Developmental Changes in the Myocardial ... - Semantic Scholar

THE ANATOMICAL RECORD 248:421–432 (1997)

Developmental Changes in the Myocardial Architecture of the Chick DAVID SEDMERA,1* TOMAS PEXIEDER,1 NORMAN HU,2 AND EDWARD B. CLARK2 1University of Lausanne, Institute of Histology and Embryology, Lausanne, Switzerland 2National Institutes of Health, Specialized Center of Research in Pediatric Cardiovascular Diseases, Strong Children’s Research Center, Department of Pediatrics, University of Rochester School of Medicine & Dentistry, Rochester, New York

ABSTRACT Background: Numerous studies describing myocardial architecture have been performed on the adult heart but considerably fewer have been made during embryonic or fetal development. To serve as a basis for interspecies comparison of ventricular morphology, and as a reference for studying the effects of experimental perturbations, we examined the development of chick throughout the entire incubation period. Methods: Chick hearts from stage 14 (day 2) to stage 46 (day 21) were perfusion-fixed, and sectioned in transverse, frontal and sagittal planes. The ventricular myocardial architecture was examined and photographed in the scanning electron microscope. Results: At embryonic stage 16 and earlier, the smooth-walled heart loop had an outer myocardial mantle, cardiac jelly, and endocardium. From stage 18, there was an outer compact and inner trabeculated myocardium. Trabeculated myocardium could be subdivided into the outer (basal) portion adjacent to the compact layer and the central (luminal) part. The outer basal layer could be distinguished from the inner luminal by shorter and finer trabeculae with small, round intertrabecular spaces. From stage 24, the patterns of trabeculae and intertrabecular spaces were ventriclespecific. Between stages 24 to 31, abundant trabeculations were present throughout both ventricular cavities. The trabeculae were initially radially arranged, but later adopted a spiral course, which persisted in a simplified form into adulthood. Conclusions: The ventricular myocardium undergoes distinctive morphogenesis, characterized by changes in trabecular patterning and orientation. We speculate that the embryonic trabecular architecture reflects the directions of the main stresses. Unlike fetal and adult hearts, which rely mostly on the compact myocardial layer, the trabeculae play a crucial role in the contractile function of the embryonic heart. Anat. Rec. 248:421–432, 1997. r 1997 Wiley-Liss, Inc. Key words: chick embryonic heart; trabecular pattern; perfusion-fixation; SEM; contractility The developmental processes that transform the primitive tubular heart, with its peristaltoid contractions, into the mature four-chambered heart of higher vertebrates, have long since attracted the attention of investigators (Vesling, 1664). Events during the embryonic period, when the ventricular myocardium has a sponge-like appearance, thus resembling that of lower vertebrates, have often been studied (Ben-Shachar et al., 1985; Icardo and Fernandez-Teran, 1987). However, substantially less is known about its transformation into the mature form and no systematic study throughout prenatal development has been made. The early phases of development of ventricular trabeculation in the chick embryo have been described by Icardo and Fernandez-Teran (1987). They proposed that the mechanism of trabecular formation involves r 1997 WILEY-LISS, INC.

endocardial invasion disaggregating the initially compact myocardial mantle. However, this study neither focused on the changes in trabecular pattern nor described the stages beyond day 5 (HH stage 27; Hamburger and Hamilton, 1951), after which many of these changes occurred. Ben-Shachar et al. (1985) studied chick hearts between HH stages 16–39 and described the initial trabecular arrangement as radial. They emphasized the importance of trabecular coalescence in Contract grant sponsor: the Swiss National Science Foundation; contract grant number: 31-38889.92; contract grant sponsor: NIH; contract grant numbers: P50-HL51498 and HL-42151. *Correspondence to: David Sedmera, Institute of Physiology, University of Lausanne, Rue du Bugnon 7, CH-1005 Lausanne, Switzerland. E-mail: [email protected] Received 9 October 1996; accepted 4 February 1997.

