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European Journal of Morphology
1998, Vol. 36, No. 2, pp. 105-119
A QUANTITATIVE STUDY
THE
0924-3860/98/3602-0105$12.00
0 Swets & Zeitlinger
OF THE
V ENTRICULAR M YOARCHITECTURE
IN
STAGE 21-29 CHICK E MBRYO FOLLOWING D ECREASED LOADING
2
David Sedmeral, Tomas Pexiederl, Norman Hu2 and Edward B. Clark
lInstitute of Histology and Embryology, University of Lausanne, 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 14642, U.S.A.
ABSTRACT
During the early developmental period, ventricular myoarchitecture undergoes a transition from a smooth-walled
cardiac tube, to left and right ventricular chambers filled with a sponge-like network of trabecular struts. We
measured the quantitative changes of ventricular myocardium properties in normal stage 21-29 chick embryos and
after chronic verapamil suffusion, which is known to decrease work load and decelerate ventricular growth. The
morphologic parametres (compact layer thickness, ventricular wall composition, porosity of different layers and
trabecular orientation) were determined from scanning electron micrographs of transversely dissected perfusionfixed hearts. A vascular bed of stage 21 chick embryos was suffused with 1 ng of verapamil at 1 pl per hour up to
stages 24, 27 and 29 via a miniosmotic pump. From stage 24, the thickness of the compact myocardium in the left
ventricle was greater than that of the right. The increase in thickness was minimal between stages 24 and 27, while
the predominantly radially arranged trabeculae comprised up to 75% of total myocardial mass. The ratio of intertrabecular spaces to trabeculae (local porosity) decreased from the ventricular center (70%) towards the compact
myocardium (0%). In verapamil-treated embryos, the hearts were smaller and showed delayed development. The
compact myocardium was thinner than normal, and the proportion of trabeculae was higher than in controls. The
local porosity values were similar in control and experimental groups. Decreased load resulted in delayed growth and
morphogenesis, expressed as a persistence of trabeculae and a thinner compact myocardium. Embryonic heart
pumping function is largely based on extensively developed trabeculation with regionally different properties.
KEYWORDS: Compact myocardium, image analysis, perfusion-fixation, trabecular orientation, chronic verapamil
suffusion.
ABBREVIATIONS: AV, atrioventricular junction; AVCa, anterior atrioventricular cushion; AVCp, posterior atrioventricular cushion; BT, basal trabecular layer; Co, compact layer of the ventricular wall; CLT, compact layer
thickness; Ct, conotruncus; En, endocardium; Ep, epicardium; IS, interventricular septum; ITS, intertrabecular
space; LA, left atrium; LT, luminal trabecular layer; LV, left ventricle; RA, right atrium; RV, right ventricle; SEM,
scanning electron microscopy; Tr, trabecula; V, ventricle
INTRODUCTION
The existence of a specific pattern of cardiomyocyte
assembly in different regions of the heart is clear from
Address correspondence to: David Sedmera, Institute of
Physiology, University of Lausanne, Rue du Bugnon 7, CH1005 Lausanne, Switzerland. Fax +41 21 692 5505; e-mail
[email protected]
either macroscopic (Streeter, 1979) or microscopic
(Usson et al., 1994) examination of the ventricular
wall. During the embryonic period, the trabeculae are
radially arranged in the ventricles (Challice &
Viragh, 1973; Ben-Shachar et al., 1985; Icardo &
Fernandez-Teran, 1987). Together with biomechanical engineers (Taber et al., 1993), we believe that this
architecture is of crucial importance for the heart’s
106
D. SEDMERA ET AL.
pumping function (de Jong et al., 1992; Broekhuisen
et al., 199.5; Hu & Keller, 1995). The patterns of
trabecular architecture are modified during the course
of development (Rychterova, 1971; Sedmera et al.,
1997) and are influenced by haemodynamics (Rychter
& Rychterova, 1981; our unpublished data). Modifications of the myocardial architecture were observed
in the retinoic acid-treated mouse embryos (Pexieder
et al., 1992), mice lacking the normal RXRa gene
(Kastner et al., 1994; Sucov et al., 1994), as well as
some other recently produced mutants (e.g. Kitsukawa et al., 1995). These studies prompted us to develop a simple method for systematic, both qualitative and quantitative, description of the embryonic
myoarchitecture.
Several investigations have been performed on the
myocardial architecture of human fetal and adult
hearts (Usson et al., 1994; Sanchez-Quintana et al.,
1995), and various methods have been developed for
studying muscle fibre orientation (for review see
Streeter, 1979). Jouk et al. (1995) have recently provided a new computer-based method enabling threedimensional reconstruction and tracing of individual
muscle fibres. In the developing mouse heart, semiautomated technique based on serial sections was described by McLean et al. (1989), and demonstrated
the gradual development of three-layered system.
However, only few qualitative descriptive studies
have been performed on the embryonic chick heart
(Rychter & Rychterova, 1981; Steding et al., 1982;
Ben-Shachar et al., 1985; Icardo & Fernandez-Teran,
1987). Wenink (1992) contributed to the study of human embryonic left and right trabecular patterns, and
the quantification of volume proportions of compact
and trabeculated myocardium was done by Blausen et
al. (1990). Unlike that of mature bird or mammalian
hearts where the vast majority of muscle mass is contained in the compact myocardium, the embryonic
myocardium is a sponge-like structure with most myofibrils localised in the trabeculae (Markwald, 1969;
Challice & Viragh, 1973; Tokuyasu, 1990).
