HNK-1 expression patterns in the embryonic rat heart distinguish between sinuatrial tissues and atrial myocardium.

HNK-1 expression was studied by immunohistochemistry in serial sections of embryonic and fetal rat hearts from 11.5 to 16.5 embryonic days. Graphic reconstructions were made to obtain detailed 3D information on the localization of immunoreactive tissues. The antibody used appeared to stain most parts of the venous sinus and the sinuatrial transitional zone as well as the atrioventricular transitional zone, but the patterns varied through the different developmental stages. At 11.5 days, positive myocardium was found in the right atrium and on top of the ventricular septal primordium. At 13.5 days, the left venous valve and the posterior atrial wall containing the orifice of the pulmonary vein were immunoreactive, and so were the right venous valve, the septum spurium and the superior, right-lateral and inferior parts of the atrioventricular canal. From the latter, immunoreactivity continued onto the crest of the ventricular septum. At 15.5 days, HNK-1 positivity in the two venous valves had become continuous, whereas the right-lateral part of the atrioventricular canal had lost its positivity, thus making the positive areas in the superior and inferior parts of this canal discontinuous. From the venous valves immunoreaction continued into the venous sinus septum but this area remained discontinuous with the inferior part of the atrioventricular canal. It is concluded that the entirety of venous sinus and sinuatrial transitional zone expresses the HNK-1 antigen and that the orifice of the pulmonary vein belongs to this complex, rather than to the embryonic atrium proper, which is HNK-1 negative. Extrapolation of these data to the adult human atrium leads to the conclusion hat most "atrial septal structures" are of sinuatrial origin, leaving the flap valve of the oval fossa (atrial septum primum) as the only really atrial structure. It is suggested that the atrioventricular node is derived from the inferior portion of the atrioventricular canal, and that two expansions of sinuatrial tissue form the substrate for anterior and posterior atrionodal inputs which in the literature have been described as internodal tracts.

Development of the atrioventricular valve tension apparatus in the human heart.

Using various microscopical techniques we studied the development of the atrioventricular valves in human hearts between 5 and 19 weeks of development. Within the atrioventricular cushions two different layers could be recognized that remained present in all ages studied. The atrial layer, being present at the side of the atrioventricular orifice, was positive for laminin while the ventricular layer, that was connected to the myocardium, was positive for fibronectin and collagen III. Fate-mapping of these two layers, morphometrics, and scanning electron microscopy, supplemented with in vivo labeling of cushion tissue in chicken hearts have lead to new insights in the process of valve development. The cushions became freely movable prevalvular leaflets by delamination of ventricular myocardium underneath the cushion tissue. This myocardium gradually retracted towards annulus and papillary muscles and finally disappeared, resulting in fibrous, non-myocardial valves. The atrial layer of the cushions remained present as a jelly-like surface on the valve leaflets while the ventricular layer of the cushions became the compact fibrous tissue of the leaflets and the chords. Chordal development was first visible at 10 weeks of development when gaps were formed in the ventricular layer of the cushions on top of the papillary muscles. These gaps enlarged into the interchordal spaces while the cushion tissue in between the gaps lengthened to form the chords. We conclude that the leaflets as well as the chords of the atrioventricular valves are derived from atrioventricular cushion tissue. Myocardium is only important for loosening of the leaflets while keeping connection with the developing papillary muscles. Errors in delamination or retraction of myocardium or remodeling of cushion tissue into chords form the basis for various congenital valve anomalies.

Diminished growth of atrioventricular cushion tissue in stage 24 retinoic acid-treated chicken embryos.

