Development of the inlet portion of the right ventricle in the embryonic rat heart: The basis for tricuspid valve development

Background: Before septation the entire atrioventricular canal is connected with the ventricular inlet segment (primitive left ventricle), wheres the mature heart exhibits an exclusive connection of the right atrium to the right ventricule. The process which is responsible for this change is controversial. Methods: Graphic reconstructions of serially sectioned embryonic rat hearts as well as scanning electron micrographs of similar specimens were made. Results: The first indication of a right atrioventricular connection was seen as a groove in the atrioventricular junctional myocardium to the right of the inferior endocardial cushion. This groove expanded to form the right ventricular inlet portion. The right, inferior, and superior walls of this newly formed cavity were formed from junctional myocardium, which demarcated it from the trabeculated right ventricular portion in all developmental stages. The left wall equally developed from this junctional myocardium and formed the ventricular inlet septum. The junctional myocardium between right ventricular inlet and trabeculated portions was seen to develop into the tricuspid valve and its tension apparatus. Conclusions: The preseptation embryonic heart has no inlet portion to the right ventricle. This new cavity is created by remodelling of atrioventricular junctional myocardium. This myocardium also provides the material contribution to the tricuspid valve and its tension apparatus. Malformations of the right ventricular inlet portion and of the tricuspid valve are indissolubly linked. © 1994 Wiley-Liss, Inc.     

The Atrioventricular Region of the Teleost Heart. A Distinct Heart Segment

This article reports the morphological characteristics of the atrioventricular (AV) region of the teleost heart. A total of 29 teleost species belonging to 19 families and 10 orders were analyzed. The study includes hearts with entirely trabeculated ventricles and hearts possessing a ventricular compacta. In all cases, the AV region is formed by a ring of compact myocardium surrounded by a connective tissue ring. The myocardium contains vessels in most species having entirely trabeculated ventricles, and in all species possessing a compacta. The ring of connective tissue is rich in collagen and elastin, partially isolating the AV muscle from the surrounding musculature. Continuity between the AV muscle and the atrial and ventricular musculature is always observed. The AV muscle supports the AV valves, which are formed by two leaflets, lack papillary muscles, show a collagenous fibrosa on the atrial side, and present a variable number of cells and a variable amount of extracellular material. Ventricular trabeculae attach to the AV muscle. These trabeculae may help to bear the stress generated by ventricular contraction and may constitute a preferential pathway for the conduction of electrical impulses. In all teleosts studied, the AV region can be recognized as a distinct morphological segment interposed between the atrium and the ventricle. The morphological characteristics of the AV segment appear to be species specific. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

The vomeronasal organ in the human embryo, studied by means of three-dimensional computer reconstruction

The human vomeronasal organ is of interest because of its potential role in sex pheromone detection. Due to the scarcity of early human material, studies of its development have concentrated on fetal rather than embryonic stages. The availability of embryonic specimens in the Walmsley Collection has enabled us to study the development of the vomeronasal organ (VNO) in human embryos between Carnegie Stages 17 and 23. Embryos at Carnegie Stage 17 or below showed no evidence of a VNO. One embryo with characteristics intermediate between Carnegie stages 17 and 18 was the earliest to show evidence of a VNO, in the form of a shallow indentation. All embryos at Carnegie Stages 18 or later had VNOs. Three-dimensional computer reconstructions were made of the VNO in each specimen where this was possible. This in part depended on the plane of section. The total volume and lumen volume were measured from these reconstructions and the volume of the vomeronasal epithelium was calculated by subtraction. A generally consistent increase in total volume and epithelial volume was observed with increasing developmental stage. The lumen contributed rather little to the total volume at these stages.     

