndrome (HLHS) is one of the most challenging forms of congenital heart diseases. Previous studies were mainly focused on intrinsic defects in myocardium. However, this does not sufficiently explain the abnormal development of the cardiac valve, septum, and vasculature, known to originate from the endocardium. Here, using single-cell transcriptomic profiling, induced pluripotent stem cells (iPSC) derived endocardial cells (iEECs), human fetal heart tissue with underdeveloped left ventricle, as well as a Xenopus model, we identified a developmentally impaired endocardial population in HLHS. The intrinsic endocardial deficits contributed to abnormal endothelial to mesenchymal transition, NOTCH signaling, and extracellular matrix organization, all of which are key factors in valve formation. Consequently, in an endocardium-myocardium co-culture system, we found that endocardial abnormalities conferred reduced proliferation and maturation of iPSC derived cardiomyocyte (iPSC-CMs) judged by Ki67 staining, contractility, sarcomere organization, and related gene expressions through a disrupted fibronectin (FN1)-integrin interaction. Several recently described HLHS de novo mutations such as ETS1 and CHD7 showed reduced binding to FN1 promoter and enhancer in HLHS vs. control iEECs based on ChIP-qPCR analysis. Additionally, we found that suppression of the ETS1 in Xenopus caused reduced endocardial FN1 expression and impaired heart development. Supplementation of FN1 or ETS1 over-expression in HLHS iEECs could rescue dysfunctions in both endocardium and myocardium in HLHS. Our studies reveal a critical role of endocardial abnormality in causing HLHS, and provide a rationale for improving endocardial function in future regenerative strategies. Schematic illustration of the endocardial and myocardial defects in HLHS.

Download Full-text