Stage 34 chicken hearts have shown a spectrum of looping disturbances, changed hemodynamics, and changed growth of both right ventricular myocardium and atrioventricular cushion tissue after retinoic acid treatment. To obtain more information about the onset of the malformations we studied stage 24, the stage between the previously studied stage 34 and the moment of treatment. Sixteen stage 24 chicken embryos were examined after treatment with 1 microg all-trans retinoic acid at stage 15 and compared with 6 sham operated embryos. Morphological examination was supported by graphic reconstructions. Absolute volumes of atrial, atrioventricular, and ventricular myocardia were measured by a point counting method. The absolute volumes of the endocardial cushions were measured as well. Fifteen (15/16) retinoic acid-treated hearts did not show marked malformations as far as could be detected with our current macroscopic and microscopic techniques. One (1/16) retinoic acid-treated heart showed an abnormal tubular C-shape with a less bended inner curvature and with an abnormal horizontally oriented atrioventricular canal. The dorsal cushion tissue of this atrioventricular canal was discontinuous with the dorsal mesocardium and covered the malpositioned myocardial border between the atrium and the atrioventricular canal. The volume measurements did show a difference between retinoic acid treatment and sham operations. The retinoic acid-treated hearts showed a significant volume decrease of the atrioventricular cushions. No significant differences were found in the volumes of the ventricular myocardium compared to the sham operated embryos. We hypothesize that, between stages 15 and 24, retinoic acid directly affects the myocardial wall and the cushion tissue formation. In the present material this has resulted in decreased atrioventricular cushion growth, in changed hemodynamics, and in a severe looping disturbance of one embryo. We further hypothesize that, between stages 24 and 34, the malformations with minor looping disturbances will become apparent. Thus, development beyond stage 24 would result in the spectrum of looping disturbances as has been found at stage 34. These latter morphological malformations would lead to increasing hemodynamic changes, resulting in changes in growth as a secondary effect.

Development of the papillary muscles of the mitral valve: morphogenetic background of parachute-like asymmetric mitral valves and other mitral valve anomalies.

OBJECTIVES: To understand papillary muscle malformations, such as in parachute mitral valves or parachute-like asymmetric mitral valves, we studied the development of papillary muscles. METHODS: Normal human hearts at between 5 and 19 weeks of development were studied with immunohistochemistry, three-dimensional reconstructions, and gross inspection. Scanning electron microscopy was used to study human and rat hearts. RESULTS: In embryonic hearts a prominent horseshoe-shaped myocardial ridge runs from the anterior wall through the apex to the posterior wall of the left ventricle. In the atrioventricular region this ridge is continuous with atrial myocardium and covered with cushion tissue. The anterior and posterior parts of the trabecular ridge enlarge and loosen their connections with the atrial myocardium. Their lateral sides gradually delaminate from the left ventricular wall, and the continuity between the two parts is incorporated in the apical trabecular network. In this way the anterior and posterior parts of the ridge transform into the anterolateral and the posteromedial papillary muscles, respectively. Simultaneously, the cushions remodel into valve leaflets and chordae. Only the chordal part of the cushions remains attached to the developing papillary muscles. CONCLUSIONS: Disturbed delamination of the anterior or posterior part of the trabecular ridge from the ventricular wall, combined with underdevelopment of chordae, seems to be the cause of asymmetric mitral valves. Parachute valves, however, develop when the connection between the posterior and anterior part of the ridge condenses to form one single papillary muscle. Thus parachute valves and parachute-like asymmetric mitral valves originate in different ways.

The parachute-like asymmetric mitral valve and its two papillary muscles.

OBJECTIVES: The morphologic features of parachute-like asymmetric mitral valves are described to discriminate this anomaly from parachute mitral valves. BACKGROUND: Mitral valves with unifocal attachment of chords have been called "parachute valves," independent of the number of papillary muscles. Therefore the anomaly involving two papillary muscles has not received separate attention. METHODS: The gross anatomy of 29 mitral valves with focalized attachment of chords was studied. RESULTS: In 28 of the autopsy specimens asymmetric mitral valves with two papillary muscles were present, and one of the muscles was elongated, located higher in the left ventricle with its tip reaching to the anulus, and attached at both its base and lateral side to the left ventricular wall. The valve leaflets could be directly attached to this abnormal muscle that received few chords or, in three hearts, no chords at all, resulting in an oblique and eccentric orifice. Because of the focalized attachment of chords to one of the two papillary muscles, we call this malformation "parachute-like asymmetric mitral valve," We found only one "true parachute mitral valve," that is, one having a single papillary muscle that received all chords. CONCLUSIONS: The morphologic features of asymmetric mitral valves are essentially different from those of true parachute valves. A distinction between these two anomalies will contribute to recognition by the pediatric cardiologist and surgeon.

Ultrastructural changes of the myocardium in the embryonic rat heart.