Structure and vascularization of the ventricular myocardium in Holocephali: their evolutionary significance

It was generally assumed that the ventricle of the primitive vertebrate heart was composed of trabeculated, or spongy, myocardium, supplied by oxygen‐poor luminal blood. In addition, it was presumed that the mixed ventricular myocardium, consisting of a compacta and a spongiosa, and its supply through coronary arteries appeared several times throughout fish evolution. Recent work has suggested, however, that a fully vascularized, mixed myocardium may be the primitive condition in gnathostomes. The present study of the heart ventricles of four holocephalan species aimed to clarify this controversy. Our observations showed that the ventricular myocardium of Chimaera monstrosa and Harriotta raleighana consists of a very thin compacta overlying a widespread spongiosa. The ventricle of Hydrolagus affinis is composed exclusively of trabeculated myocardium. In these three species there is a well‐developed coronary artery system. The main coronary artery trunks run along the outflow tract, giving off subepicardial ventricular arteries. The trabeculae of the spongiosa are irrigated by branches of the subepicardial arteries and by penetrating arterial vessels arising directly from the main coronary trunks at the level of the conoventricular junction. The ventricle of Rhinochimaera atlantica has only spongy myocardium supplied by luminal blood. Small coronary arterial vessels are present in the subepicardium, but they do not enter the myocardial trabeculae. The present findings show for the first time that in a wild living vertebrate species, specifically H. affinis, an extensive coronary artery system supplying the whole cardiac ventricle exists in the absence of a well‐developed compact ventricular myocardium. This is consistent with the notion derived from experimental work that myocardial cell proliferation and coronary vascular growth rely on distinct developmental programs. Our observations, together with data in the literature on elasmobranchs, support the view that the mixed ventricular myocardium is primitive for chondrichthyans. The reduction or even lack of compacta in holocephali has to be regarded as a derived anatomical trait. Our findings also fit in with the view that the mixed myocardium was the primitive condition in gnathostomes, and that the absence of compact ventricular myocardium in different actinopterygian groups is the result of a repeated loss of such type of cardiac muscle during fish evolution.     

Evidence for the expression of neonatal skeletal myosin heavy chain in primary myocardium and cardiac conduction tissue in the developing chick heart.

We isolated a neonatal skeletal myosin heavy chain (MHC) cDNA clone, CV11E1, from a cDNA library of embryonic chick ventricle. At early cardiogenesis, diffuse expression of neonatal skeletal MHC mRNA was first detected in the heart tube at stage 10. During subsequent embryonic stages, the expression of the mRNA in the atrium was upregulated until shortly after birth. It then diminished, dramatically, and disappeared in the adult. On the other hand, in the ventricle, only a trace of the expression was detected throughout embryonic life and in the adult. However, transient expression of mRNA in the ventricle was observed, post-hatching. At the protein level, during the embryonic stage, the atrial myocardium was stained diffusely with monoclonal antibody 2E9, specific for chick neonatal skeletal MHC, whereas the ventricles showed weak reactivity with 2E9. At the late embryonic and newly hatched stages, 2E9-positive cells were located clearly in the subendocardial layer, and around the blood vessels of the atrial and ventricular myocardium. These results provide the first evidence that the neonatal skeletal MHC gene is expressed in developing chick hearts. This MHC appears during early cardiogenesis and is then localized in cardiac conduction cells. Dev Dyn 2000;217:37-49.     

Quantitative volumetric analysis of cardiac morphogenesis assessed through micro-computed tomography.

We present a method to generate quantitative embryonic cardiovascular volumes at extremely high resolution without tissue shrinkage using micro-computed tomography (Micro-CT). A CT dense polymer (Microfil, Flow Tech, Inc.) was used to perfuse avian embryonic hearts from Hamburger and Hamilton stage (HH) 15 through HH36, which solidified to create a cast within the luminal space. Hearts were then scanned at 10.5 mum(3) voxel resolution using a VivaCT scanner, digital slices were contoured for regions of interest, and computational analysis was conducted to quantify morphogenetic parameters. The three-dimensional morphology was compared with that of scanning electron microscopy (SEM) images and serial section reconstruction of similarly staged hearts. We report that Microfil-perfused hearts swelled to maximum end-diastolic volume with negligible shrinking after polymerization. Comparison to SEM revealed good agreement of cardiac chamber proportions and intracardiac tissue structures (i.e., valves and septa) at the stages of development assessed. Quantification of changes in chamber volume over development revealed several notable results that confirm earlier hypotheses. Heart chamber volumes grow over two orders of magnitude during the 1-week developmental period analyzed. The atrioventricular canal comprised a significant proportion of the early heart volume. While left atrium/left ventricular volume ratios approached 1 in later development, right atrium/right ventricle ratios increase to over 2.5. Quantification of trabeculation patterns confirmed that the right and left ventricles are similarly trabeculated before HH27, after which the right ventricle became quantitatively coarser than that of the left ventricle. These results demonstrate that Micro-CT can be used to image and quantify cardiovascular structures during development.     

