Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates.

BACKGROUND: Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are 2 distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. METHODS: To understand how intramyocardial coronary arteries are generated to connect these 2 coronary vascular populations, we combined genetic lineage tracing, light sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. RESULTS: We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 (methyltransferase-like protein 3) and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. CONCLUSIONS: A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.

The transcription factor Sox7 modulates endocardiac cushion formation contributed to atrioventricular septal defect through Wnt4/Bmp2 signaling.

Cardiac septum malformations account for the largest proportion in congenital heart defects. The transcription factor Sox7 has critical functions in the vascular development and angiogenesis. It is unclear whether Sox7 also contributes to cardiac septation development. We identified a de novo 8p23.1 deletion with Sox7 haploinsufficiency in an atrioventricular septal defect (AVSD) patient using whole exome sequencing in 100 AVSD patients. Then, multiple Sox7 conditional loss-of-function mice models were generated to explore the role of Sox7 in atrioventricular cushion development. Sox7 deficiency mice embryos exhibited partial AVSD and impaired endothelial to mesenchymal transition (EndMT). Transcriptome analysis revealed BMP signaling pathway was significantly downregulated in Sox7 deficiency atrioventricular cushions. Mechanistically, Sox7 deficiency reduced the expressions of Bmp2 in atrioventricular canal myocardium and Wnt4 in endocardium, and Sox7 binds to Wnt4 and Bmp2 directly. Furthermore, WNT4 or BMP2 protein could partially rescue the impaired EndMT process caused by Sox7 deficiency, and inhibition of BMP2 by Noggin could attenuate the effect of WNT4 protein. In summary, our findings identify Sox7 as a novel AVSD pathogenic candidate gene, and it can regulate the EndMT involved in atrioventricular cushion morphogenesis through Wnt4-Bmp2 signaling. This study contributes new strategies to the diagnosis and treatment of congenital heart defects.

Cardiogenesis with a focus on vasculogenesis and angiogenesis.

The initial intraembryonic vasculogenesis occurs in the cardiogenic mesoderm. Here, a cell population of proendocardial cells detaches from the mesoderm that subsequently generates the single endocardial tube by forming vascular plexuses. In the course of embryogenesis, the endocardium retains vasculogenic, angiogenic and haematopoietic potential. The coronary blood vessels that sustain the rapidly expanding myocardium develop in the course of the formation of the cardiac loop by vasculogenesis and angiogenesis from progenitor cells of the proepicardial serosa at the venous pole of the heart as well as from the endocardium and endothelial cells of the sinus venosus. Prospective coronary endothelial cells and progenitor cells of the coronary blood vessel walls (smooth muscle cells, perivascular cells) originate from different cell populations that are in close spatial as well as regulatory connection with each other. Vasculo- and angiogenesis of the coronary blood vessels are for a large part regulated by the epicardium and epicardium-derived cells. Vasculogenic and angiogenic signalling pathways include the vascular endothelial growth factors, the angiopoietins and the fibroblast growth factors and their receptors.

Identification of Human Very Small Embryonic like Stem Cells (VSELS) in Human Heart Tissue Among Young and Old Individuals.

Very Small Embryonic-Like (VSEL) stem cells are a proposed pluripotent population, residing in adult tissues. VSELs have been described in multiple tissues including bone marrow, cord blood, and gonads. They exhibit multiple characteristics of embryonic stem cells including the ability to differentiate into cellular lineages of all three germ layers, including cardiomyocytes and vascular endothelial cells. However, their presence in adult solid organs such as heart in humans has not been established. VSELs are valuable source of stem cells for tissue regeneration and replacement of cells for turnover and usual wear-and-tear. The purpose of our study was to explore the existence of human VSELs (huVSELs) in human heart tissue and examine the changes in their prevalence with aging and cardiac disease. Human heart tissue, collected from healthy and ischemic heart disease subjects was examined for the prevalence of VSELS, defined as CD45-/CD133+/SSEA4+. Both epicardial and endocardial tissues were examined comparing VSEL numbers across different age groups. Our data confirm the existence of huVSELs in adult hearts with decreasing prevalence during aging. This is the first evidence of huVSELs in adult cardiac tissue. Cardiac huVSELs could be further explored in future studies to characterize their primitive potential and therapeutic potential in regenerative studies.