BACKGROUND: Ultrastructural changes of the embryonic heart have been described, and quantitative studies have reported the changes of cellular organelles in late fetal and postnatal development. However, no specific data are available on the quantitative morphology of the individual segments and intersegmental junctions of the early embryonic heart, although these components must have different functions. METHODS: We measured the absolute volumes of glycogen, Golgi complex, myofibrils, mitochondria, and the surface areas of the rough endoplasmic reticulum and mitochondrial cristae in the different regions of the embryonic rat heart by using stereological tools. RESULTS: During embryonic development, the cardiac segments and intersegmental junctions increase their glycogen volume. The sinoatrial junction and primary fold show a more rapid increase than all the other cardiac regions, whereas the atrioventricular canal shows a high level of glycogen content throughout the period studied. The Golgi complex and rough endoplasmic reticulum show a conspicuous decrease from day 15 onward. The cellular content of myofibrils and mitochondria and the surface area of the mitochondrial cristae show a gradual increase from day 11 to day 17 of development, but full maturation apparently takes place in late fetal and early postnatal stages. At day 15 of development, the cellular volumes of myofibrils and mitochondria show a temporary decrease. CONCLUSIONS: The glycogen content cannot be explained on the basis of metabolism alone. The storage of glycogen is hypothesized to serve mechanical cell stability and may also be related to a target mechanism for ingrowing nerves. Myofibrillar and mitochondrial contents of the myocytes indicate a relatively late differentiation of the venous pole of the heart. Uninterrupted maturation is only started at the time of septation.

Stereological study of stage 34 chicken hearts with looping disturbances after retinoic acid treatment: disturbed growth of myocardium and atrioventricular cushion tissue.

BACKGROUND: In a previous study retinoic acid treatment of chicken hearts has resulted in a spectrum of looping disturbances. Because of a decrease in contraction force of these hearts, the myocardial volume was hypothesized to be altered. Because retinoic acid has been suggested to influence endocardial cushion volumes, these were estimated as well. METHODS: The previously studied hearts were used for estimating the absolute volumes of the atrial and ventricular myocardium and of the endocardial cushions by means of Cavalieri's principle. To measure the surface density of the trabeculations according to the isector method, we used retinoic acid treated hearts, which were perfusion fixed and in which the sections were isotropic uniform random. The volumes and surface densities found in the three morphologically distinguished groups, i.e., intact septum, isolated ventricular septal defect and double outlet right ventricle, were compared with those in shams. RESULTS: A significant volume decrease was found in the right ventricular free wall myocardium of the double outlet right ventricle. No significant differences were found in the surface densities of the trabeculae. The volume of the atrioventricular cushion tissue in the double outlet right ventricle hearts was significantly increased. The morphological spectrum observed previously was also expressed in the right ventricular myocardial volume, which appeared to decrease from the least to the most malformed hearts, and in the volume of the atrioventricular cushion tissue, which appeared to increase. CONCLUSIONS: Several studies have shown pathology in myocardial and cushion tissue after retinoic acid treatment. In this study we have found a decreased growth of the right ventricular myocardium and an increased growth of the atrioventricular cushion tissue. We suggest that the previously found looping disturbance causes changed hemodynamics, as reported elsewhere, and that these result in changes in growth. We cannot exclude a direct effect of retinoic acid on the myocardium, which has to explain the looping disturbance.

Development of myocardial fiber organization in the rat heart.

Confocal laser-scanning microscopy of phalloidine-stained actin fibers is a relatively new tool for studying the development of myocardial fiber organization. It seems to show orientation of myocytes in rather early embryonic stages. To further evaluate the differentiation of the myocardium, this technique was compared with transmission electron microscopy in rat embryos aged between 11 and 18 days. Although the confocal images of actin filament patterns pointed to early myocyte orientation, the electron micrographs revealed that even at 17 days the ventricular myocardium was far from mature. Myofibrils never completely filled the myocytes, and lack of organization was the rule rather than the exception. The organized structure as revealed by confocal microscopy was based on cell-to-cell continuity, whereas electron microscopy showed crossing and disarray within individual myocytes. Exceptions were in the ventricular trabeculations, which showed precocious myofiber differentiation. The trabeculations probably support ventricular systole in those stages in which the free walls do not yet provide efficient contractions. The other exception was the wall of the outflow tract, which showed well-oriented myofibrils from early stages onwards. Apparently, the outflow tract has a different function in these stages. The differences found between confocal microscopy and electron microscopy suggest that some caution is indicated in the interpretation of fluorescent images of relatively low magnification.

