Endocardial Cushion Tissue Research Articles

Loss of myocardial Hey2/Hrt2 function disrupts rightward shift of atrioventricular cushion tissue and causes tricuspid atresia.

Endocardial cushion tissue is primordia of the valves and septa of the adult heart, and its malformation causes various congenital heart diseases (CHDs). Tricuspid atresia (TA) is defined as congenital absence or agenesis of the tricuspid valve caused by endocardial cushion defects. However, little is known about what type of endocardial cushion defect causes TA. Using three-dimensional volume rendering image analysis, we demonstrated morphological changes of endocardial cushion tissue in developing Hey2/Hrt2 KO mouse embryos that showed malformation of the tricuspid valve, which resembled human TA at neonatal period. In control embryos, atrioventricular (AV) endocardial cushion tissues showed rightward shift to form a tricuspid valve. However, the rightward shift of endocardial cushion tissue was disrupted in Hey2/Hrt2 KO embryos, leading to the misalignment of AV cushions. We also found that muscular tissue filled up the space between the right atrium and ventricle, resulting in the absence of the tricuspid valve. Moreover, analysis using tissue-specific conditional KO mice showed that HEY2/HRT2-expressing myocardium may physically regulate the AV shift. Disruption of rightward cushion movement is an initial cue of TA phenotype, and myocardial HEY2/HRT2 is necessary for the regulation of proper alignment of AV endocardial cushion tissue.

Knowing Real Cardiac Anatomy Enhances Understanding of Cardiac Development

New ideas about cardiac chamber formation, interatrial septum formation and development of the outflow region can be better understood by knowing real cardiac anatomy. The definitive chambers of the heart can be regionalized into components that provide a basis for understanding their formation during prenatal embryonic development. Each atrium has a venous inlet, a body, an appendage and a vestibule, while each ventricle consists of an inlet, an apical trabeculated portion and an outlet. During development several precursors combine to form the components of definitive chambers. Recent studies have challenged the classical version of interatrial septum formation, particularly the presence of a secondary atrial septum (septum secundum), and the closure of the primary atrial foramen (ostium primum). These studies suggest that the primary atrial septum (septum primum) merges with a fold of myocardium from the roof of the right atrium rather than with a second septum. This fold can be demonstrated in the dissection laboratory. Closure of the primary atrial foramen is accomplished by fusion of the ingrowing vestibular spine fusing with the mesenchymal cap of the primary septum and the merged AV endocardial cushions. This forms the anterior inferior atrial buttress that might be considered a secondary atrial septum. Finally, formation of the outflow region can be better understood using a tripartite explanation rather that the traditional bipartite version. During cardiac looping, the outflow region elongates and develops a bend in the middle. This flexure can define the outflow region into proximal, intermediate and distal segments. From these segments the outflow regions of the ventricles, the aortic valves and the intrapericardial roots of the great vessels are formed. The septum dividing the proximal segment is formed exclusively from endocardial cushion tissue, while the septum that partitions the intermediate and distal outflow regions is derived from cushion tissue and neural crest ectomesenchyme. Traditionally the aorticopulmonary septum was described as partitioning the entire outflow region. More recent examination of outflow tract development suggests that the aorticopulmonary septum only partitions the distal portion of the outflow region and the aortic sac. The intrapericardial and extrapericardial roots of the great vessels form from the division of the distal outflow region and the aortic sac.

Abstract 16895: Lrp1, an Endocytic Vesicle Trafficking Protein, is Associated With Congenital Heart Diseases - Through a "non-Autonomous" Action