Remodeling of chick embryonic ventricular myoarchitecture under experimentally changed loading conditions

Adult myocardium adapts to changing functional demands by hyper‐ or hypotrophy while the developing heart reacts by hyper‐ or hypoplasia. How embryonic myocardial architecture adjusts to experimentally altered loading is not known. We subjected the chick embryonic hearts to mechanically altered loading to study its influence upon ventricular myoarchitecture. Chick embryonic hearts were subjected to conotruncal banding (increased afterload model), or left atrial ligation or clipping, creating a combined model of increased preload in right ventricle and decreased preload in left ventricle. Modifications of myocardial architecture were studied by scanning electron microscopy and histology with morphometry. In the conotruncal banded group, there was a mild to moderate ventricular dilatation, thickening of the compact myocardium and trabeculae, and spiraling of trabecular course in the left ventricle. Right atrioventricular valve morphology was altered from normal muscular flap towards a bicuspid structure. Left atrial ligation or clipping resulted in hypoplasia of the left heart structures with compensatory overdevelopment on the right side. Hypoplastic left ventricle had decreased myocardial volume and showed accelerated trabecular compaction. Increased volume load in the right ventricle was compensated primarily by chamber dilatation with altered trabecular pattern, and by trabecular proliferation and thickening of the compact myocardium at the later stages. A ventricular septal defect was noted in all conotruncal banded, and 25% of left atrial ligated hearts. Increasing pressure load is a main stimulus for embryonic myocardial growth, while increased volume load is compensated primarily by dilatation. Adequate loading is important for normal cardiac morphogenesis and the development of typical myocardial patterns. Anat Rec 254:238–252, 1999. © 1999 Wiley‐Liss, Inc.     

Conus arteriosus of the teleost heart: dismissed, but not missed

The heart outflow tract (OFT) of primitive fish is formed by two portions: a proximal conus arteriosus and a distal bulbus arteriosus. The OFT of modern teleosts is considered to be formed by a single component, the bulbus, the conus having been lost through evolution. This article challenges the concept of the disappearance of the conus arteriosus in the teleost heart. A total of 28 teleost species belonging to 19 families and 10 orders were analyzed. The hearts were divided into two large groups: those having entirely trabeculated ventricles, and those possessing a compacta. In the hearts having entirely trabeculated ventricles, the conus arteriosus appears as a distinct segment interposed between the ventricle and the bulbus arteriosus, being formed by compact vascularized myocardium. However, the conus of several species lacks vessels. In these cases, the conus presents large intercellular spaces bounded by collagen. In the hearts possessing a ventricular compacta, the conus either appears as a muscular ring of variable length connecting the ventricle and the bulbus or forms a crown or ring of myocardium apposed to the ventricular base. In all the teleosts studied, the conus can be recognized as an anatomic entity different from the ventricle. Furthermore, the conus appears as a distinct heart segment in the developing fish. Therefore, the conus arteriosus has not been lost in evolution and constitutes a fundamental part of the teleost OFT. In all the species studied, the conus supports the OFT valves, which should properly be named conus valves.     