MRI appearance of a double inlet and double outlet right ventricle with supero-inferior ventricular relationship.

This patient was diagnosed with a double inlet and double outlet RV with supero-inferior ventricular relationship, ventricular inversion was diagnosed on the basis of left-handed topology of the RV, and the straddling of the right-sided mitral valve over an anterior VSD, with its tension apparatus extending into the outflow tract of the RV. MRI was found to be superior to color Doppler echocardiography and contrast ventriculography in the segmental analysis leading to a full understanding of this complex case.

Nuclear and cellular size of myocytes in different segments of the developing rat heart.

BACKGROUND: In the embryonic heart, the individual cardiac segments show different growth rates. For the analysis of changing form in relation with changing function, data on number and shape of cardiomyocytes are necessary. Such data will give insight into the process of hypertrophy and/or hyperplasia as they may take place in the myocardium in the embryonic period. METHODS: We have measured the volumes of the nuclei and myocytes as well as the surface areas of the nuclear envelope and cellular membrane using stereological tools in rat embryos from 11 days postcoitum to 17 days postcoitum. From the data of the cellular volume of the myocytes and the myocardial volume of the individual segments, we have calculated the total number of myocytes during the developmental period. RESULTS: It is shown that the sinus venosus, sinu-atrial junction, and atrium increase their cellular volume during development, whereas the other cardiac segments show no difference in cellular volume. Similarly, the surface area of the cell membrane of the sinus venosus and sinu-atrial junction had increased during development. The nuclear volume and the surface area of the nuclear envelope did not differ during the period studied. The total number of myocytes showed a conspicuously smaller increase in the atrio-ventricular canal and distal outlet segment than in the other segments. CONCLUSIONS: The increase of the cellular volume in the segments sinus venosus and sinu-atrial junction seems to be due to a late differentiation process. In general, however, the increase of the myocardial volume in the individual cardiac segments is caused by hyperplasia of the cardiomyocytes in these segments and not by hypertrophy. The surface area of cells has a fixed relationship with cell volume, indicating that no important changes take place in the developmental period studied.

Spectrum of looping disturbances in stage 34 chicken hearts after retinoic acid treatment.

BACKGROUND: In a recently developed chick model the teratogen retinoic acid has appeared to induce a spectrum of double outlet right ventricle, which needs further detailed evaluation. It is known that retinoic acid is able to induce cardiac malformations. Although the exact mechanism is not known, an interaction with neural crest cell function is thought to exist. METHODS: After treatment with 1 microgram all-trans retinoic acid at Hamburger and Hamilton stage 15 and reincubation until stage 34 of development 41 chicken embryos were evaluated macroscopically and microscopically, supported by graphic reconstructions. These retinoic acid treated embryos were compared with a control group (n = 8). RESULTS: The retinoic acid treated embryos could be divided in three groups. Group 1 (23/41) had an intact septum, group 2 (11/41) had an isolated ventricular septal defect (VSD), and group 3 (7/41) had a double outlet right ventricle (DORV). Besides, in the group with an intact septum 11 hearts showed an abnormal course of the subaortic outflow tract. In the group with DORV a straddling tricuspid orifice (7/8) and a double inlet left ventricle (1/8) could be distinguished. Considering the external contour, the hearts in the DORV group all showed a dextroposed arterial pole. Malformed pharyngeal arch arteries were found in all three groups (11/41) and with a great diversity. CONCLUSIONS: The present cardiac malformations in the chicken as a result of retinoic acid treatment are part of a continuous spectrum, varying from hearts with an intact ventricular septum and a normal course of the subaortic outflow tract to a double outlet right ventricle with a straddling tricuspid orifice or even a double inlet left ventricle. A remarkable observation in this spectrum concerns the correlation of malformations of the inflow and outflow tracts, which is explained as a cardiac looping disturbance. The disturbance of the looping process seems to lead to malalignment of septal components, although, in the chick, retinoic acid does not in general interfere with the formation of these septal components themselves.

In normal development pulmonary veins are connected to the sinus venosus segment in the left atrium.