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ventricular septation and did not consider in detail the fate or possible functions of the remaining trabeculae. Wenink (1992) analyzed quantitatively the trabecular patterns of early human hearts, and reported differences between the left and right ventricle, which change during development. The role of trabeculation in propagation of excitation was demonstrated by de Jong et al. (1992) who showed that they are responsible for rapid propagation from central septal structures to the ventricular apexes, which is important for proper coordination of ventricular contraction. The later development of right ventricular trabeculation (between incubation days 6–14) was studied by Rychterova (1971). She described the process of fusion of the basal portions of the trabeculae in the right ventricle and demonstrated their important contribution to the thickening of the compact myocardium. The differences in trabecular pattern between left and right avian ventricle are well documented in both embryonic (Rychter and Rychterova, 1981; Ben-Shachar et al., 1985; Manasek et al., 1986; Icardo and Fernandez-Teran, 1987) and adult chick (Komarek et al., 1982; King and McLelland, 1984). The geometrical difference between the left and right ventricle is one of the factors responsible for cardiac failure in transposition of great arteries (Parrish et al., 1983; Castaneda et al., 1994). Important differences between different heart regions also exist at the molecular level (Zimmer, 1994; Barton et al., 1995). The pattern of trabeculation is influenced by genetic and epigenetic factors. Myocardial architecture has been altered in RXR alpha knockout transgenic mice (Sucov et al., 1994; Kastner et al., 1994). Abnormalities of the ventricular myocardium in the retinoic acidtreated mouse embryos are an example of chemical teratogenesis (Pexieder et al., 1992). Broekhuizen et al. (1995) reported abnormal function of hearts in retinoic acid-treated chick embryos, which might be related to abnormalities in the myoarchitecture, such as thinning of the compact myocardium (our unpublished observations). Haemodynamic forces affect heart morphogenesis, and abnormal myocardial architecture is produced in the perturbated flow patterns (Rychter et al., 1979; Rychter and Rychterova, 1981). These and other similar studies prompted us to reexamine in detail the

Abbreviations Ao AL At AV Co Ct DL En IS LA LV LVP Mi mp My Pu RA RV RVP RVS SV Tr

aorta ascending loop atrium atrioventricular junction compact layer of the ventricular wall conotruncus descending loop endocardium interventricular septum left atrium left ventricle left ventricle, parietal view mitral valve papillary muscle myocardium pulmonary artery right atrium right ventricle right ventricle, parietal view right ventricle, septal view sinus venosus trabecula

Fig. 1. HH stage 14. a: Frontal view of looped tubular heart indicating level of section and face viewed (arrowheads) in the next figure. b: Cross section shows the smooth inner relief and three-layered structure of the wall (outside to inside): myocardial mantle (My), cardiac jelly (star), and endocardium (En). Scale bar 5 100 µm.

normal development of myocardial architecture. The aim of this study is to describe the course of normal development of ventricular myocardial architecture in the chick heart. These results will serve as a standard (norm) for future experimental studies. They will also provide a background for quantitative evaluation of biomechanical parameters, necessary to be able to develop a mathematical model of trabeculated heart. MATERIALS AND METHODS

Fertilized White Leghorn chicken eggs were incubated in a forced-draft 37.5°C incubator with four rotations per day from stage 14 (2 days) to stage 46 (21 days, hatching; Hamburger and Hamilton, 1951). We studied stages 14 to 24 at an interval of 12 hours, stages 27 to 36 at an interval of 24 hours, and stages 39 to 46 at an interval of 3 days. At least three hearts per stage were examined.

CHICK VENTRICULAR TRABECULATION

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Fig. 2. HH stage 18. a: In a frontal view, primitive atrium (At), ventricle (V), and conotruncus (Ct) can be distinguished. b: Primary trabeculae (Tr) in the apex are arranged as dorso-ventral ridges. The compact layer (Co) is thin and remnants of cardiac jelly (star) are

present in the area of atrioventricular junction. c: The caudo-cranial view of the successive slice shows the smooth inner wall of the atrium, sinus venosus (SV), and conotruncus. Scale bar 5 100 µm.