Biomechanical signaling within the developing zebrafish heart attunes endocardial growth to myocardial chamber dimensions.

Intra-organ communication guides morphogenetic processes that are essential for an organ to carry out complex physiological functions. In the heart, the growth of the myocardium is tightly coupled to that of the endocardium, a specialized endothelial tissue that lines its interior. Several molecular pathways have been implicated in the communication between these tissues including secreted factors, components of the extracellular matrix, or proteins involved in cell-cell communication. Yet, it is unknown how the growth of the endocardium is coordinated with that of the myocardium. Here, we show that an increased expansion of the myocardial atrial chamber volume generates higher junctional forces within endocardial cells. This leads to biomechanical signaling involving VE-cadherin, triggering nuclear localization of the Hippo pathway transcriptional regulator Yap1 and endocardial proliferation. Our work suggests that the growth of the endocardium results from myocardial chamber volume expansion and ends when the tension on the tissue is relaxed.

Tie2 regulates endocardial sprouting and myocardial trabeculation.

The ang1-Tie2 pathway is required for normal vascular development, but its molecular effectors are not well-defined during cardiac ontogeny. Here we show that endocardial specific attenuation of Tie2 results in mid-gestation lethality due to heart defects associated with a hyperplastic but simplified trabecular meshwork (fewer but thicker trabeculae). Reduced proliferation and production of endocardial cells (ECs) following endocardial loss of Tie2 results in decreased endocardial sprouting required for trabecular assembly and extension. The hyperplastic trabeculae result from enhanced proliferation of trabecular cardiomyocyte (CMs), which is associated with upregulation of Bmp10, increased retinoic acid (RA) signaling, and Erk1/2 hyperphosphorylation in the myocardium. Intriguingly, myocardial phenotypes in Tie2-cko hearts could be partially rescued by inhibiting in utero RA signaling with pan-retinoic acid receptor antagonist BMS493. These findings reveal two complimentary functions of endocardial Tie2 during ventricular chamber formation: ensuring normal trabeculation by supporting EC proliferation and sprouting, and preventing hypertrabeculation via suppression of RA signaling in trabecular CMs.

Endocardial Hippo signaling regulates myocardial growth and cardiogenesis.

The Hippo signaling pathway has been implicated in control of cell and organ size, proliferation, and endothelial-mesenchymal transformation. This pathway impacts upon two partially redundant transcription cofactors, Yap and Taz, that interact with other factors, including members of the Tead family, to affect expression of downstream genes. Yap and Taz have been shown to regulate, in a cell-autonomous manner, myocardial proliferation, myocardial hypertrophy, regenerative potential, and overall size of the heart. Here, we show that Yap and Taz also play an instructive, non-cell-autonomous role in the endocardium of the developing heart to regulate myocardial growth through release of the paracrine factor, neuregulin. Without endocardial Yap and Taz, myocardial growth is impaired causing early post-natal lethality. Thus, the Hippo signaling pathway regulates cell size via both cell-autonomous and non-cell-autonomous mechanisms. Furthermore, these data suggest that Hippo may regulate organ size via a sensing and paracrine function in endothelial cells.

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo.

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

Endocardial Cell Plasticity in Cardiac Development, Diseases and Regeneration.

Endocardial cells are specialized endothelial cells that form the innermost layer of the heart wall. By virtue of genetic lineage-tracing technology, many of the unexpected roles of endocardium during murine heart development, diseases, and regeneration have been identified recently. In addition to heart valves developed from the well-known endothelial to mesenchymal transition, recent fate-mapping studies using mouse models reveal that multiple cardiac cell lineages are also originated from the endocardium. This review focuses on a variety of different cell types that are recently reported to be endocardium derived during murine heart development, diseases, and regeneration. These multiple cell fates underpin the unprecedented roles of endocardial progenitors in function, pathological progression, and regeneration of the heart. Because emerging studies suggest that developmental mechanisms can be redeployed and recapitulated in promoting heart disease development and also cardiac repair and regeneration, understanding the mechanistic regulation of endocardial plasticity and modulation of their cell fate conversion may uncover new therapeutic potential in facilitating heart regeneration.

