Prenatal ethanol exposure impairs the conduction delay at the atrioventricular junction in the looping heart.

The etiology of ethanol-related congenital heart defects has been the focus of much study, but most research has concentrated on cellular and molecular mechanisms. We have shown with optical coherence tomography (OCT) that ethanol exposure led to increased retrograde flow and smaller atrioventricular (AV) cushions compared with controls. Since AV cushions play a role in patterning the conduction delay at the atrioventricular junction (AVJ), this study aims to investigate whether ethanol exposure alters the AVJ conduction in early looping hearts and whether this alteration is related to the decreased cushion size. Quail embryos were exposed to a single dose of ethanol at gastrulation, and Hamburger-Hamilton stage 19-20 hearts were dissected for imaging. Cardiac conduction was measured using an optical mapping microscope and we imaged the endocardial cushions using OCT. Our results showed that, compared with controls, ethanol-exposed embryos exhibited abnormally fast AVJ conduction and reduced cushion size. However, this increased conduction velocity (CV) did not strictly correlate with decreased cushion volume and thickness. By matching the CV map to the cushion-size map along the inflow heart tube, we found that the slowest conduction location was consistently at the atrial side of the AVJ, which had the thinner cushions, not at the thickest cushion location at the ventricular side as expected. Our findings reveal regional differences in the AVJ myocardium even at this early stage in heart development. These findings reveal the early steps leading to the heterogeneity and complexity of conduction at the mature AVJ, a site where arrhythmias can be initiated.NEW & NOTEWORTHY To the best of our knowledge, this is the first study investigating the impact of ethanol exposure on the early cardiac conduction system. Our results showed that ethanol-exposed embryos exhibited abnormally fast atrioventricular conduction. In addition, our findings, in CV measurements and endocardial cushion thickness, reveal regional differences in the AVJ myocardium even at this early stage in heart development, suggesting that the differentiation and maturation at this site are complex and warrant further studies.

Wnt/ß-catenin signaling enables developmental transitions during valvulogenesis.

Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated and stratified valves. Wnt signaling and its canonical effector ß-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/ß-catenin signaling by chemically inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Furthermore, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/ß-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/ß-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects.

BMP-2 induces versican and hyaluronan that contribute to post-EMT AV cushion cell migration.

Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.

Muscleblind-like 1 is a negative regulator of TGF-beta-dependent epithelial-mesenchymal transition of atrioventricular canal endocardial cells.

The development of the valves and septa of the heart depends on the formation and remodeling of endocardial cushions. Here, we report that the alternative splicing regulator muscleblind-like 1 (MBNL1) exhibits a regionally restricted pattern of expression in canal region endocardium and ventricular myocardium during endocardial cushion development in chicken. Knockdown of MBNL1 in atrioventricular explants leads to a transforming growth factor beta-dependent increase in epithelial-mesenchymal transition (EMT) of endocardial cells. This reveals a novel role for MBNL1 during embryonic development, and represents the first evidence that an alternative splicing regulator is a key player in endocardial cushion development.

Chondroitin sulfate expression is required for cardiac atrioventricular canal formation.

Defects in cardiac valvulogenesis are a common cause of congenital heart disease, and the study of this process promises to provide mechanistic insights and lead to novel therapeutics. Normal valve development involves multiple signaling pathways, and recently roles have been identified for extracellular matrix components, including glycosaminoglycans. We, therefore, explored the role of the glycosaminoglycan chondroitin sulfate during zebrafish cardiac development. Beginning at 33 hr, there is a distinct zone of chondroitin sulfate expression in the atrioventricular (AV) boundary, in the cardiac jelly between the endocardium and myocardium. This expression is both spatially and temporally restricted, and is undetectable after 48 hr. Chemical as well as genetic inhibition of chondroitin synthesis results in AV canal (AVC) defects, including loss of the atrioventricular constriction, blood regurgitation, and failure of circulation. Lack of chondroitin disrupts a marker of cell migration, results in a loss of myocardial and endothelial markers of valvulogenesis, and misregulates bone morphogenetic protein expression, supporting an early role in AVC development. In summary, we have defined a requirement for chondroitin sulfate expression in the normal patterning of the AV boundary, suggesting that this component of the cardiac jelly provides a necessary signal in this critical transition in vertebrate cardiogenesis.