A tapered glass cannula with diameter of 30 to 160 µm was inserted into the ventricle, and the hearts were perfusion-fixed at high flow low pressure (Pexieder, 1981) with 2% glutaraldehyde-1% formaldehyde in isotonic 0.1 M cacodylate buffer. Direct ventricular perfusion washed out the blood and arrested the hearts in end-diastole. The hearts were then postfixed in 1% osmium tetroxide, and photographed in 3 standard projections (frontal view, left and right profiles) under a M400 photomacroscope (Wild, Heerbrugg, Switzerland). The specimens were cut transversely to the long axis with thinned microdissection scissors into 0.15– 0.25 mm thick slices. Additional dissection in frontal and sagittal planes was also performed. The slices were ethanol-dehydrated, and critical point dried using freons in a CPD 030 critical point dryer (Balzers, Liechtenstein). After mounting on stubs with colloidal silver, the specimens were coated with 300 nm of gold in a S150 sputter coater (Edwards, Zivy, Basel, Switzerland), and photographed in a JSM 630OF scanning electron microscope (JEOL, Tokyo, Japan).

mantle of myocardium with a basal membrane, an acellular extracellular matrix (cardiac jelly), and a single cell layer thick endocardium (Fig. 1). Although the inner relief was smooth, the endocardium contained numerous fenestrations. The first signs of trabeculation could be found at stage 16 as oval pits in the inner surface of the apical part of the cardiac loop (future left ventricle). At stage 17, a pattern of distinct dorsoventrally aligned ridges or projections was apparent in the ventricular apex. The ellipsoidal spaces among these projections (lacunae) started to be enclosed at stage 18 (Fig. 2). This was the first occurrence of trabecular branching into the lumen, and the ventricular wall started to have a sponge-like appearance. The arrangement of the primitive trabeculation, which was the first morphological sign of ventricular differentiation, is schematically depicted in Figure 3. The cardiac jelly disappeared during this process in areas other than developing cardiac cushions. The trabeculation progressed in an apicobasal direction. The wall of conotruncus always remained smooth. From stage 21 onwards, the trabecular pattern specific to the future right ventricle was distinguished in the right lateral wall (Fig. 4). However, the interventricular septum was still morphologically undemar-

RESULTS

At stage 14, the heart was a smooth-walled, looped tube composed of three layers: a thin compact outer

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Fig. 3. Schematic representation of primary trabeculation shows that the process is limited to the primitive ventricle (V), while the inner relief of the primitive atrium (At) and conotruncus (Ct) is smooth.

cated. Trabeculation continued to increase, and by stage 24, a fine trabecular network occupied almost entirely both ventricular cavities. The trabeculae or more precisely trabecular sheets by this stage were oriented perpendicular to the outer compact layer in the frontal plane. In transverse sections, they curved parallel to the lateral wall or the interventricular septum, which was well distinguishable by stage 24. The trabeculated myocardium showed regional differences between its central and peripheral portion. The trabeculae close to the compact layer were fine, short, and oriented perpendicular to it. The intertrabecular spaces were small and round, and their pattern was similar in both right and left ventricle. The trabeculae in the central (luminal) area were thicker, and the intertrabecular spaces were oblong in shape and considerably larger than in the basal area. Trabeculae-free lumina existed only in the basal parts of the ventricles. The compact layer, relatively thin at these stages, was bounded by the outermost extent of the endocardium. The left and right ventricles were distinguishable by their trabeculation patterns (Fig. 5). In the left ventricle, the central (luminal) trabeculae were thicker than in the right ventricle, and were interconnected only in the apical part. In the right ventricle, the luminal trabeculae were shorter due to the high num-