Ventricular myocardial architecture in marine fishes

The fiber architecture of the ventricular myocardium has been studied in elasmobranch (Isurus oxyrhinchus, Galeorhinus galeus, Prionace glauca) and teleost (Xiphias gladius, Thunnus thynnus, Thunnus alalunga) fish species with hearts displaying mixed types of ventricular musculature (compact and trabecular). In all cases, the compact myocardium is organized in layers of fiber bundles with an orderly arrangement within the ventricular walls. The number of these layers appears to be dependent on the relative thickness of the compact myocardium. Differences in the pattern of myocardial fiber arrangement were observed among the different fish species. In elasmobranchs the compact myocardium at the level of the atrioventricular orifice is continuous with the trabeculated myocardium. Furthermore, in elasmobranchs the trabeculated myocardium displays a precise arrangement in arcuate trabeculae running from the auriculoventricular to the conoventricular orifices. In teleosts, the compact myocardium is independent of the trabeculated myocardium and a large number of fibers insert into the bulboventricular fibrous ring. The trabeculated myocardium in these species displays an anarchic arrangement except at the level of the bulboventricular orifice, where the fibers tend to be aligned longitudinally, also being inserted into the fibrous ring. Minor differences, consisting mainly of the presence of extra bundles of fibers, were also observed among different individuals of the same species. The possible relationship between myocardial fiber architecture and ventricular shape is discussed.     

Higher spatial resolution improves the interpretation of the extent of ventricular trabeculation

The ventricular walls of the human heart comprise an outer compact layer and an inner trabecular layer. In the context of an increased pre‐test probability, diagnosis left ventricular noncompaction cardiomyopathy is given when the left ventricle is excessively trabeculated in volume (trabecular vol >25% of total LV wall volume) or thickness (trabecular/compact (T/C) >2.3). Here, we investigated whether higher spatial resolution affects the detection of trabeculation and thus the assessment of normal and excessively trabeculated wall morphology. First, we screened left ventricles in 1112 post‐natal autopsy hearts. We identified five excessively trabeculated hearts and this low prevalence of excessive trabeculation is in agreement with pathology reports but contrasts the prevalence of approximately 10% of the population found by in vivo non‐invasive imaging. Using macroscopy, histology and low‐ and high‐resolution MRI, the five excessively trabeculated hearts were compared with six normal hearts and seven abnormally trabeculated and excessive trabeculation‐negative hearts. Some abnormally trabeculated hearts could be considered excessively trabeculated macroscopically because of a trabecular outflow or an excessive number of trabeculations, but they were excessive trabeculation‐negative when assessed with MRI‐based measurements (T/C <2.3 and vol <25%). The number of detected trabeculations and T/C ratio were positively correlated with higher spatial resolution. Using measurements on high resolution MRI and with histological validation, we could not replicate the correlation between trabeculations of the left and right ventricle that has been previously reported. In conclusion, higher spatial resolution may affect the sensitivity of diagnostic measurements and in addition could allow for novel measurements such as counting of trabeculations.     

Ventricular system and choroid plexuses of the human brain during the embryonic period proper

This morphological study, based on serial sections and graphic reconstructions at 4–8 postovulatory weeks (stages 11–23), is believed to be the first account of the ventricular system in staged human embryos. Closure of the caudal neuropore at stage 12 heralds the onset of the ventricular system and separates the ependymal from the amniotic fluid. After the appearance of the optic ventricle at stage 11, the cavity of the telencephalon medium is discernible at stage 13. At stage 14 the future cerebral hemispheres and lateral ventricles begin, and the rhomboid fossa becomes apparent. The medial and lateral ventricular eminences cause indentations in the lateral ventricle by stage 15. The hypothalamic sulcus is evident at stage 16. At stages 17–18 the interventricular foramina are becoming relatively smaller, and cellular accumulations indicate the future choroid villi of the fourth and lateral ventricies. The areae membranaceae rostralis and caudalis are visible in the roof of the fourth ventricle at state 18, and the paraphysis is appearing. At stage 19 choroid villi are seen in the fourth ventricle, and a mesencephalic evagination (Blindsack) is detectable. Choroid villi are noticeable in the lateral ventricle at state 20. An olfactory ventricle is present by stage 21. At about stages 21–23 the lateral ventricle has become C -shaped, so that anterior and inferior horns are visible. Several recesses, e.g., the optic, infundibular, and pineal, develop in the third ventricle during the embryonic period. Features of the ventricular system that do not become apparent until the fetal period include the posterior horn of the lateral ventricle, choroid plexus of the third ventricle, suprapineal recess, interthalamic adhesion, aqueduct, and apertures in the roof of the fourth ventricle.     