ROCK1 expression is regulated by TGFbeta3 and ALK2 during valvuloseptal endocardial cushion formation.

During early heart development at the looped heart stage, endothelial cells in the outflow tract and atrioventricular (AV) regions transform into mesenchyme to generate endocardial cushion tissue. This endocardial epithelial-mesenchymal transition (EMT) is regulated by several regulatory pathways, including the transforming growth factor-beta (TGFbeta), bone morphogenetic protein (BMP), and Rho-ROCK pathways. Here, we investigated the spatiotemporal expression pattern of ROCK1 mRNA during EMT in chick and examined whether TGFbeta or BMP could induce the expression of ROCK1. At the onset of EMT, ROCK1 expression was up-regulated in endothelial/mesenchymal cells. A three-dimensional collagen gel assay was used to examine the mechanisms regulating the expression of ROCK1. In AV endocardium co-cultured with associated myocardium, ROCK1 expression was inhibited by either anti-TGFbeta3 antibody, anti-ALK2 antibody or noggin, but not SB431542 (ALK5 inhibitor). In cultured preactivated AV endocardium, TGFbeta3 protein induced the expression of ROCK1, but BMP did not. AV endothelial cells that were cultured in medium supplemented with TGFbeta3 plus anti-ALK2 antibody failed to express ROCK1. These results suggest that the expression of ROCK1 is up-regulated at the onset of EMT and that signaling mediated by TGFbeta3/ALK2 together with BMP is involved in the expression of ROCK1.

BMP-2 induces cell migration and periostin expression during atrioventricular valvulogenesis.

Atrioventricular (AV) endocardium transforms into the cushion mesenchyme, the primordia of the valves and membranous septa, through epithelial-mesenchymal transformation (EMT). While bone morphogenetic protein (BMP)-2 is known to be critical for AV EMT, the role of BMP-2 in post-EMT AV valvulogenesis remains to be elucidated. To find BMP signaling loops, we first localized Type I BMP receptors (BMPRs), BMPR-1A (ALK3), -1B (ALK6) and ALK2 in AV cushion mesenchyme in stage-24 chick embryos. Based on the BMP receptor expression pattern, we examined the functional roles of BMP-2 and BMP signaling in post-EMT valvulogenesis by using stage-24 AV cushion mesenchymal cell aggregates cultured on 3D-collagen gels. Exogenous BMP-2 or constitutively active (ca) BMPR-1B (ALK6)-virus treatments induced migration of the mesenchymal cells into the collagen gels, whereas noggin, an antagonist of BMPs, or dominant-negative (dn) BMPR-1 B (ALK6)-virus treatments reduced cell migration from the mesenchymal cell aggregates. Exogenous BMP-2 or caBMPR-1B (ALK6) treatments significantly promoted expression of an extracellular matrix (ECM) protein, periostin, a known valvulogenic matrix maturation mediator, at both mRNA and protein levels, whereas periostin expression was repressed by adding noggin or dnBMPR-1B (ALK6)-virus to the culture. Moreover, transcripts of Twist and Id1, which have been implicated in cell migration in embryogenesis and activation of the periostin promoter, were induced by BMP-2 but repressed by noggin in cushion mesenchymal cell cultures. These data provide evidence that BMP-2 and BMP signaling induce biological processes involved in early AV valvulogenesis, i.e. mesenchymal cell migration and expression of periostin, indicating critical roles for BMP signaling in post-EMT AV cushion tissue maturation and differentiation.