The Development of the Atrioventricular Junction in the Human Heart
Abstract The histogenesis of the separation between atrial and ventricular myocardium at the atrioventricular junction in the developing human heart has been investigated immunohistochemically by using monoclonal antibodies specific for atrioventricular cushion tissue, mesenchymal cells, atrial and ventricular myocardium, and myocardium of the primary ring. It was found that the insulation between the muscle masses of atrium and ventricle is established by the fusion of the tissues of the atrioventricular sulcus (located at the epicardial side of the junctional myocardium) with those of the atrioventricular cushions (located at the endocardial side of the junctional myocardium). This process takes place at the ventricular margin of the myocardium of the atrioventricular canal. The separation of atrial and ventricular myocardium starts at ≈7 weeks of development in the anteromedial portion of the right atrioventricular junction and is largely completed around the 12th week of development. The only remaining myocardial continuity between atrial and ventricular myocardium is the atrioventricular axis of conduction. Our findings show that the nonmuscular part of the developing leaflets of the atrioventricular valves derives from the atrioventricular cushions and that the tissues of the atrioventricular groove do not contribute to the development of these leaflets.
One of the intriguing aspects of cardiac development is the manner in which the tubular embryonic myocardium, functioning initially as a fingerpump without one-way valves, eventually transforms into the complex adult four-chambered pump with a well-developed valvar apparatus and fibrous skeleton, capable of supporting two separate blood circulations after birth. In earlier studies, we have demonstrated how the processes responsible for this transformation—such as ventricular septation, the development of the atrioventricular valves, and the development of the atrioventricular conduction system—are closely related to one another.1 2 3 4 5 In this contribution, we focus on the morphological events producing the necessary insulation of the atrial from the ventricular myocardium.
It is well established that during early cardiac development the ordinary atrial myocardium is continuous with myocardium of the ventricles throughout the atrioventricular junction.2 6 7 At these stages, the physical insulation by fibrous tissue is not essential owing to the intrinsic property of slow propagation of the cardiac impulse of the atrioventricular junctional myocardium.8 In the late fetal, neonatal, and adult heart, the only remaining myocardial continuity between atrial and ventricular myocardium is the atrioventricular axis of conduction, comprising the specialized myocardium of the atrioventricular node and bundle.9 10 Myocardial atrioventricular continuities outside this main axis of conduction can occur. These (abnormal) pathways are known as accessory atrioventricular muscular connections.11 12 13 14 15 16 Although it is generally believed that these pathways reflect the incomplete insulation of the atrial from the ventricular myocardium, the histogenesis is only poorly understood. It was anticipated that a good insight into the processes that lead to the normal insulation occurring during development of the atrioventricular junctions would provide a better understanding of the histogenesis of accessory atrioventricular connections.
Agreement exists about the different types of tissues participating in the development of the fibrous atrioventricular junctions. These are the atrial and ventricular myocardium, the myocardium of the atrioventricular canal, the atrioventricular cushion tissue, and the tissue of the atrioventricular groove.7 17 Controversies exist, however, concerning the contributions of the tissues involved and hence concerning the exact development of the atrioventricular insulation. Two different hypotheses have been proposed (Fig 1⇓). In the first, atrioventricular insulation is held to be established by invagination of the atrioventricular sulcus tissue into the myocardium of the atrioventricular canal.17 18 In this hypothesis (Fig 1b⇓), the atrioventricular valves develop as a result of the invagination of the sulcus combined with a process called myocardial undermining. In this model the cushions do not contribute to the formation of the fibrous annulus and play only a minor role in the formation of the leaflets of the atrioventricular valves. In the second hypothesis (Fig 1c⇓), sulcus tissue and cushion tissue both contribute to the establishment of the atrioventricular insulation, with the atrioventricular cushions contributing significantly to the formation of the leaflets of the atrioventricular valves.19 20
In the past, adequate description of the development of cardiac structures has often been hindered by the difficulty of distinguishing the subpopulations of cells that contribute to the formation of these structures (see Reference 21). With regard to the atrioventricular junctions, the development of the fibrous skeleton and the development of the leaflets of the atrioventricular valves have been contentious topics, in part because of different interpretations of serial sections stained with classical histological dyes, such as hematoxylin-azofloxin.17 18 22 Recently, we have identified specific antibodies for the tissues participating in the insulation of the atrial and ventricular muscle masses. In addition, we report here two new monoclonal antibodies that facilitate the study of the developing human heart. Together with the antibodies used in previous studies,1 2 3 these antibodies have allowed us to test the two hypotheses of atrioventricular insulation.