BMP-SMAD signalling output is highly regionalized in cardiovascular and lymphatic endothelial networks.

BACKGROUND: Bone morphogenetic protein (BMP) signalling has emerged as a fundamental pathway in endothelial cell biology and deregulation of this pathway is implicated in several vascular disorders. BMP signalling output in endothelial cells is highly context- and dose-dependent. Phosphorylation of the BMP intracellular effectors, SMAD1/5/9, is routinely used to monitor BMP signalling activity. To better understand the in vivo context-dependency of BMP-SMAD signalling, we investigated differences in BMP-SMAD transcriptional activity in different vascular beds during mouse embryonic and postnatal stages. For this, we used the BRE::gfp BMP signalling reporter mouse in which the BMP response element (BRE) from the ID1-promotor, a SMAD1/5/9 target gene, drives the expression of GFP. RESULTS: A mosaic pattern of GFP was present in various angiogenic sprouting plexuses and in endocardium of cardiac cushions and trabeculae in the heart. High calibre veins seemed to be more BRE::gfp transcriptionally active than arteries, and ubiquitous activity was present in embryonic lymphatic vasculature. Postnatal lymphatic vessels showed however only discrete micro-domains of transcriptional activity. Dynamic shifts in transcriptional activity were also observed in the endocardium of the developing heart, with a general decrease in activity over time. Surprisingly, proliferative endothelial cells were almost never GFP-positive. Patches of transcriptional activity seemed to correlate with vasculature undergoing hemodynamic alterations. CONCLUSION: The BRE::gfp mouse allows to investigate selective context-dependent aspects of BMP-SMAD signalling. Our data reveals the highly dynamic nature of BMP-SMAD mediated transcriptional regulation in time and space throughout the vascular tree, supporting that BMP-SMAD signalling can be a source of phenotypic diversity in some, but not all, healthy endothelium. This knowledge can provide insight in vascular bed or organ-specific diseases and phenotypic heterogeneity within an endothelial cell population.

The roadmap of WT1 protein expression in the human fetal heart.

The transcription factor Wilms' Tumor-1 (WT1) is essential for cardiac development. Deletion of Wt1 in mice results in disturbed epicardial and myocardial formation and lack of cardiac vasculature, causing embryonic lethality. Little is known about the role of WT1 in the human fetal heart. Therefore, as a first step, we analyzed the expression pattern of WT1 protein during human cardiac development from week 4 till week 20. WT1 expression was apparent in epicardial, endothelial and endocardial cells in a spatiotemporal manner. The expression of WT1 follows a pattern starting at the epicardium and extending towards the lumen of the heart, with differences in timing and expression levels between the atria and ventricles. The expression of WT1 in cardiac arterial endothelial cells reduces in time, whereas WT1 expression in the endothelial cells of cardiac veins and capillaries remains present at all stages studied. This study provides for the first time a detailed description of the expression of WT1 protein during human cardiac development, which indicates an important role for WT1 also in human cardiogenesis.

Endocardial Notch Signaling in Cardiac Development and Disease.

The Notch signaling pathway is an ancient and highly conserved signaling pathway that controls cell fate specification and tissue patterning in the embryo and in the adult. Region-specific endocardial Notch activity regulates heart morphogenesis through the interaction with multiple myocardial-, epicardial-, and neural crest-derived signals. Mutations in NOTCH signaling elements cause congenital heart disease in humans and mice, demonstrating its essential role in cardiac development. Studies in model systems have provided mechanistic understanding of Notch function in cardiac development, congenital heart disease, and heart regeneration. Notch patterns the embryonic endocardium into prospective territories for valve and chamber formation, and later regulates the signaling processes leading to outflow tract and valve morphogenesis and ventricular trabeculae compaction. Alterations in NOTCH signaling in the endocardium result in congenital structural malformations that can lead to disease in the neonate and adult heart.

Genetic interaction between pku300 and fbn2b controls endocardial cell proliferation and valve development in zebrafish.