ber of interconnecting segments, and radiated from the interventricular septum in a fan-like pattern. The intertrabecular spaces were considerably smaller than those of the left ventricle, and their shape was less oblong. This trabecular arrangement (Fig. 6) did not change qualitatively until stage 29. From stage 31 (day 7) onward, the trabeculae started to change to long, thick bundles (trabeculae carneae) that attached to the ventricular wall by their ends and along the entire length (Fig. 7). The extent of tertiary trabeculation was confined to the ventricular walls, and trabeculae-free lumina were present in both ventricles. The pattern of trabeculation was ventricle-specific. In the left ventricle, the preferential orientation of the trabeculae changed to longitudinal from the apex to the mitral orifice, with some oblique connecting segments between the main trabecular bundles (Fig. 8). In the right ventricle, the trabeculae thickened as well, and their radial arrangement started to spiral from HH stage 36 (day 10). Fine trabeculations (trabeculae tendineae) were observed in the apices of both ventricles (Figs. 8, 9, 10). Both sides of the interventricular septum and the outflow tract of the left ventricle were smooth. The thickness of the compact myocardium was gradually increasing. From stage 34, subepicardial sinusoids forming the coronary vascular bed were present. By stage 39, the radial arrangement of the trabeculae in the right ventricle became clearly spiral., with the origin on the upper part of the muscular interventricular septum turning basally in a counterclockwise direction (Fig. 8). The thickness of trabeculae increased in a basopical direction, but they were generally finer than in the left ventricle. There were still numerous connecting segments between the main bundles. In the left ventricle, similar spiralling of longitudinal apicobasal ridges was also observed (Fig. 9). By stage 42, the heart had further increased in size and the ventricles expanded considerably. However, there was no substantial change in the pattern of trabeculation, and the spiral systems were distinctly present in both ventricles. The compact myocardium formed most of the ventricular mass by that time. During further development, the interconnecting segments became much less abundant, which gave the trabeculae a coarser, ropelike appearance (Fig. 10). The fine trabeculation in the basal region of the right ventricle could be found even in the most advanced stages studied. Both the right and left sides of the interventricular septum adjacent to the atrioventricular valves were smooth. By this time, the trabecular pattern, schematically depicted in Figure 11, was essentially the same as in the adult chick. DISCUSSION

The onset of ventricular trabeculation has been described in detail by Icardo and Fernandez-Teran (1987), and our observations of the early trabecular pattern on the organ level correspond well with theirs. As a mechanism setting off trabeculation, they have suggested endocardial invasion into the cardiac jelly and myocardium. The further development of trabeculation is effected by apposition from the periphery, where mitotic activity is highest (Rychterova, 1971; Tokuyasu, 1990). This was confirmed by DNA-labelling studies of Thompson et al. (1995), who demonstrated that the


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Fig. 4. HH stage 21. a: A frontal view shows still undivided ventricle (V), atrium (At), and conotruncus (Ct). b: Inside, there is a fine three-dimensional network of secondary trabeculation, which is starting to occupy the lumen. Compact (Co) and trabeculated myocardium can be distinguished on the cross-section. Trabeculae (Tr) (arrow-

heads) do not yet fill the entire ventricular cavity. There is left to right gradient in trabecular thickness. c: In the cranial slice, the trabeculae of the lateral wall, which is about to belong to the right ventricle, have predominantly radial arrangement. Stars, atrioventricular cushions. Scale bar 5 100 µm.

myocytes in trabeculae are mitotically quiescent in comparison with the compact layer, and by retroviral lineage tracing studies (Mikawa et al., 1992), which showed that the inner trabeculated layer grows at slower rate than the outer compact myocardium in the second half of incubation. The development of ventricular trabeculation progresses through three stages that are similar to human or mouse embryos (Vuillemin and Pexieder, 1989; Jouk and Pexieder, unpublished data; Si Minh Pham, personal communication). The Type I (primary) trabeculation is characterized as trabeculae attached full length to the ventricular wall. BenShachar et al. (1985) described the orientation of the primitive trabeculation as radial, which corresponds well with our results and the initial stages of apical trabeculation in mammalian heart (Vuillemin and Pexieder, 1989). Type II (secondary) trabeculation can be found at stages 21 to 31. This consists of fine trabeculae with abundant branching, filling almost the entire ventricles. The cranial portion of the trabeculation marks the proximal boundary of the conotruncus, which has a smooth inner relief (Pexieder, 1978). In mammalian heart, the trabeculae show more pronounced regional differences in thickness between their basal and lumi-