Development of the atrioventricular valve region in the human embryo

Severe cardiac malformations may involve the atrioventricular valve region, but the sequence of embryonic development of this important area has been little studied. In particular, the basis of atrioventricular muscular discontinuity, except at the conduction system, has remained unexplained. To examine this question, serial histologic sections of human embryos from the Carnegie Embryological Collection and from the Hopkins Pathology Collection were studied and six embryos were reconstructed. The atrioventricular sulcus can be identified in Carnegie stage 10 as an indentation or crease on the right side separating the heart tube from the umbilical vein. By stage 12 the sulcus has deepened and rotated anteriorly as the atria appear and the heart tube elongates rapidly within the confining pericardial space. Selective accumulation of cardiac jelly on the endocardial aspect of the constriction of the heart tube produced by the atrioventricular sulcus is pronounced by stage 14. By stage 16 the separation of the atrioventricular orifice into right and left components is well advanced, and by stage 18 the septation of the atria and ventricles is largely complete. The muscular connection between the atria and the ventricles becomes interrupted around most of the artioventricular sulcus, except for the His bundle, during the latter part of the embryonic period. The topography of the original sulcus assumes a catenoidal or saddle-shaped configuration, i.e., convex in one plane and concave in the perpendicular plane. The tension and pressure relationships in such a structure would favor cardiac jelly accumulation and the eventual disintegration of lines of myocyte connections passing across the groove. The preservation of the His bundle connection is explained by the failure of the sulcus to completely encircle the heart.     

Muscle Fiber Orientation in the Development and Regression of Right Ventricular Hypertrophy in Pigs

The development and regression of right ventricular hypertrophy was investigated in 12 pigs with special reference to changes in ventricular function and myocardial fiber orientation. Nine ventricles were pressure -loaded by banding the pulmonary artery for 28–81 days, and four of them were then released from the load by removing the band. Right ventricular systolic pressure (RVSP), end-diastolic pressure (RVEDP) and end systolic volume index (ESVI) increased significantly during banding and decreased after debanding. End diastolic volume index (EDVI) and stroke volume index (SVI) showed no significant change during banding and after debanding. The weight of the right ventricle relative to both ventricles (RV/TV) and the thickness of muscle fibers were increased significantly in the loaded ventricles, and reduced again to the control level in ventricles released from the load. The intramyocardial distribution of angles ( θ ) of inclination of muscle fibers from the transverse plane of the outflow tract was estimated histometrically. There was a significantly larger proportion of circularly oriented fibers (|θ|≦30) in the pressure loaded ventricles than in the control, whereas these fibers decreased again to the control level after removal of the pressure load. The present findings indicates that 1) the right ventricular hypertrophy induced by pressure loading is characterized not only by an increase in ventricular weight and muscle fiber thickness, but also by a change in intramyocardial fiber orientation, and 2) the hypertrophic right ventricle can regress both functionally and morphologically to a normal state after removal of the pressure load.     