Materials and Methods
Tissue Sources and Preparation
Fifteen human embryos and fetuses (a minimum of two for each stage) from Carnegie Stage 14 onward were studied. Some of the embryos were also used in our studies on the spatial distribution of creatine kinase and myosin heavy chain isoforms1 2 and in our studies on the development of the conduction system.3 4 The embryos and fetuses were obtained from terminations of pregnancy at the Academic Medical Center of Amsterdam and at the Postgraduate Medical School in Budapest. The studies were approved by the respective local medical-ethical committees. The Carnegie Stage of development of the embryos was estimated by comparison of the observed external landmarks and cardiac morphology with literature data.23 No morphological abnormalities were observed in the specimens described. Embryos and isolated hearts from fetuses were fixed at room temperature in a mixture of methanol/acetone/acetic acid/water (35:35:5:25 [per volume]) for a minimum of 2 hours, dehydrated in a graded series of ethanol, cleared in chloroform, and embedded in Paraplast Plus (Monoject). The specimens were cut into 5-μm-thick serial sections and mounted on microscope slides coated with poly-l-lysine.
To detect the binding of the specific monoclonal antibodies with the respective antigens on paraplast sections, the indirect unconjugated PAP technique was applied as described in detail previously.1 2
The production and characterization of anti–α-MHC (249-5A4), anti–β-MHC (169-1D5), and anti-GlN2 have been described in detail in previous papers.2 3 The monoclonal antibody recognizing both the α-MHC and β-MHC isoform (149-23A2) was raised against MHC extracted from atrial myocardium of adult rat heart. Anti–Leu-7 (anti–HNK-1) was purchased from Becton Dickinson and reacts in the human heart identical to anti-GlN2.3 4 Anti–human desmin was purchased from Sanbio. The monoclonal antibodies reacting specifically with the atrioventricular cushion tissue (249-9G9), designated anti–cushion tissue antigen (anti-CTA), and the monoclonal antibody reacting with endothelial and mesenchymal cells (249-5E9), designated anti–mesenchymal cell antigen (anti-MCA), were obtained as byproducts during the procedure in which the anti–α-MHC antibody was produced.2 Because the amount of the antibodies 249-9G9 and 249-5E9 available was limited, extensive biochemical characterization of the antigens unfortunately could not be performed. A single Western blot analysis, using protein extracted from human adult ventricle, indicated that anti-MCA recognizes a protein with an apparent molecular weight of 75 kD. Characteristics of the antibodies used in experiments are detailed in Table 1⇓.
The Embryonic Stages
Carnegie Stages 14 and 15 (31 to 38 Days)
In the youngest specimens investigated (Carnegie Stage 14), α-MHC is expressed in both the embryonic atrial and ventricular myocardium and in the myocardium of the atrioventricular canal (Fig 2a⇓). β-MHC, however, is expressed only in the embryonic ventricle and in the lower part of the wall of the atrioventricular canal (Fig 2b⇓; see also Reference 2). At this stage, the myocardial continuity between atrium and ventricle throughout the atrioventricular junctions is most clearly illustrated by the expression of α-MHC. The lower part of the right portion of the atrioventricular canal is characterized by the expression of GlN2/Leu-7 (Fig 2c⇓).3 4 This part of the wall of the right atrioventricular canal belongs to the “primary ring” of myocardium that surrounds the primary interventricular foramen and will partly develop into atrioventricular conduction tissue3 (see also Fig 3⇓). Most of the myocardium of the wall of the atrioventricular canal, joining the atrial and ventricular muscle masses, is sandwiched between the tissue of the atrioventricular groove encircling the atrioventricular canal completely on the epicardial side and the tissue of the (anterosuperior and posteroinferior) atrioventricular cushions on the endocardial side (Fig 2d⇓). A small area of the internal relief of the atrioventricular canal is not covered with cushion material since, at this stage, the right and left lateral atrioventricular cushions have yet to be formed. Hence, in this area the endocardium is in direct contact with myocardium (Fig 2e⇓ through 2h).