Abnormal cardiac valve morphogenesis is a common cause of human congenital heart disease. The molecular mechanisms regulating endocardial cell proliferation and differentiation into cardiac valves remain largely unknown, although great progress has been made on the endocardial contribution to the atrioventricular cushion and valve formation. We found that scotch tape(te382) (sco(te382)) encodes a novel transmembrane protein that is crucial for endocardial cell proliferation and heart valve development. The zebrafish sco(te382) mutant showed diminished endocardial cell proliferation, lack of heart valve leaflets and abnormal common cardinal and caudal veins. Positional cloning revealed a C946T nonsense mutation of a novel gene pku300 in the sco(te382) locus, which encoded a 540-amino-acid protein on cell membranes with one putative transmembrane domain and three IgG domains. A known G3935T missense mutation of fbn2b was also found ∼570 kb away from pku300 in sco(te382) mutants. The genetic mutant sco(pku300), derived from sco(te382), only had the C946T mutation of pku300 and showed reduced numbers of atrial endocardial cells and an abnormal common cardinal vein. Morpholino knockdown of fbn2b led to fewer atrial endocardial cells and an abnormal caudal vein. Knockdown of both pku300 and fbn2b phenocopied these phenotypes in sco(te382) genetic mutants. pku300 transgenic expression in endocardial and endothelial cells, but not myocardial cells, partially rescued the atrial endocardial defects in sco(te382) mutants. Mechanistically, pku300 and fbn2b were required for endocardial cell proliferation, endocardial Notch signaling and the proper formation of endocardial cell adhesion and tight junctions, all of which are crucial for cardiac valve development. We conclude that pku300 and fbn2b represent the few genes capable of regulating endocardial cell proliferation and signaling in zebrafish cardiac valve development.

Development of the endocardium.

The endocardium, the endothelial lining of the heart, plays complex and critical roles in heart development, particularly in the formation of the cardiac valves and septa, the division of the truncus arteriosus into the aortic and pulmonary trunks, the development of Purkinje fibers that form the cardiac conduction system, and the formation of trabecular myocardium. Current data suggest that the endocardium is a regionally specialized endothelium that arises through a process of de novo vasculogenesis from a distinct population of mesodermal cardiogenic precursors in the cardiac crescent. In this article, we review recent developments in the understanding of the embryonic origins of the endocardium. Specifically, we summarize vasculogenesis and specification of endothelial cells from mesodermal precursors, and we review the transcriptional pathways involved in these processes. We discuss the lineage relationships between the endocardium and other endothelial populations and between the endocardium and the myocardium. Finally, we explore unresolved questions about the lineage relationships between the endocardium and the myocardium. One of the central questions involves the timing with which mesodermal cells, which arise in the primitive streak and migrate to the cardiac crescent, become committed to an endocardial fate. Two competing conceptual models of endocardial specification have been proposed. In the first, mesodermal precursor cells in the cardiac crescent are prespecified to become either endocardial or myocardial cells, while in the second, fate plasticity is retained by bipotential cardiogenic cells in the cardiac crescent. We propose a third model that reconciles these two views and suggest future experiments that might resolve this question.

Molecular morphology of the chick heart visualized by MALDI imaging mass spectrometry.

Utilization of MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) for tissue imaging is a relatively new proteomic technique that simultaneously maps the spatial distribution of multiple proteins directly within a single frozen tissue section. Here, we report the development of a methodology to apply MALDI tissue imaging to chick heart tissue sections acquired from fixed and paraffin-embedded samples. This protocol produces molecular images that can be related to the high-quality histological tissue sections. Perfused term chick hearts were fixed in acidic ethanol and embedded in paraffin wax. Tissue sections (15 microm) were collected onto conductive slides, deparaffinized with xylene, and transitioned into water with graded ethanol washes and allowed to air dry. In separate experiments, three different MALDI matrices were applied to chick heart tissue sections through repeated cycles from a glass nebulizer. Tissue sections were then analyzed by MALDI mass spectrometry using a raster step-size of 75-100 microm, and molecular images for specific m/z ratios reconstituted. MALDI tissue imaging revealed spatially resolved protein signals within single heart sections that are specific to structures or regions of the heart, for example, vessels, valves, endocardium, myocardium, or septa. Moreover, no prior knowledge of protein expression is required as is the case for immunohistochemistry and in situ hybridization methodologies. The ability to simultaneously localize a large number of unique protein signals within a single tissue section, with good preservation of histological features, provides cardiovascular researchers a new tool to give insight into the molecular mechanisms underlying normal and pathological conditions.