Muscle fiber orientation in the development and regression of right ventricular hypertrophy in pigs

The development and regression of right ventricular hypertrophy was investigated in 12 pigs with special reference to changes in ventricular function and myocardial fiber orientation. Nine ventricles were pressure-loaded by banding the pulmonary artery for 28-81 days, and four of them were then released from the load by removing the band. Right ventricular systolic pressure (RVSP), end-diastolic pressure (RVEDP) and end-systolic volume index (ESVI) increased significantly during banding and decreased after debanding. End-diastolic volume index (EDVI) and stroke volume index (SVI) showed no significant change during banding and after debanding. The weight of the right ventricle relative to both ventricles (RV/TV) and the thickness of muscle fibers were increased significantly in the loaded ventricles, and reduced again to the control level in ventricles released from the load. The intramyocardial distribution of angles (theta) of inclination of muscle fibers from the transverse plane of the outflow tract was estimated histometrically. There was a significantly larger proporation of circularly oriented fibers (magnitue of theta less than or equal to 30 degrees) in the pressure-loaded ventricles than in the control, whereas these fibers decreased again to the control level after removal of the pressure load. The present findings indicates that 1) the right ventricular hypertrophy induced by pressure loading is characterized not only by an increase in ventricular weight and muscle fiber thickness, but also by a change in intramyocardial fiber orientation, and 2) the hypertrophic right ventricle can regress both functionally and morphologically to a normal state after removal of the pressure load.     

大鼠胚心室肌心房肽样物质定位的研究

本文用免疫组织化学方法研究了大鼠胚(13～19天)心房肽样免疫反应物质在心室肌细胞的定位,并在电镜下观察了大鼠胚心室肌细胞的超微结构。所得结果表明,心房肽类物质不仅存在于心房肌,亦在心室肌细胞内。含心房肽样免疫反应物质的细胞主要位于左心室,尤其是靠内膜侧的小梁状或网格状结构内。左右心室壁内这种细胞很少。心室肌细胞内所含心房肽样免疫反应颗粒的数量较同期心房肌细胞少。在电子显微镜下,见部分心室肌细胞含有与心房特殊颗粒(含心房肽类物质)相似的电子致密颗粒,它们大多靠近高尔基复合体。部分细胞不含这种颗粒,前者可能为心房肽样免疫反应阳性细胞,后者可能为阴性细胞。     

Morphogenesis of the human excretory lacrimal system

The aim of this study was to determine the principal developmental stages in the formation of the excretory lacrimal system in humans and to establish its morphogenetic period. The study was performed using light microscopy on serial sections of 51 human specimens: 33 embryos and 18 fetuses ranging from 8 to 137 mm crown-rump length (CR; 5-16 weeks of development). Three stages were identified in the morphogenesis of the excretory lacrimal system: (1) the formative stage of the lacrimal lamina (Carnegie stages 16-18); (2) the formative stage of the lacrimal cord (Carnegie stages 19-23); and (3) the maturative stage of the excretory lacrimal system, from the 9th week of development onward. A three-dimensional reconstruction of the excretory lacrimal system was performed from serial sections of an embryo at the end of the embryonic period (27 mm CR).     

Spatial distribution of "tissue-specific" antigens in the developing human heart and skeletal muscle. III. An immunohistochemical analysis of the distribution of the neural tissue antigen G1N2 in the embryonic heart; implications for the development of the atrioventricular conduction system.

A monoclonal antibody raised against an extract from the Ganglion Nodosum of the chick and designated G1N2 proves to bind specifically to a subpopulation of cardiomyocytes in the embryonic human heart. In the youngest stage examined (Carnegie stage 14, i.e., 4 1/2 weeks of development) these G1N2-expressing cells are localized in the myocardium that surrounds the foramen between the embryonic left and right ventricle. In the lesser curvature of the cardiac loop this "primary" ring occupies the lower part of the wall of the atrioventricular canal. During subsequent development, G1N2-expressing cells continue to identify the entrance to the right ventricle, but the shape of the ring changes as a result of the tissue remodelling that underlies cardiac septation. During the initial phases of this process the staining remains recognizable as a continuous band of cells in the myocardium that surrounds the developing right portion of the atrioventricular canal, subendocardially in the developing interventricular septum and around the junction of the embryonic left ventricle with the subaortic portion of the outflow tract. During the later stages of cardiac septation, the latter part of the ring discontinues to express G1N2, while upon the completion of septation, no G1N2-expressing cardiomyocytes can be detected anymore. The topographic distribution pattern of G1N suggests that the definitive ventricular conduction system derives from a ring of cells that initially surrounds the "primary" interventricular foramen. The results indicate that the atrioventricular bundle and bundle branches develop from G1N2-expressing myocytes in the interventricular septum, while the "compact" atrioventricular node develops at the junction of the band of G1N2-positive cells in the right atrioventricular junction (the right atrioventricular ring bundle) and the ("penetrating") atrioventricular bundle. A "dead-end tract" represents remnants of conductive tissue in the anterior part of the top of the interventricular septum. The location of the various components of the avian conduction system is topographically homologous with that of the G1N2-ring in the human embryonic heart, indicating a phylogenetically conserved origin of the conduction system in vertebrates.     