BACKGROUND: Classic theories describe that the common pulmonary vein develops as an outgrowth from either the sinus venosus or atrial segment. Recent studies show that the pulmonary veins are connected to the sinu-atrial region before its differentiation into a sinus venosus and atrial segment. METHODS: The development of the sinu-atrial region with regard to the developing common pulmonary vein and the growth of the atrial septum was investigated in avian embryos, using both scanning electron microscopy and immunohistochemistry. Embryos ranging between stage HH12 and HH28 were incubated with QH-1 that recognizes quail endothelial cells and precursors, HNK-1, that appears in this study to detect the myocardium of the sinus venosus, or with HHF-35, being specific for muscle actins. Also vascular casts of the heart were produced by injecting prepolymerized Mercox into the vascular system. RESULTS: In preseptation stages the common pulmonary vein drains into the left part of the sinus venosus, that is clearly demarcated by the sinu-atrial fold and HNK-1 expression. During atrial septation the left part of the sinus venosus, in contrast to the right part, loses its HNK-1 antigen from stage HH23 onwards, while at the same time the sinu-atrial fold in the left atrial dorsal wall flattens and disappears. From stage HH25 onwards HNK-1 expression is restricted to the right part of the sinus venosus, which contributes to the right atrium. The myocardial atrial septum never expresses the HNK-1 antigen, suggesting that the septum is of atrial origin. DISCUSSION: It appeared that the sinus venosus does not only contribute to the sinus venarum of the right atrium, but also to the left atrium.

Growth of the myocardial volumes of the individual cardiac segments in the rat embryo.

BACKGROUND: Although the growth of the developing heart in relation to an increase of ventricular systolic pressure and the growth of the entire embryo during development has been described, no data are available on the growth of the individual segments and intersegmental junctions. Because these different portions are known to function differently, the need for data on their individual development is obvious. METHODS: We have measured the volumes of these different compartments by Cavalieri's point counting method in rat embryos from 11 to 17 days. RESULTS: It is shown that sinus venosus and sinu-atrial junction as well as the main compartments atrium, inlet, and proximal outlet segment grow roughly proportional to the total myocardial volume. Atrio-ventricular canal and distal outlet segment show a restricted growth and their proportional volumes decrease in time. The inlet segment is the most important part of the ventricular mass at 11 days of gestation, when it is still larger than the proximal outlet segment and, thus, takes the greater part in systolic action of the ventricular mass. The growth of the primary fold increases from day 13 onwards and can be considered as part of the wall of the inlet segment which gives rise to the main part of the ventricular septum. CONCLUSIONS: The timing of the septal volume increase fits with qualitative descriptions of ventricular septation. The atrio-ventricular canal and distal outlet segment have an important constrictive function in early stages, when valves are not yet present. Slow conduction and contraction patterns have been reported to be a characteristic feature of these portions of the embryonic heart. With development of valves these segments are loosing their mechanical function and, thus, their proportional volume declines.

Origin of subepicardial cells in rat embryos.

BACKGROUND: The role of the villi and vesicles of the epicardium primordium in the formation of the epicardium has been extensively studied over the last decades. With regard to the cellular contents of the villi and vesicles of the epicardium primordium, in quail the presence of mesenchymal cells in the villi recently has been described. In the present study, we have determined whether the villi and vesicles of the epicardium primordium in rat embryos contain mesenchymal cells that originate from the transverse septum and if so, whether these cells will become part of the subepicardium. METHODS: Mesenchymal cells in the transverse septum of rat embryos were labelled by a method consisting of in vitro whole embryo culture and labelling of the ectoderm and its daughter cells, using wheat germ agglutinin-gold (WGA-Au) as a marker. RESULTS: In concordance with our observations in the standard noncultured rat embryos, labelled cells were present in the transverse septum, extending from the umbilical ring, i.e., the transition of amniotic epithelium to ectoderm, up to the villi, in the villi and vesicles, and subepicardially. CONCLUSIONS: These observations suggest that the epicardium primordium contains mesenchymal cells derived from the transverse septum. These cells reach the subepicardium, using the villi and vesicles of the epicardium primordium as their vehicle.

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) whereas the mature heart exhibits an exclusive connection of the right atrium to the right ventricle. 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.

Development of the ventriculoarterial segment of the human embryonic heart: a morphometrics study.

In the literature, discussions continue on the question whether the distal portion of the cardiac outlet segment (ventriculoarterial portion, outflow tract of the embryonic heart) is subject to a shortening (absorption, retraction) during development. In 28 human embryos ranging from 4-42 mm crown-rump length, stereological estimates of volume fractions and surface densities were used to calculate the diameter and the length of the distal outlet segment and their changes during development. A significant increase was found in the wall thickness of this segment, whereas its length remained about the same. An actual shortening was not found. It is concluded that the relative change in proportions is the cause of the disagreements. It is further concluded that there is still a mechanical role for the aorticopulmonary septum in maintaining the length of the outlet segment during growth of this region.