nal parts. In mouse hearts prepared for SEM, they appear generally coarser than in the chick. Unlike in human or mouse embryos (Wenink and Gittenberger-De Groot, 1982; Vuillemin and Pexieder, 1989; Jouk and Pexieder, unpublished data, 1997; Si Minh Pham, personal communication), the arrangement of the secondary trabeculae in the chick is not uniform (isotropic), but shows a distinct pattern of orientation. This orientation is dorsoventral in the left ventricle, but radial in the right (Ben-Shachar et al., 1985; Icardo and Fernandez-Teran, 1987). Secondary trabeculation in the mouse seems to be more symmetric than in the chick. This could be due to different spatial arrangement and geometry of the embryonic mouse heart. The ventricular chambers in mouse are two ellipsoids communicating through the interventricular foramen, rather than a cylinder with a cone wrapped around it as seen in chick. An ellipsoid has more planes of symmetry than a cone. These differences are mostly marked in the embryonic period. The fetal chick and mouse hearts are more similar to one another. Wenink (1992) studied quantitatively the right and left trabecular patterns on histological series of CRL 4.0–42 mm human embryos. Initially, the trabeculae were thicker in the left ventricle, but after CRL 25 mm they became thicker in the right

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Fig. 5. Dissection of HH stage 27 heart. a: All four chambers are externally demarcated on the frontal view. b: In the apex, the number of interconnecting segments is high, and interventricular septum starts to form by coalescence of the trabeculae (arrows). c: Left and right ventricle are easily distinguishable by different trabeculation patterns. Note the fine secondary trabeculation, filling most of both ventricles. d: Trabeculae-free lumen exists only in the basal portions of the ventricles, separated by fine interventricular septum (IS). The walls are still heavily trabeculated. e: Trabeculation limits the proximal extent of septating conotruncus (Ct), which is smooth. Scale bar 5 100 µm.

ventricle. The discrepancy with our data might reflect the differences between classes, and also the importance of trabecular remodeling in the fetal period. In this respect the human heart, being mammalian, is more similar to that of mouse, which should be kept in mind when attempting to model the human heart biomechanically. There are also important regional differences within trabeculated myocardium. The basal trabeculae adjacent to the compact layer are finer, and their orientation is perpendicular to it in both ventricles. The corresponding intertrabecular spaces are small and round. During the course of development, this layer gradually regresses in a basoapical direction, as its putative nutritive function is taken over by developing coronary circulation (Rychterova, 1971; Rychter and Ostadal, 1971). The process of compaction of the basal portions of the trabeculae contributes to the increase of thickness of the compact myocardium (Rychterova, 1971). Coronary vascularization starts to develop at stage 34 (day 8) and is completed at stage 40 (day 14) (Rychter and Rychterova, 1981; Waldo et al., 1990). We believe that the secondary trabeculation is a temporary


Fig. 6. A schematic representation of the secondary ventricular trabeculation pattern at its maximal development (approximately HH stage 28) on transverse and frontal sections.

compromise between the need to increase myocardial mass so as to cope with the elevated functional requirements (Clark et al., 1986), and a lack of adequate blood supply (Tokuyasu, 1990). If the only function of all the intertrabecular spaces was to provide nutrition (Minot, 1901), one would expect them to be a spherical shape with an isotropic arrangement. In contrast, the radial arrangement of secondary trabeculation and the ellipsoidal shape of the intertrabecular spaces probably reflects the direction of stresses during contractions by analogy with the orientation of collagen fibrils in the aortic valve leaflets, i.e., perpendicular to the wall tangent at any point (Peskin and McQueen, 1994). Together with Wenink et al. (1996) we speculate that the trabeculae are the main contractile element at these stages. The arrangement of secondary trabeculation supports its role in ventricular contractility. It should also be noted that the cells in trabeculae form most of the myocardial mass during this period (Sedmera et al., 1995), and are more differentiated than the cells in the compact layer (Markwald, 1969; Thompson et al., 1995; Wenink et al., 1996). The relatively high proportion of trabeculae in embryonic hearts resembles the situation found in fishes and