Prenatal development of the human epicardiac Ganglia.

The aim of this study was to determine the developmental anatomy of intrinsic cardiac ganglia with respect to epicardiac ganglionated nerve plexus in the human fetuses at different gestation stages. Twenty fetal hearts were investigated applying a technique of histochemistry for acetylcholinesterase to visualize the epicardiac neural ganglionated plexus with its subsequent examinations on total (non-sectioned) hearts. Most epicardiac ganglia embodied multilayered neurons and were oval in shape, but some ganglia involved neurons lying in one layer or had the irregular appearance because of their extensions along inter-ganglionic nerves. The mean ganglion area of fetuses at gestation stages of 15-40 weeks was 0.03 +/- 0.008 mm(2). The largest epicardiac ganglia, reaching in area 0.4 mm(2), were concentrated on the dorsal surface of both atria. The particular fused or "dual" ganglia were identified at the gestation stages of 23-40 weeks, but they composed only 2.3 +/- 0.7% of all found epicardiac ganglia. A direct positive correlation was determined between the fetal age and the ganglion area (mm(2)) as well as between the fetal age and the number of inter-ganglionic nerves. The revealed appearance of epicardiac ganglia in the human fetuses at 15-40 weeks of gestation confirms their prenatal development and presumable intrinsic remodelling.

Endocardiogenesis in embryoid bodies: novel markers identified by gene expression profiling.

Endocardial cells and cardiomyocytes differentiate from the cardiogenic mesoderm at about the same time during development. Although in vitro embryonic stem (ES) cell systems have been used to study the differentiation of various types of cell lineages, including cardiomyocytes, smooth muscle cells, and vascular endothelial cells, differentiation of endocardial cells, or endocardiogenesis, has not been well reported, because of a lack of specific molecular markers. In our search for cardiogenesis-associated genes expressed in embryoid bodies, we found several genes expressed in the heart region of mouse embryos, but not in cardiomyocytes. To identify the cell types expressing these genes, CD31(+) cells were taken from mouse embryos on embryonic day (E)8.5 and E9.5 and sorted, then their transcripts were analyzed using quantitative RT-PCR analyses. In those embryos, Gata4 and Nfatc1, as well as newly identified Cgnl1 and Dok4 were found to be preferentially expressed in endocardial cells, but not in yolk sac endothelial cells, while Cdh5 and Kdr were expressed in both cardiac and yolk sac endothelial cells. Immunohistochemical analyses of embryoid bodies revealed that some CD31(+) cells co-expressing Gata4 and Nfatc1 were located in close proximity to cardiomyocytes. These results suggest that embryoid bodies express endocardial specific genes and likely generate endocardial cells along with cardiomyocytes. Further, they indicate that these new marker genes are useful to study the origin and induction of endocardial cells, and identify other endocardial markers.

In vitro re-endothelialization of detergent decellularized heart valves under simulated physiological dynamic conditions.

The production of viable biological heart valves is of central interest in tissue engineering (TE). The aim of this study was to generate decellularized heart valves with an intact ultra-structure and to repopulate these with endothelial cells (EC) under simulated physiological conditions. Decellularization of ovine pulmonary valve conduits was performed under agitation in detergents followed by six wash cycles. Viability of EC cultures exposed to washing solution served to prove efficiency of washing. Resulting scaffolds were free of cells with preserved extracellular matrix. Biomechanical standard tension tests demonstrated comparable parameters to native tissue. Luminal surfaces of decellularized valvular grafts were seeded with ovine jugular vein EC in dynamic bioreactors. After rolling culture for 48 h, pulsatile medium circulation with a flow of 0.1 L/min was started. The flow was incremented 0.3 L/min/day up to 2.0 L/min (cycle rate: 60 beats/min), while pH, pO2, pCO2, lactate and glucose were maintained at constant physiological levels. After 7 days, a monolayer of cells covered the inner valve surface, which expressed vWF, indicating an endothelial origin. A complete endothelialization of detergent decellularized scaffold can be achieved under simulated physiological circulation conditions using a dynamic bioreactor system, which allows continuous control of the culture environment.