New findings concerning ventricular septation in the human heart. Implications for maldevelopment.

BACKGROUND: The mechanics involved in development of the inlet component of the morphologically right ventricle are, as yet, undecided. Some argue that this component is derived from the descending limb of the ventricular loop, and that the inlet and apical trabecular components of the muscular ventricular septum have separate developmental origins. Others state that the entirety of the right ventricle grows from the ascending limb of the loop, and that the muscular septum, apart from its outer component, has a unitary origin. We now have material from human embryos at our disposal, which, we believe, solves this conundrum. METHODS AND RESULTS: We used a monoclonal antibody against an antigen to neural tissue from the chick to demarcate a ring of cells separating the descending (inlet) and ascending (outlet) limbs of the developing ventricular loop of the human heart. Preparation of serial sections of graded human embryos enabled us to trace the fate of this ring, and hence the formation of the inlet of the right ventricle, to the completion of cardiac septation. Eight embryos were studied, encompassing stages 14-23 of the Carnegie classification. The ring of cells initially separating the ascending and descending limbs of the ventricular loop were, at the conclusion of ventricular septation, located within the atrioventricular junction, sequestrated for the most part in the terminal segment of atrial myocardium. CONCLUSIONS: Our study conclusively shows that the inlet component of the morphologically right ventricle is derived from the ascending limb of the embryonic ventricular loop, and that the inlet and apical trabecular components of the muscular septum are derived from the same primary ventricular septum.

Quantitative morphology of the embryonic heart: an approach to development of the atrioventricular valves.

In 32 human embryos ranging from 4.0 to 42 mm CR-length, the volumes of the atrioventricular endocardial cushions and of the ventricular myocardium were estimated by the point counting method. The surface densities of the left and right ventricular apical trabeculations were estimated by the point and intersection counting method. It is concluded that the cushions do not grow after the 25 mm stage, by that time having reached the maximal value of 0.074 mm3. This supports the concept that the cushions do not materially contribute to the definitive atrioventricular valves. In young embryos, the left ventricular trabeculations are thicker (as concluded from their higher surface density) than the right ventricular trabeculations. Only around the 25 mm stage, the ratio becomes 1. After this stage, right ventricles have thicker trabeculations than left ventricles have. This supports the concept that the trabecular pattern is changed during the period of valve formation, which process is characterized by delamination of the inner myocardial layers.

Anterolateral muscle bundle of the left ventricle in atrioventricular septal defect: left ventricular outflow tract and subaortic stenosis.

The anatomy of the left ventricular outflow tract (LVOT) in 77 hearts with atrioventricular septal defect (AVSD), 36 with a separate A V orifice and 41 with a common A V-orifice, were investigated. In all specimens, an anterolateral muscle bundle of the left ventricle was identified between the superior bridging leaflet and the left coronary aortic cusp. It displaced the attachment of the superior bridging leaflet, resulting in its clockwise rotation. The muscle bundle frequently bulged into the LVOT, but was never prominent enough to have caused significant subaortic stenosis. Measurement of the LVOT aortic ratio was possible in 54 hearts and ranged from 36-100%. In 23 cases (43%), there was mild to moderate subaortic narrowing with a ratio ranging from 53-88%. In six cases (11%), unequivocal subaortic stenosis was present, mainly in AVSD with separate A V orifices (five of six) and iatrogenic in one case with surgically corrected complete defect. A decreased ratio was mainly due to decreased anteroposterior width of the septum in the subaortic area, with anterior displacement of the superior bridging leaflet in cases with dense septal attachment of the superior bridging leaflet (i.e., in AVSD with separate A V orifices, type A complete defect with small ventricular septal defect, or surgically corrected complete defect). Significant subaortic stenosis was caused by hypertrophy of the ventricular septum in the subaortic area with anteroseptal twist in four cases, by anomalous chordal insertion of the superior bridging leaflet in one case, and iatrogenic in one case after surgical correction with left A V valve replacement in type C complete defect.(ABSTRACT TRUNCATED AT 250 WORDS)