The configuration of the respective structures and tissues at Stage 15 (data not shown) closely resembles that of Stage 14. An important feature of the late Carnegie Stage 15 heart (36 to 38 days)2 3 is the fusion of the posteroinferior and anterosuperior atrioventricular cushions and the appearance of the lateral atrioventricular cushions.
A schematic representation of the structure of the right atrioventricular junction in hearts of Stages 14 and 15 is given in Fig 4a⇓.
Carnegie Stages 17 Through 19 (42 to 51 Days)
Except for the expression of α-MHC, which has become virtually restricted to the atria and the wall of the atrioventricular canal (Fig 5a⇓ and 5b⇓), the hearts examined at Stages 17 through 19 do not show any changes in the expression patterns of the antigens studied when compared with the hearts at Stages 14 and 15. The lower part of the atrioventricular canal characteristically expresses both cardiac MHC isoforms (Fig 5a⇓ through 5c) and, at the right side only, GlN2/Leu-7/HNK-1 (Figs 3⇑ and 5d⇓).The atrioventricular sulcus tissue is still separated from CTA-expressing endocardial cushion tissue (Fig 5f⇓) by atrioventricular junctional myocardium. However, compared with the younger hearts, the shape of the atrioventricular junction has changed. This change in morphology is due to the outward expansion of the ventricular myocardium, leading to the bulging of the “shoulders” of the ventricular free wall (see Fig 4b⇑).
At this stage, the lateral atrioventricular cushions have become prominent mesenchymal structures. The left lateral cushion contributes to the formation of the mural leaflet of the mitral valve, while the right lateral cushion (Fig 5a⇑ through 5f) contributes to the formation of the inferior leaflet of the tricuspid valve.5 The stainings with anti–β-MHC (Fig 5c⇑) and anti-CTA (Fig 5f⇑) clearly demonstrate that, at this stage, the developing leaflets of the atrioventricular valves are partly mesenchymal and, due to the delamination of the valvar tissue from the ventricular muscle mass (see also Reference 5), partly muscular in composition.
Carnegie Stages 20 Through 23 (52 to 60 days)
An interruption of the continuity between atrial and ventricular muscle masses is detected for the first time in the anterior aspect of the right atrioventricular junction in the heart of an embryo at Carnegie Stage 20 (Fig 6a⇓ through 6d). Elsewhere in this heart, the atrial and ventricular muscle masses are still continuous.
In two later embryonic specimens (Stages 22 and 23, 56 to 60 days), the separation has extended somewhat more to the lateral portion of the right atrioventricular junction (not shown). When the right atrioventricular junction is followed toward the atrioventricular nodal area, the amount of nonmuscular material separating atrium from ventricle decreases, and the muscle masses are still continuous at the posterolateral portion of the right atrioventricular junction. Furthermore, atrioventricular continuity is still observed in the left atrioventricular junctions.
3 Months of Development
At the beginning of the third month of gestation atrioventricular discontinuity is, in addition to the discontinuity observed in the right side, now also observed in the posterior region of the left atrioventricular junction (Fig 7⇓). The developing leaflets at this stage still consist partly of ventricular (ie, β-MHC–expressing) myocytes and partly of cushion-derived (ie, CTA-positive) mesenchymal cells.