Backgrounds: Congenital heart diseases affect nearly 1% of neonates per year in the United States and are the leading cause of neonatal death. Despite a strong indication of genetic contribution to congenital heart diseases, the developmental processes and genetic components are poorly understood. From the large-scale forward genetic screen using inbred mice chemically mutagenized with ethyl-nitrosourea (ENU), a novel finding of endocytic trafficking gene mutations, including Lrp1, were recovered as causing congenital heart disease phenotypes. Endocytosis plays roles in modulating cell signaling by regulating the trafficking of the internalized endocytic vesicles to different endosomal compartments, some destined for fusion with lysosomes and degradation while others are recycled to the surface by recycling endosomes. Mutations in these genes causes a spectrum of congenital heart disease comprising of outflow tract abnormalities and alignment defects. Methods and Results: The LRP1 [line 1554(MGI 96828)] mutant mouse line recovered from a forward genetic screen results from a missense (C4232R) mutation in the region encoding the epidermal growth factor (EGF) repeat domain of LRP1. The mutants exhibit a spectrum of septation and outflow tract defects including endocardial cushion defects, double outlet of right ventricle with normal related great arteries as well as DORV with D-malposition of great arteries (Taussig-Bing anomalies). Mouse embryonic fibroblasts (MEF) from LRP1 m/m mutants demonstrated decreased cell-surface expression and increased retention in the endosomes. Migration assay of MEF demonstrated decreased migration of LRP1 m/m compared with control with normal Golgi orientation. The decreasing migration LRP1 m/m also observed in the endocardial cushion tissue from E9.5-10.5 embryos. Different cell lineages crucial to cardiac development with tissue-specific genetic manipulations were used to define the roles of the LRP1 pathway in these three fields of cells during outflow tract development and septation. Cardiac neural crest cell (Wnt1-Cre) knockout of LRP1 recapitulate LRP1 m/m cardiac phenotypes as well as Nkx2.5/Tie2-cre double knockout of LRP1. RNA expression profile suggests Notch pathway and integrin pathway are associated with LRP1 during cardiac development. Conclusions: We provide the first evidence of endocytic vesicle trafficking pathway associated with the development of congenital heart diseases with malalignment/septation and outflow tract defects

Expression of the Tgfβ2 Gene During Chick Embryogenesis

We performed a comprehensive analysis of the expression of transforming growth factor (TGF) β2 during chick embryogenesis from stage 6 to 30 (Hamburger and Hamilton, J Morphol 1951;88:49-92) using in situ hybridization. During cardiogenesis, Tgfβ2 was expressed in the endothelial/mesenchymal cells of the valvulo-septal endocardial cushion tissue and in the epicardium until the end of embryogenesis. During the formation of major arteries, Tgfβ2 was localized in smooth muscle progenitors but not in the vascular endothelium. During limb development, Tgfβ2 was expressed in the mesenchymal cells in the presumptive limb regions at stage 16, and thereafter it was localized in the skeletal muscle progenitors. In addition, strong Tgfβ2 expression was seen in the mesenchymal cells in the pharyngeal arches. Tgfβ2 mRNA was also detected in other mesoderm-derived tissues, such as the developing bone and pleura. During ectoderm development, Tgfβ2 was expressed in the floor plate of the neural tube, lens, optic nerve, and otic vesicle. In addition, Tgfβ2 was expressed in the developing gut epithelium. Our results suggest that TGFβ2 plays an important role not only in epithelial-mesenchymal interactions but also in cell differentiation and migration and cell death during chick embryogenesis. We also found that chick and mouse Tgfβ2 RNA show very similar patterns of expression during embryogenesis. Chick embryos can serve as a useful model to increase our understanding in the roles of TGFβ2 in cell-cell interactions, cell differentiation, and proliferation during organogenesis.

Roles of TGFβ and BMP during valvulo–septal endocardial cushion formation

The primordia of valves and the atrioventricular septum arise from endocardial cushion tissue that is formed in the outflow tract (OFT) and in the atrioventricular (AV) regions during cardiogenesis. Abnormal development of the endocardial cushion results in various congenital heart diseases. Endocardial epithelial-mesenchymal transformation (EMT) is a critical process in cushion tissue formation and is regulated by many factors, such as growth factors, intercellular signaling molecules, transcription factors, and extracellular matrices. A signal that is produced by the myocardium of the AV and OFT regions and transferred to the adjacent endocardium across the extracellular matrix mediates EMT. Studies in vitro and genetic analyses have shown that transforming growth factor beta and bone morphogenetic protein play central roles in the regulation of EMT during cushion tissue formation.