Spatial distribution of "tissue-specific" antigens in the developing human heart and skeletal muscle. II. An immunohistochemical analysis of myosin heavy chain isoform expression patterns in the embryonic heart

The spatial distribution of alpha- and beta-myosin heavy chain isoforms (MHCs) was investigated immunohistochemically in the embryonic human heart between the 4th and the 8th week of development. The development of the overall MHC isoform expression pattern can be outlined as follows: (1) In all stages examined, beta-MHC is the predominant isoform in the ventricles and outflow tract (OFT), while alpha-MHC is the main isoform in the atria. In addition, alpha-MHC is also expressed in the ventricles at stage 14 and in the OFT from stage 14 to stage 19. This expression pattern is very reminiscent of that found in chicken and rat. (2) In the early embryonic stages the entire atrioventricular canal (AVC) wall expresses alpha-MHC whereas only the lower part expresses beta-MHC. The separation of atria and ventricles by the fibrous annulus takes place at the ventricular margin of the AVC wall. Hence, the beta-MHC expressing part of the AVC wall, including the right atrioventricular ring bundle, is eventually incorporated in the atria. (3) In the late embryonic stages (approx. 8 weeks of development) areas of alpha-MHC reappear in the ventricular myocardium, in particular in the subendocardial region at the top of the interventricular septum. These coexpressing cells are topographically related to the developing ventricular conduction system. (4) In the sinoatrial junction of all hearts examined alpha- and beta-MHC coexpressing cells are observed. In the older stages these cells are characteristically localized at the periphery of the SA node.     

Myocardial heterogeneity in permissiveness for epicardium-derived cells and endothelial precursor cells along the developing heart tube at the onset of coronary vascularization

The coronary vasculature develops from mesothelial and endothelial precursor cells (EPCs) derived from the proepicardial organ (PEO), which migrate over the heart to form the epicardium. By epithelial-mesenchymal transition (EMT), the subepicardium and epicardium-derived cells (EPDCs) are formed. EPDCs migrate into the myocardium, where they differentiate into smooth muscle cells and fibroblasts that stabilize the developing coronary vasculature and contribute to myocardial architecture. Complete PEO ablation results in embryonic lethality due to cardiac defects, including a looping disorder with a too wide inner curvature. To investigate the behavior of early coronary contributors, we analyzed normal quail embryos and found lumenized endothelial vessels in the subepicardium already at stage HH19. Furthermore, EPCs had penetrated into the myocardium of the inner curvature. To confirm that the myocardium of the inner curvature is specifically permissive for EPCs and to study early EPDC migration in more detail, chimeric chicken embryos harboring a quail PEO were analyzed. Lateral epicardial outgrowth and EMT were observed throughout, but migration into the myocardium was restricted to the inner curvature between HH19 and 22. The permissive myocardial area expanded to the atrium, atrioventricular canal, and trabeculated ventricle at stage HH23-24. In contrast, outflow tract myocardium was never found to be permissive for EPDCs and EPCs until HH30, not even when the quail PEO was attached directly onto it. We conclude that early coronary formation starts in the inner curvature and hypothesize that the presence of PEO-derived cells is essential for the maturation of the inner curvature and subsequent looping of the heart tube.