At the end of the third month of gestation, the atrioventricular valves have obtained their mature morphology, more or less (Fig 8⇓). CTA is expressed in the leaflets of the atrioventricular and arterial valves as well as in the central fibrous body. Only a few myocardial cells can still be detected within the leaflets of the atrioventricular valves at this stage (Fig 8a⇓, 8d⇓, and 8e⇓). At both the left and right atrioventricular junctions, the atrial myocardium and ventricular myocardium are now almost completely separated due to the fusion of the tissues of the atrioventricular sulcus and cushions. In most areas, nonetheless, the atrial myocardium and ventricular myocardium are still in very close proximity, and isolated muscular atrioventricular connections can still be observed. At the right atrioventricular junction, the area of fusion of the CTA-expressing, cushion-derived tissue and the tissue of the sulcus is located at the ventricular margin of the right atrioventricular ring. Although the ring no longer expresses GlN2/Leu-7, it remains easily distinguishable from its surroundings by its peculiar myocardial arrangement.3 24 25 The location of the zone of fusion between sulcus tissue and cushion-derived tissue within the left atrioventricular junction seems to be much deeper than the zone of fusion in the right atrioventricular junction. This is due to the fact that, in addition to the outward growth of the ventricular wall, a prominent ventricular thickening has developed toward the luminal side in the left ventricle. This development is schematically depicted in Fig 9⇓.
4 Months of Development
At this fetal stage, the atrial and ventricular muscle masses at the right atrioventricular junction are separated from each other by a well-formed thick layer of connective tissue (Fig 10a⇓ and 10c⇓). The leaflets of the atrioventricular valves and the central fibrous body still express CTA (Fig 10b⇓ and 10d⇓). At the parietal margin of the right atrioventricular junction, the insertion of the anterosuperior leaflet of the tricuspid valve is located at the ventricular side of the right atrioventricular ring (Fig 10d⇓). The valvar tissue is in continuity with the sulcus tissue, which remains confined to the epicardial side of the atrioventricular ring. At the left atrioventricular junction, the separation between atrial and ventricular muscle masses is as yet not so clear, and occasionally strands of myocardium continue to cross the presumptive line of fusion between sulcus and cushion tissue.
The Development of the Atrioventricular Junction in the Normal Heart
We have studied the morphogenesis of the tissues producing atrioventricular insulation in the human heart by using specific antibodies reacting with atrial and ventricular myocardium, the myocardium of the atrioventricular canal, the atrioventricular cushion tissue, and mesenchymal cells in the atrioventricular sulcus. The data presented demonstrate unambiguously that the separation of the atrial and ventricular muscle masses is associated with “fusion” of the tissues of the atrioventricular sulcus with the atrioventricular cushions. The interruption of the atrioventricular continuity, ie, the line of fusion of the nonmyocardial tissues, lies at the ventricular margin of the atrioventricular junctional myocardium. As a consequence, the entire embryonic atrioventricular junctional myocardium, as defined by its specific pattern of gene expression, extensively described in previous papers,1 2 3 4 26 becomes incorporated into the definitive atrium. The atrioventricular axis of conduction tissue persists as the only muscular atrioventricular continuity in the healthy adult human heart as a result of the lack of fusion of the tissues of the atrioventricular sulcus and the dorsal atrioventricular cushion.
On the basis of the material studied, we conclude that the process of separation of atrial and ventricular myocardium starts at ≈7 weeks of development at the anteromedial portion of the right atrioventricular junction. This is much earlier than suggested in 1939 by Odgers,19 who argued that the atrioventricular junctional myocardium becomes interrupted for the first time at 4 months of development. Although we observed that the process of fusion between the tissues of the atrioventricular sulcus and atrioventricular cushions is largely completed around the 12th week of development, in all the fetal hearts we studied, at the line of fusion between sulcus and atrioventricular cushion tissue, isolated strands of cardiomyocytes were observed that were possibly connecting the atrial with the ventricular muscle mass. Interestingly, examination of serial sections of neonatal human hearts, previously described in a study on the distribution of myosin heavy chain isoforms,25 revealed that these isolated strands of cardiomyocytes can still be observed in these otherwise normal hearts (Fig 11⇓).