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amphibians. The ventricular compartments of lower vertebrates are extensively trabeculated, and have only a thin compact layer (Van Mierop and Kutsche, 1984). All these hearts generate relatively low pressures, and are supplied by not completely oxygenated blood, so this arrangement might be important for nutrition and oxygenation of the working myocardium. The dorsoventrally aligned, sickle-like folds observed in chick embryonic left ventricle may serve the same purpose (blood compartmentalization; Hogers et al., 1995) as those seen in the non-septated hearts of lower vertebrates such as frog. They also form the muscular part of the interventricular septum (Ben-Shachar et al., 1985). As shown by de Jong et al. (1992), they are also an important element of ventricular conduction at stages 23–31. Type III (tertiary) trabeculation starts from stage 31 (day 7) onward, and these trabeculae are long, thick bundles attached to the wall by their ends and along their entire length. The only previous study concerning this period was that of Rychterova (1971), who described the fate of right ventricular trabeculation between incubation days 6 (stage 29) and 14 (stage 40), but in less detail than the current study. The extent of tertiary trabeculation is limited mainly to the walls, so free lumen is present in both ventricles by this time. The pattern of tertiary trabeculation is again ventriclespecific. In the left ventricle, it is composed of longitudinal, slightly spiraled ridges (trabeculae carneae), stretched between the apex and the mitral orifice, where they end as papillary muscles. There are also oblique connecting segments between them. Both sides of the interventricular septum, together with the outflow tract of the left ventricle, are smooth. This arrangement could serve several functions. They provide support for the mitral valve leaflets and enable better apposition of the inner wall in systole (similar to the slits and scallops in the mitral valve: Solomon and Nayak, 1994). They might also participate directly in the contractile function of the left ventricle, since the direction of trabeculae is the same as that of the muscle fibres in the compact layer (Jouk et al., 95). The tertiary trabeculae are also associated with the terminal branches of the conduction system (Anderson and Becker, 1980), thus providing the morphological substrate for coordinated contraction of the left ventricle. Fine trabeculations (trabeculae tendineae), found in the ventricular apexes, are composed purely of the conduction tissue. The pattern of trabeculae in the right ventricle is more complicated. Their contribution to total myocardial mass is more substantial than in the left ventricle. They are arranged in an anticlockwise spiral, which begins at the cranial end of muscular interventricular septum and runs first to the apex and then towards the conotruncus. This spiral course again resembles the arrangement of muscle fibres in adult (Streeter, 1979) or fetal (Jouk et al., 1995) heart, and the bellows-like pattern of trabeculae is probably important for the contractility (Castaneda et al., 1994). Another possible function is the strengthening of the comparatively thinner ventricular wall. Similarly, as in the human fetuses (Jouk and Pexieder, unpublished data), the pattern of tertiary trabeculation can be directly matched with the adult one. On the other hand, mouse embryos close to term show much more abundant trabeculations (Vuillemin and Pexieder, 1989; Si

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Fig. 7. HH stage 31. a: Frontal view showing nearly adult external morphology, with all chambers demarcated. b: In the apical part of the left ventricle, the trabecular arrangement is becoming apico-basal. The apical part of the right ventricle contains small, rounded intertrabecular spaces. c: In the left ventricle, the extent of trabeculation starts to be limited to the lateral wall. The muscular interventricular septum, apparently belonging mostly to the left ventricle, can also be seen. d: The area of trabeculae-free lumen increased. In the LV, the compact layer forms most of the myocardium, and the papillary muscles (mp) can be seen in cross section. The trabeculae still form a significant proportion of the right ventricular (RV) wall. The muscular interventricular septum by this stage. e: The last slice shows the inflow and outflow tracts of both ventricles. Scale bar 5 100 µm.