A combination of right ventricular hypertrabeculation/noncompaction and Ebstein's anomaly

A 35-year-old man admitted with iterative extremities edema for three months and mild chest complaint associated with activity without chest distress for five days. He underwent an initial 2DE that did not confirm HVM/NVM and 3DE that strengthened the diagnosis of HVM/NVM. The limitations of 2DE may lead to misinterpretation of prominent trabeculations as “false tendons,” and may also lead to underestimation of the severity of HVM/NVM. In contrast to 2DE, 3DE provides for pyramid-shaped datasets that encompass the entire right ventricular. Specifically, right ventricular can be sectioned in userselected planes and an unlimited number and angles of such planes can be used. Intracavitary echodensities that are suspicious for trabeculations can be tracked in multiple directions from base to apex. To our knowledge, this is the first report on only right ventricular HVM/NVM associated with Ebstein's malformation. The leaflets of the tricuspid valve develop from endocardial cushion tissue and the myocardium. The tricuspid valve leaflets become freely movable after delamination of the tension apparatus within the myocardium. Although the coexistence of right ventricular HVM/NVM and Ebstein's malformation could be a coincidence, I believe that both defects could be caused by a developmental arrest of the right ventricular myocardium. That is to say, the genetic change that is responsible for Ebstein's anomaly also plays a role in the differentiation of the right myocardium.

Latent Transforming Growth Factor-β-Binding Protein-4 Regulates Transforming Growth Factor-β1 Bioavailability for Activation by Fibrogenic Lung Fibroblasts in Response to Bleomycin

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (-) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-beta activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-beta1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-beta activation, we examined the role of latent TGF-beta-binding proteins (LTBPs), key regulators of TGF-beta bioavailability and activation, in TGF-beta1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-beta1 complexes in Thy-1 (-) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-beta1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-beta1. Expression of LTBP-4 restored TGF-beta1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-beta-binding site or only the TGF-beta-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-beta activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-beta1 activation in bleomycin-induced lung fibrosis.

ROCK1 Expression is Regulated by TGFβ3 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.

Accessory Atrioventricular Pathways

By definition, accessory atrioventricular pathways are aberrant muscle bundles that connect the atrium to a ventricle outside of the regular atrioventricular conduction system. Clinically, they may manifest as substrates for ventricular preexcitation. The first accessory pathway in a patient who suffered from Wolff-Parkinson-White syndrome was described in 1943 by Wood, Wolferth, and Geckler.1 Shortly after, Ohnell created a reconstruction of an accessory pathway that very elegantly showed the close proximity of the pathway to the fibrous attachment of the mitral valve and its relationship with the sulcus coronarius (Figure, A).2 Subsequent histological studies have demonstrated unequivocally that these pathways are the anatomic substrates for the classical Wolff-Parkinson-White variety of preexcitation. Figure. A, Ohnell’s depiction of an accessory bundle in the left atrioventricular groove.2 Reprinted with permission from Blackwell Publishing. B, Histological section stained with Masson’s trichrome (fibrous tissue in green and myocardium in red) to show an accessory left-sided pathway (arrow) skirting the mitral annulus. C, Histological section showing a broad right-sided accessory pathway (arrow) that is formed by a myocardial pouch extending from the right atrial appendage to the ventricle and ultimately traced to a small right ventricular vein. This pathway is some distance from the fibrous annulus of the tricuspid valve. *Valvar annulus. Article p 1508 Accessory atrioventricular pathways are found most often in the parietal atrioventricular junctional areas, including the paraseptal areas. They breach the insulation provided by the fibrofatty tissues of the atrioventricular groove (sulcus tissue) and the hingelines (fibrous annulus) of the valves. They are rarely found in the area of fibrous continuity between the aortic and mitral valves because in this area, there is usually a wide gap between the atrial myocardium and ventricular myocardium to accommodate the aortic outflow tract. On the left parietal side, the accessory pathways tend …

Accessory mitral valve as a potential source of cardioembolism

An accessory mitral valve (AMV) is considered to arise from abnormal development of endocardial cushion tissue. It is a very rare entity, commonly diagnosed in childhood and associated with symptomatic left ventricular outflow tract (LVOT) obstruction. Here we describe the presence of AMV in a 58-year old patient who presented with a transient ischemic attack. Transesophageal echocardiography visualized a spherical structure attached to the ventricular aspect of the anterior mitral valve leaflet.