Structure of the conus arteriosus of the sturgeon (Acipenser naccarii) heart. I: the conus valves and the subendocardium.

Sturgeons are bony fish that retain structural traits typical of the more primitive Chondrostei. From an evolutionary viewpoint, sturgeons are considered relic fish. However, they show remarkable ecological plasticity and are well adapted to contemporary environmental conditions. Although development of the cardiovascular system is critical for all organs and systems, and is affected by evolutionary changes, the structure of the sturgeon heart has been mostly overlooked. This is also true for the conus arteriosus, which, as in Chondrostei, is endowed with several rows of valves and a layer of contractile myocardium. This work reports on the structure of the valves, the endocardium, and the subendocardium of the conus arteriosus of the sturgeon (Acipenser naccarii) heart. It is part of a broader study that aims to cover the entire structure of the sturgeon heart. The conus arteriosus of 15 A. naccarii hearts, ranging in age from juveniles to sexually-differentiated adults, has been studied by conventional light, transmission (TEM), and scanning electron microscopy (SEM). In addition, maceration of the soft tissues with NaOH, and actin localization by fluorescent phalloidin has been used. The conus is a tubular chamber that arises from the right ventricular side and presents two constrictions at the conus-ventricle and conus-aorta junctions. The conus is endowed with three rows of valves: one distal and two proximal. The segment of the conus located between the distal and the two proximal rows is devoid of valvular structures. The distal row has four leaflets, while the two proximal rows show the greatest variation in leaflet number, size, and shape. All leaflets have collagenous chordae tendineae arising from the free border and from the parietal side of the leaflets. The endocardium is a flat endothelium which shows a thick, irregular basement membrane. The leaflet body is formed by a loose connective tissue which blends with the subendocardium. The subendocardium is a connective tissue consisting of myofibroblasts, collagen, and elastin. It is divided into two distinct areas: one proximal, which shows little elastin and poorly organized collagen; and one distal, which is rich in elastin, with cells and extracellular fibers organized into layers that are oriented in alternative circumferential and longitudinal directions. The present report is the first systematic analysis of the structure of the sturgeon conus. Descriptions of the conus valves should recognize the existence of three valve rows only. The variability in valve morphology, and the loose structure of the leaflet tissue make it unlikely that the valves play an effective role in preventing blood backflow. In this regard, the ventricle-conus constriction may act as a sphincter. The subendocardium is an elastic coat capable of actively sustaining the tissue deformation that accompanies the heart contractile cycle. Further comparative studies are needed to provide deeper insight into the structural changes that accompany phyletic diversification.

Development of the human pulmonary vein and its incorporation in the morphologically left atrium.

OBJECTIVE: Using a newly acquired archive of previously prepared material, we sought to re-examine the origin of the pulmonary vein in the human heart, aiming to determine whether it originates from the systemic venous sinus ("sinus venosus"), or appears as a new structure draining to the left atrium. In addition, we examined the temporal sequence of incorporation of the initially solitary pulmonary vein to the stage at which four venous orifices opened to the left atrium. METHODS: We studied 26 normal human embryos, ranging from 3.8 mm to 112 mm crown-rump length, and representing the period from the 12th Carnegie stage to 15 weeks of gestation. RESULTS: The pulmonary vein canalised as a solitary vessel within the mediastinal tissues so as to connect the intraparenchymal pulmonary venous networks to the heart, using the regressing dorsal mesocardium as its portal of cardiac entry. The vein was always distinct from the tributaries of the embryonic systemic venous sinus. The orifice of the solitary vein became committed to the left atrium by growth of the vestibular spine. During development, a marked disparity was seen between the temporal and morphological patterns of incorporation of the left-sided and right-sided veins into the left atrium. The pattern of the primary bifurcation was asymmetrical, a much longer tributary being formed on the left than on the right. Contact between the atrial wall and the venous tributary on the left initially produced a shelf, which became effaced with incorporation of the two left-sided veins into the atrium. CONCLUSIONS: The initial process of formation of the human pulmonary vein is very similar to that seen in animal models. The walls of the initially solitary vein in humans become incorporated by a morphologically asymmetric process so that four pulmonary veins eventually drain independently into the left atrium. Failure of incorporation on the left side may provide the substrate for congenital division of the left atrium.