Minh Pham, personal communication), which corresponds with their less advanced development at birth compared to chick. The differences of trabecular arrangement can be attributed to different left/right ventricular geometry (a prolate ellipsoid rather than a cone, or more precisely a crescent), and the contraction pattern (circular rather than bellows-like) (Hutchins et al., 1978; Castaneda et al., 1994). The left ventricle is proportionally larger in adult birds than in mammals probably because a greater blood supply is needed for the powerful flight muscles (Komarek et al., 1982; King and McLelland, 1984). The avian left ventricular compact layer is about five times thicker than the right one, while in mammals this ratio is about 3:1. The proportion of trabeculae in the right ventricle is higher than in the left one, which relies mostly on its compact layer. The maturation of the right ventricle is characterized by the gradual


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Fig. 8. HH stage 39. a: Frontal view indicating cuts and directions of observation (arrowheads). b–d: Sagittal dissection of the heart. In the parietal part of the right ventricle (RVP), the trabeculae have a spiral course and numerous cross bridges. In the septal part (RVS), the origin of the trabecular radiation at the end of muscular interventricular septum can be seen. Star indicates the pulmonary trunk. In the parietal part of the left ventricle (LVP), trabeculae can be observed in a longitudinal pattern, merging with the papillary muscles. Trabeculae tendineae, formed by conduction tissue, can be found in the apical part (arrow in d). IS, part of the interventricular septum with a bit of the right ventricle (RV). Scale bar 5 1 mm.

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Fig. 9. HH stage 42. a: In the apex of the left ventricle can be seen the origin of major spiraled bundles of tertiary trabeculation, which end as papillary muscles. b: The section at the midportion level shows the limited extent of tertiary trabeculation in the left ventricle (LV) and the spiral system in the apex of the right (RV). Scale bar 5 1 mm.

Fig. 10. HH stage 45, frontal dissection. The heart increased in size, mainly in longitudinal axis. Both dorsal (a) and ventral (b) halves of the heart are shown, which gives an idea about three-dimensional organization of trabeculation, very similar to the adult pattern.

Apicobasal counterclockwise spiral systems can be recognized in both ventricles. Arrow, interatrial septum; star, right AV valve. Scale bar 5 1 mm.

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In this study, we have presented a qualitative morphological description of the development of the chick ventricular trabeculation. This method and data serve as the basis for interspecies comparisons of ventricular morphology and as a reference for studying the effects of experimental modifications such as conotruncal banding, drug treatment, or genetic manipulations. ACKNOWLEDGMENTS

Our thanks are due to Mrs. Ariane Gerber for excellent technical help, Dr. Penny Thomas (National Heart and Lung Institute, London, UK) and Dr. Bradley B. Keller (University of Rochester, Rochester, NY) for challenging discussions and kind revision of the manuscript. We also appreciate inspiring discussions with Dr. Si Minh Pham, who made available to us his unpublished data concerning normal development of mouse ventricular trabeculation. This work was supported by Swiss National Science Foundation grant 31-38889.92 (T.P.), and NIH grants P50-HL51498 and HL-42151 (E.B.C., N.H.). LITERATURE CITED

Fig. 11. Schematic representation of tertiary trabecular pattern. In the left ventricle, only two of the principal bundles are shown for clarity. In both ventricles, the trabeculae can be followed in a counterclockwise apicobasal spiral (when viewed from base towards the apex), so the right-left differences can be mainly attributed to different ventricular geometry (cone/crescent vs. cylinder/prolate ellipsoid).

thickening of existing trabeculae, the spiralling of their courses, and the reduction in their number (Rychterova, 1971). We believe that the remaining trabeculae serve as a support against excessive expansion since the compact layer of the ventricular wall is quite thin. In summary, when viewed from the base towards the apex, the trabeculae form apicobasal counterclockwise spirals in both ventricles. Its correspondence with the muscle fibre orientation in the compact layer (Jouk et al., 1995; Sanchez-Quintana et al., 1995) could be advantageous for the wringing action during contractions (Gibbonskroeker et al., 1993). The myocardial architecture can be influenced genetically (Sucov et al., 1994; Kastner et al., 1994) or by epigenetic mechanisms (Clark et al., 1984; Pexieder et al., 1992). Rychter et al. (1979) proposed that modifications in haemodynamics had an influence on trabecular pattern. They described distinct changes in the internal relief (coarsening of the trabeculae) of the right ventricle induced experimentally by clipping the left atrium. Preliminary data (our unpublished results) from hearts that underwent conotruncal banding (Clark et al., 1984) suggest that the normal trabecular pattern is affected under altered haemodynamic conditions. Thus, mechanical forces might be one of the stimuli to the establishment of the trabecular pattern.

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