It is noteworthy that the atrioventricular insulation at the right side develops and “matures” first during development, but that the left-sided fibrous annulus is better developed in the adult heart.27 The ontogenesis of the atrioventricular junction is schematically depicted in Table 2⇓.
Accessory Atrioventricular Connections
Our description of the formation of the fibrous insulating tissues offers new insights on the possible morphogenesis of parietal accessory atrioventricular muscular connections. It seems possible that atrioventricular connections may persist into adult life as a result of an incomplete fusion of sulcus and cushion tissue (Fig 12b⇓). Incomplete fusion would well account for the intimate relationship of the muscular connections to the fibrous annulus supporting the leaflets of the mitral valve.28 It is also of interest to note that in the right-sided atrioventricular junction, parietal accessory atrioventricular connections with special histological characteristics have been described,11 15 classified previously as “parietal specialized accessory atrioventricular muscle bundles.”12 These connections are almost certainly related to the specialized ring tissue.
Considering the development of the fibrous annulus, as described above, it is unlikely that the parietal accessory atrioventricular bundles located subepicardially and “crossing” the adipose part of the fibrous annulus (Fig 12c⇑) at the epicardial aspect (ie, through the sulcus tissue) do present persisting embryonic atrioventricular connections, since they are not located in the area of the embryonic junctional myocardium. Moreover, as these bundles are not observed in the sulcus tissue of embryonic hearts, the data presented in this paper indicate that they have to be “acquired” during fetal and/or postnatal life.
Formation of the Leaflets of the Atrioventricular Valves
Our study lends no support to the concept that ingrowth of the tissue of the atrioventricular sulcus produces the larger component of the mesenchymal portion of the leaflets of the atrioventricular valves. This concept was derived from studies of serial sections stained with classical histological dyes. In these studies, structures within the sections were discriminated on the basis of their morphological appearance, using terms like “poorly staining appearance” and “more readily stainable.”22 It was stated that the insulation of the atrial and ventricular muscle masses became established by simple invagination of the extracardiac atrioventricular sulcus tissue17 18 (see Fig 1⇑). The material contribution of the atrioventricular cushion tissue to the formation of the fibrous skeleton and the atrioventricular valvar leaflets was thought to be very small.6 17 18 29 Our results, in contrast, show that the leaflets of the valves derive equally from the atrioventricular cushions and myocardium.5 By means of the antibody reacting specifically with the cushions in the early stages, we were able to follow their fate as development proceeded. Moreover, we have not been able to confirm the presence of remnants of the cushions in the apical part of the parietal leaflet of the tricuspid valve, the so-called “noduli albini,” mentioned for the first time by Bernays in 1876 (see Reference 19) and reiterated more than a century later by others.17 18 Our results, therefore, support the more classical accounts, which accorded for a more important role of the cushions in the formation of the fibrous annulus and valves.19 20 21 30
Thus, our immunohistochemical results show convincingly that no true inward growth of atrioventricular sulcus tissue takes place. The impression of active invagination of the atrioventricular groove is given by three closely related processes (Fig 4⇑). The first is the thickening of the ventricular free wall. This process takes place around the fifth week of development. The second is the bulging of the shoulder of the free wall of the ventricle, which gives rise to the formation of the “shark-teeth”–shaped appearance of the atrioventricular groove (Fig 4b⇑). The third process is the increase in the mass of the sulcus tissue “filling up” the atrioventricular groove. This occurs around the sixth week of gestation. The insulation between atrial myocardium and ventricular myocardium is established by fusion of the tissues of the atrioventricular sulcus and the atrioventricular cushion (Fig 4c⇑).
This work was supported by grant 90-066 of the Netherlands Heart Foundation. The authors gratefully acknowledge A.A. van Horssen for his informative drawings and C. Gravemeijer for excellent photography.
Reprint requests to Andy Wessels, PhD, Department of Cell Biology and Anatomy, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425-2204. E-mail firstname.lastname@example.org.
- Received March 22, 1995.
- Accepted August 30, 1995.
- © 1996 American Heart Association, Inc.
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