Loss of Fibulin-2 Expression Is Associated with Breast Cancer Progression

Fibulin-2, an extracellular matrix protein expressed by normal epithelia, was found to be down-regulated in several breast cancer cell lines. Fibulin-2 protein expression was also decreased in breast cancer tissue samples as evaluated by immunohistochemistry. Reintroduction of Fibulin-2 into breast cancer cell lines that do not express Fibulin-2 reduced cancer cell motility and invasion in vitro but had no effect on cell growth and adhesion properties. Together with evidence that Fibulin-2 contributes to wound healing and inhibits smooth muscle cell migration, our findings suggest that loss of Fibulin-2 expression may facilitate migration and invasion in breast cancer.

The Matricellular Protein CCN1 Is Essential for Cardiac Development

The matricellular protein CCN1 (formerly named CYR61) regulates cell adhesion, migration, proliferation, survival, and differentiation through binding to integrin receptors and heparan sulfate proteoglycans. Here we show that Ccn1-null mice are impaired in cardiac valvuloseptal morphogenesis, resulting in severe atrioventricular septal defects (AVSD). Remarkably, haploinsufficiency for Ccn1 also results in delayed formation of the ventricular septum in the embryo and persistent ostium primum atrial septal defects (ASD) in approximately 20% of adults. Mechanistically, Ccn1 is not required for epithelial-to-mesenchymal transformation or cell proliferation and differentiation in the endocardial cushion tissue. However, Ccn1 deficiency leads to precocious apoptosis in the atrial junction of the cushion tissue and impaired gelatinase activities in the muscular component of the interventricular septum at embryonic day 12.5, when fusion between the endocardial cushion tissue and the atrial and ventricular septa occurs, indicating that these defects may underlie the observed AVSD. Moreover, human CCN1 maps to 1p21-p31, the chromosomal location of an AVSD susceptibility gene. Together, these results provide evidence that deficiency in matrix signaling can lead to autosomal dominant AVSD, identify Ccn1(+/-) mice as a genetic model for ostium primum ASD, and implicate CCN1 as a candidate gene for AVSD in humans.

Chick embryos exposed to trichloroethylene in an ex ovo culture model show selective defects in early endocardial cushion tissue formation

Formation of the primitive heart is a critical step for establishing a competent circulatory system necessary for continued morphogenesis, and as such has significant potential as a target for environmental insult. The goal of this study was to identify the initial cellular events that precede more superficially observable abnormalities resulting from exposing early chick embryos to trichloroethylene (TCE). A whole embryo culture method was used to assess the susceptibility of endocardial epithelial-mesenchymal transformation in the early chick heart to TCE. This method has the benefits of maintaining the anatomical relationships of developing tissues and organs, instantaneously exposing precisely staged embryos to quantifiable levels of TCE in a protein-free medium, and the ability to directly monitor developmental morphology. A minority of embryos (Hamburger and Hamilton [HH] stage 13-14) exposed to TCE (10-80 ppm) were not viable after 24 hr in culture and exhibited a variety of gross malformations in a dose-dependent fashion. However, the majority of treated embryos remained viable and developed into HH stage 17 embryos that were superficially indistinguishable from vehicle-treated controls. Further analysis of the hearts of these superficially normal embryos by whole-mount confocal microscopy revealed selective reduction in the number of atrioventricular canal mesenchymal cells. Additionally, those mesenchymal cells that did develop migrated abnormally as long thin cords of adherent cells. The regional selectivity of these effects in the chick heart suggests a critical window of susceptibility to TCE in the epithelial-mesenchymal transformation of atrioventricular canal endocardium.

