Functional BMP receptor in endocardial cells is required in atrioventricular cushion mesenchymal cell formation in chick

Transformation of atrioventricular (AV) canal endocardium into invasive mesenchyme correlates spatially and temporally with the expression of bone morphogenetic protein (BMP)-2 in the AV myocardium. We revealed the presence of mRNA of Type I BMP receptors, BMPR-1A (ALK3), BMPR-1B (ALK6) and ALK2 in chick AV endocardium at stage-14(-), the onset of epithelial to mesenchymal transformation (EMT), by RT-PCR and localized BMPR-1B mRNA in the endocardium by in situ hybridization. To circumvent the functional redundancies among the Type I BMP receptors, we applied dominant-negative (dn) BMPR-1B-viruses to chick AV explants and whole-chick embryo cultures to specifically block BMP signaling in AV endocardium during EMT. dnBMPR-1B-virus infection of AV endocardial cells abolished BMP-2-supported AV endocardial EMT. Conversely, caBMPR-1B-virus infection promoted AV endocardial EMT in the absence of AV myocardium. Moreover, dnBMPR-1B-virus treatments significantly reduced myocardially supported EMT in AV endocardial-myocardial co-culture. AV cushion mesenchymal cell markers, alpha-smooth muscle actin (SMA), and TGFbeta3 in the endocardial cells were promoted by caBMPR-1B and reduced by dnBMPR-1B infection. Microinjection of the virus into the cardiac jelly in the AV canal at stage-13 in vivo (ovo) revealed that the dnBMPR-1B-virus-infected cells remained in the endocardial epithelium, whereas caBMPR-1B-infected cells invaded deep into the cushions. These results provide evidence that BMP signaling through the AV endocardium is required for the EMT and the activation of the BMP receptor in the endocardium can promote AV EMT in the chick.     

An unusual case of complete atrioventricular block causing Takotsubo syndrome

Complete atrioventricular (AV) block in association with Takotsubo syndrome (TS) has been well recognized, but the cause and effect relationship has not been elucidated. We describe a 78-year-old female who presented with complete AV block but one week later developed new-onset, diffuse T-wave inversions, QT prolongation, and acceleration of junctional escape rate. Left ventriculogram revealed features typical of TS. One year after permanent pacemaker implantation, complete AV block persisted despite the reversal of wall motion defects implying that conduction abnormality was the trigger of TS rather than its consequence.     

Early myocardial function affects endocardial cushion development in zebrafish

Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih^−/−) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk^−/−) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk^−/− embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfk^s11 variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.     

Transmembrane protein 2 (Tmem2) is required to regionally restrict atrioventricular canal boundary and endocardial cushion development

The atrioventricular canal (AVC) physically separates the atrial and ventricular chambers of the heart and plays a crucial role in the development of the valves and septa. Defects in AVC development result in aberrant heart morphogenesis and are a significant cause of congenital heart malformations. We have used a forward genetic screen in zebrafish to identify novel regulators of cardiac morphogenesis. We isolated a mutant, named wickham (wkm), that was indistinguishable from siblings at the linear heart tube stage but exhibited a specific loss of cardiac looping at later developmental stages. Positional cloning revealed that the wkm locus encodes transmembrane protein 2 (Tmem2), a single-pass transmembrane protein of previously unknown function. Expression analysis demonstrated myocardial and endocardial expression of tmem2 in zebrafish and conserved expression in the endocardium of mouse embryos. Detailed phenotypic analysis of the wkm mutant identified an expansion of expression of known myocardial and endocardial AVC markers, including bmp4 and has2. By contrast, a reduction in the expression of spp1, a marker of the maturing valvular primordia, was observed, suggesting that an expansion of immature AVC is detrimental to later valve maturation. Finally, we show that immature AVC expansion in wkm mutants is rescued by depleting Bmp4, indicating that Tmem2 restricts bmp4 expression to delimit the AVC primordium during cardiac development.     

Bone morphogenetic protein receptor 1A signaling is dispensable for hematopoietic development but essential for vessel and atrioventricular endocardial cushion formation

Bone morphogenetic protein 4 (BMP4) is crucial for the formation of FLK1-expressing (FLK1(+)) mesodermal cells. To further define the requirement for BMP signaling in the differentiation of blood, endothelial and smooth muscle cells from FLK1(+) mesoderm, we inactivated Alk3 (Bmpr1a) in FLK1(+) cells by crossing Alk3(floxed/floxed) and Flk1(+/Cre)Alk3(+/floxed) mice. Alk3 conditional knockout (CKO) mice died between E10.5 and E11.5. Unexpectedly, Alk3 CKO embryos did not show any hematopoietic defects. However, Alk3 CKO embryos displayed multiple abnormalities in vascular development, including vessel remodeling and maturation, which contributed to severe abdominal hemorrhage. Alk3 CKO embryos also displayed defects in atrioventricular canal (AVC) endocardial cushion formation in the heart. Collectively, our studies indicate a crucial role for ALK3 in vessel remodeling, vessel integrity and endocardial cushion formation during the development of the circulation system.     