Expression patterns of tbx genes during chick atrial development

T-box gene family encodes a set of transcription factors that are involved in various developmental processes. We cloned T-box genes, tbx2, −5 and −20, from chick embryonic heart, and examined their expression patterns in the developing atrium from stage 14 to 27 by in situ hybridization. In the looped heart at stages 14 – 16, tbx2, −5 and −20 were expressed in the atrioventricular (AV) canal, in which valvuloseptal endocardial cushion tissue would later develop. These three genes were also expressed in the dorsal mesentery, through which pulmonary veins connect to the left atrium. During the atrial septation at later stages, tbx5 was expressed in the primitive auricles, sinus venosus and caval veins. In the developing atrial septum, tbx5 and −20 were expressed in the spina vestibuli, and tbx2 and −5 in the primary septum. tbx20 was expressed in the AV endocardial cushion tissue. Results suggest that differentially expressed tbx2, −5, −20 may play an important role in the atrial development, such as septation and pulmonary venous development.

Rho kinases regulate endothelial invasion and migration during valvuloseptal endocardial cushion tissue formation

Rho-associated kinase (ROCK) is a downstream effector of small Rho-GTPases, and phosphorylates several substrates to regulate cell functions, including actin cytoskeletal reorganization and cellular motility. Endothelial-mesenchymal transformation (EMT) is a critical event in the formation of valves and septa during cardiogenesis. It has been reported that ROCK plays an important role in the regulation of endocardial cell differentiation and migration during mouse cardiogenesis (Zhao and Rivkees [2004] Dev. Biol. 275:183-191). Immunohistochemistry showed that, during chick cardiogenesis, ROCK1 and -2 were expressed in the transforming and migrating endothelial/mesenchymal cells in the outflow tract (OT) and atrioventricular (AV) canal regions from which valvuloseptal endocardial cushion tissue would later develop. Treatment with Y27632, a specific ROCK inhibitor, of cultured AV explants or AV endothelial monolayers of stage 14-minus heart (preactivated stage for EMT) on three-dimensional collagen gel perturbed the seeding of mesenchymal cells into the gel lattice. In these experiments, Y27632 did not suppress the expression of an early transformation marker, smooth muscle alpha-actin. Moreover, Y27632 inhibited the mesenchymal invasion in stage 14-18 AV explants, in which endothelial cells had committed to undergo EMT. ML-9, a myosin light chain kinase inhibitor, also inhibited the mesenchymal invasion in cultured AV explants. These results suggest that ROCKs have a critical role in the mesenchymal cell invasion/migration that occurs at the late onset of EMT.

Understanding heart development and congenital heart defects through developmental biology: A segmental approach

ABSTRACT The heart is the first organ to form and function during development. In the pregastrula chick embryo, cells contributing to the heart are found in the postero-lateral epiblast. During the pregastrula stages, interaction between the posterior epiblast and hypoblast is required for the anterior lateral plate mesoderm (ALM) to form, from which the heart will later develop. This tissue interaction is replaced by an Activin-like signal in culture. During gastrulation, the ALM is committed to the heart lineage by endoderm-secreted BMP and subsequently differentiates into cardiomyocyte. The right and left precardiac mesoderms migrate toward the ventral midline to form the beating primitive heart tube. Then, the heart tube generates a right-side bend, and the d-loop and presumptive heart segments begin to appear segmentally: outflow tract (OT), right ventricle, left ventricle, atrioventricular (AV) canal, atrium and sinus venosus. T-box transcription factors are involved in the formation of the heart segments: Tbx5 identifies the left ventricle and Tbx20 the right ventricle. After the formation of the heart segments, endothelial cells in the OT and AV regions transform into mesenchyme and generate valvuloseptal endocardial cushion tissue. This phenomenon is called endocardial EMT (epithelial-mesenchymal transformation) and is regulated mainly by BMP and TGFbeta. Finally, heart septa that have developed in the OT, ventricle, AV canal and atrium come into alignment and fuse, resulting in the completion of the four-chambered heart. Altered development seen in the cardiogenetic process is involved in the pathogenesis of congenital heart defects. Therefore, understanding the molecular nature regulating the 'nodal point' during heart development is important in order to understand the etiology of congenital heart defects, as well as normal heart development.

Essential roles of Meltrin β (ADAM19) in heart development

Morphogenesis of the heart requires development of the endocardial cushion tissue that gives rise to the membranous septa and valves. Here we show that Meltrin β/ADAM19, a novel metalloprotease-disintegrin, participates in the development of the endocardial cushion. Mice lacking Meltrin β exhibit ventricular septal defect (VSD) and immature valves, and most of the animals die soon after birth. During development of the endocardial cushion, epithelial–mesenchymal transformation (EMT) of endocardial epithelial cells generates most of the cushion mesenchymes that constitute the main components of the septa and valves. Meltrin β is expressed in both the epithelia and the mesenchymes of the endocardial cushion. In the absence of Meltrin β, the cushion is small or thin in the septum-forming region and show poor remodeling of cardiac jelly components; both of these characteristics suggest impaired growth and differentiation of the endocardial cushion. When embryonic fibroblasts are cultured sparsely, Meltrin β-lacking cells exhibit aberrant ectodomain shedding of type I Neuregulin, one of the ErbB ligands expressed in endocardial cells. These results suggest the necessity of proteolytic regulation of ErbB ligands by Meltrin β for proper heart development.

Prominent expression of lysyl oxidase during mouse embryonic cardiovascular development.

By mRNA differential display in mouse hearts, lysyl oxidase (Lox), a key enzyme catalyzing cross-links in elastin and collagens, was found to be up-regulated between embryonic days 11 (E11) and 13 (E13). This was confirmed by semiquantitative RT-PCR. We analyzed its spatio-temporal expression pattern by in situ hybridization in regard to the development of myocardial cells, endocardial cushion tissue, aortic arch vessels, and epicardium.

Expression of mOb1, a novel atypical 73 amino acid K50-homeodomain protein, during mouse development

We report the initial characterization of mOb1 ( Odd homeo Box 1), which encodes an atypical 73 amino acid K50-homeodomain protein localised in the cytoplasm and absent from nuclei during mouse development. Conserved orthologues were present in man, rat, cow, pig and chicken, but not in fish, amphibians or invertebrates. Temporo-spatial patterns of mOb1 transcript and mOb1 protein expression were coincident in developing mouse embryos. Cardiac expression was first observed at E8.25 in linear heart tube myocardium and briefly in both horns of the sinus venosus. Myocardial expression peaked at E13.5, where after it diminished and was not detectable above background by adulthood. At no stage was expression observed in endocardium, endocardial cushion tissue, the coronary arteries or great vessels. At E13.5 and E15.5, mOb1 expression broadened to include skeletal muscle, stratified epithelium (upper aerodigestive tract and skin), epithelium of developing airways, vibrissae, midbrain/hindbrain junction, meninges, mesenchymal cellular condensations that preceded cartilage formation and chondrocytes.

Embryogenesis of the rat heart: the expression of collagenases.

Little is known about extracellular matrix (ECM) remodeling during heart development. Matrix degrading metalloproteinases are possible candidates contributing to degradation of ECM during these complex biological events. We described here different forms of MMPs, based on their substrate specificity, molecular weight, immunolocalization and in situ zymography within embryonic rat myocardium at different stages of heart development (from embryonic day - ED12 until ED21). Murine collagenase-3 (MMP-13), stromelysin (MMP-3) and gelatinases A&B (MMP-2 & -9) were expressed in prenatal hearts, as demonstrated by quantitative zymography and immunohistochemistry. MMP-2, -3 and -9 were found within myocardium of avascular (ED12) and vascularized heart (ED14-21). An extensive immunolabeling over the heart trabeculae, epicardial tissue and a weaker labeling in the endocardial and truncoconal cushion tissue was observed at all stages of the heart development. Utilizing quantitative zymography we found that MMP-13 activity gradually increased from ED14-ED16 reaching a plateau from ED16-ED21, while MMP-2 activity demonstrated a transient increase starting at ED13, peaked at ED16 and declined thereafter. As to MMP-9 activity, it was seen only between ED16 and ED 18. In situ zymography with gelatin as a substrate represented activity of MMPs within the myocardium of the atria and the ventricles and a very strong activity in the interstitial tissue of the endocardial and the conotruncal cushion tissue. MMPs expressed in embryonic heart correspond to all major classes of these enzymes. They may contribute to embryonic remodeling of the heart.