Mechanically induced complete atrioventricular block during retrograde catheter ablation of a left-sided incessant tachycardia

Concern for potentially lethal complications related to transseptal catheterisation, such as perforation of the aorta, has made this approach less attractive over the last decades. Nevertheless, this method is now increasingly being used for ablation of left-sided arrhythmias. We report a transient complication of a retrograde procedure in a patient with a left-sided ''Coumel-type'' incessant tachycardia. We had to proceed to transseptal catheterisation, as the complication recurred during the second attempt even when using a less rigid transaortic radiofrequency catheter. The transseptal approach using a less stiff cryoenergy catheter was performed without complications.     

Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect

The gap junction protein connexin45-deficient (Cx45-KO) mice die shortly after the hearts begin to beat. In addition to the heart defect, they also show defective vascular development which may be closely related with the cardiac phenotype. Therefore, we created mice whose floxed-Cx45 locus could be removed conditionally. We utilized cardiac alpha-actin-Cre transgenic mice to investigate the specific cardiac muscular function of Cx45 in vivo. The resultant conditional mutants were lethal, showing conduction block similar to that of the Cx45-KO mice. Unlike Cx45-KO, development of the endocardial cushion was not disrupted in the conditional mutants. X-gal staining was detected in the embryonic cardiac myocytes as a hallmark of Cre-loxP mediated floxed-Cx45 deletion. These results reconfirm the requirement of Cx45 for developing cardiac myocytes. These also indicate that establishing the first contractions is a crucial task for the early hearts.     

Effects of vitamin A on endocardial cushion development in the mouse heart

Retinoic acid (RA) (78mg/kg) administered to ICR mice on days 9.0,9.5 and 10.0 of pregnancy (plug day = day 1), resulted in cardiac malformations in 37.6% of the surviving fetuses, including transposition of the great arteries, ventricular septal defects, and double outlet right ventricle. Histological examination of the hearts of embryos observed 24 hours after in vivo or in vitro exposure to RA on day 9 revealed abnormalities in endocardial cushion tissue. The volume of the atrioventricular endocardial was reduced in treated embryos as was the ratio of the size of the cushions to the size of the heart. The endothelial layer of the atrioventricular endocardial cushions appeared to be unaffected by the retinoic acid, however, the mesenchymal cushion cells were significantly reduced in number when compared with controls. Labeling with [3H]-thymidine indicated that the mitotic activity of the mesenchymal cell population was significantly decreased while that of the endothelial cells was comparable to control levels. The extracellular matrix or cardiac jelly of the endocardial cushions also appeared to be affected by RA exposure, as shown by studies utilizing colloidal iron to stain GAGs, which revealed a decrease in the amount of stainable material in treated cushions. Two possible cause for the reduced thymidine index of the cushion mesenchyme are discussed, namely, a direct effect of RA on the mesenchymal cells or an indirect effect via the altered extracellular matrix of the cushion tissue.     

Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation

The development of enteric and sympathetic neurons from neural crest precursor cells is regulated by signals produced by the embryonic environments to which the cells migrate. Bone morphogenetic proteins (BMPs) are present in the developing embryo and act to induce neuronal differentiation and noradrenergic properties of neural crest cells. We have investigated the role of BMP2 in regulating the appearance of distinct populations of autonomic neurons from postmigratory, HNK-1-positive neural crest precursor cells. BMP2 promotes neuronal differentiation of sympathetic and enteric precursor cells isolated from E14.5 rat. The effects of BMP2 change over time, resulting in a decrease in neuron number that can be attributed to apoptotic cell death. BMP2-dependent neuron death is rescued by gut-derived factors that provide trophic support to maturing neurons, indicating that BMP2 regulates the acquisition of trophic dependence of developing peripheral neurons. In addition to regulating neuron number, BMP2 promotes both panneuronal maturation and the acquisition of an enteric phenotype, as measured by lineage-specific changes in the expression of tyrosine hydroxylase and MASH-1. While BMP2 is sufficient to induce neuronal differentiation and panneuronal development, these results suggest that additional factors in the environment must collaborate with BMP2 to promote the final noradrenergic phenotype of sympathetic neurons.     

Hedgehog signalling controls sinoatrial node development and atrioventricular cushion formation

Smoothened is a key receptor of the hedgehog pathway, but the roles of Smoothened in cardiac development remain incompletely understood. In this study, we found that the conditional knockout of Smoothened from the mesoderm impaired the development of the venous pole of the heart and resulted in hypoplasia of the atrium/inflow tract (IFT) and a low heart rate. The blockage of Smoothened led to reduced expression of genes critical for sinoatrial node (SAN) development in the IFT. In a cardiac cell culture model, we identified a Gli2–Tbx5–Hcn4 pathway that controls SAN development. In the mutant embryos, the endocardial-to-mesenchymal transition (EndMT) in the atrioventricular cushion failed, and Bmp signalling was downregulated. The addition of Bmp2 rescued the EndMT in mutant explant cultures. Furthermore, we analysed Gli2⁺ scRNAseq and Tbx5^−/− RNAseq data and explored the potential genes downstream of hedgehog signalling in posterior second heart field derivatives. In conclusion, our study reveals that Smoothened-mediated hedgehog signalling controls posterior cardiac progenitor commitment, which suggests that the mutation of Smoothened might be involved in the aetiology of congenital heart diseases related to the cardiac conduction system and heart valves.     

Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function) pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development.