The trabeculation in the embryonic ventricle serves
several purposes distinct from those in adult trabeculation. It increases contractile myocardial mass in the
absence of coronary circulation (Tokuyasu, 1990),
participates in the compartmentalization of blood in
the pre-septation heart (Hogers et al., 1995), and contributes to the formation of the muscular interventricular septum (Ben-Shachar et al., 1985; De La Cruz
et al., 1997). The trabeculae are postulated to be an
important element of ventricular conduction (De Jong
et al., 1992). Chuck et al. (1997) have recently described the development of ventricular activation pattern in the chick embryo. At tubular stages, in most
hearts it is baso-apical, corresponding with the peristaltoid contraction pattern. In the majority of trabeculated hearts (stages 24-3 I), the left ventricular activation is almost simultaneous, pointing out the role of
the radially arranged trabeculae in excitation spread.
The adult apico-basal activation pattern predominates
from stage 31 onwards. The compaction of the basal
parts of the trabeculae contributes substantially to the
thickening of the compact myocardium (Rychterova,
1971). Remodeling of the remaining luminal parts
gives rise to the adult trabeculation pattern and the
papillary muscles (Sedmera et al., 1997).
Our goal is to analyze the developmental changes
of embryonic ventricular myocardial architecture. We
performed quantitative analysis of the composition of
the ventricular wall, local porosity and trabecular orientation in different areas in early chick embryonic
hearts using techniques of perfusion fixation and SEM
(Moscoso & Pexieder, 1990) followed by planimetry
and image analysis. The relevance of measured quantitative parameters was tested under experimentally
reduced load conditions induced by chronic verapamil
suffusion (Clark et al., 1991). Verapamil is a calcium
antagonist (class IV antiarythmic drug), decreasing
the heart contractility and pressure via vasodialatation. Its acute administration in the chick embryo exhibits dose-dependent lethality (Ostadal et al., 1987)
and results in general growth retardation. It is not yet
clear whether it is a direct systemic effect (Jaffee &
Jaffee, 1989) or a result of hypoperfusion and therefore decreased tissue metabolism. Chronic administration in dose used by Clark et al. (199 1) and in the
present study decreases significantly heart rate, systolic blood pressure, ventricular dP/dt and dorsal aortic
blood flow at matched developmental stages for 2472 hours after beginning of suffusion at stage 21,
therefore indicating a direct and specific influence on
the developing cardiovascular system.
Our results indicate that normal chick heart development between stages 21-31 is characterized by a
gradual increase in proportion and thickness of the
compact myocardium; this is more pronounced in the
left ventricle than the right. The hearts of verapamiltreated embryos are generally smaller and have less
compact myocardium and more normally oriented
trabeculae than their normal counterparts. This shows
CHICK VENTRICULAR MYOARCHITECTURE
the correlation between cardiac growth and structual
adaptations to developmentally increasing or experimentally changed load conditions.
MATERIAL AND METHODS
White leghorn eggs were incubated in a forced-draft
incubator at 37.5”C and 70% relative humidity. The
embryos were sampled at HH stages (Hamburger and
Hamilton, 1951) 21, 24, 27 and 29. The hearts were
perfusion-fixed with 2% glutaraldehyde- 1% formaldehyde in isotonic (280 mOsmol/l) 0.1 M cacodylate
Fig. 1. The schematic representation of part of the ventricular wall
with an illustration of the measurements taken. The wall
cross-section is subdivided into the compact myocardium
(dark gray), and the basal (BT) and luminal (LT) parts of
the trabeculae (light gray). Their surface areas, together
with the intertrabecular spaces (ITS), were used for the
evaluation of the ventricular wall’s composition. The diameter (feret) of one intertrabecular space measured at 0”
and its length and orientation are also shown. CLT, compact layer thickness.
107
buffer and postfixed in 1% osmium tetroxide (Pexieder, 1981). They were photographed in 3 standard
projections (frontal, right and left profiles) using a
M400 Wild Photomakroskop. This fixation removes
all blood, arresting the hearts in end diastole. The
hearts were then cut transversely with specially
thinned microdissection scissors into slices of 0.250.50 mm thickness. These were then ethanol-dehydrated and critical point dried (Balzers CPD 030) using Freon. After mounting on stubs with colloidal
silver, the specimens were sputter coated (Edwards S
150) with 300 nm of gold and examined using a JEOL
JSM 630 OF scanning electron microscope.
For image analysis on a Quantimet 970 we used
line drawings of intertrabecular spaces traced from
SEM micrographs because variations in the grey
scales of the prints prevented reliable automatic detection of tissue/space boundaries. The structure of a
cross section of the chick embryonic ventricle is rather complex. From outside\ to inside, it consists of epicardium, compact myocardial mantle, a meshwork of
trabeculae covered with endocardium and, in some
areas, trabecula-free lumen. To determine the relative
volumes of the three main wall components: the compact myocardium, trabeculae and intertrabecular spaces, we measured the area contribution of each of these
components, which together compose 100% of the
wall cross section (Fig. 1). The histograms of ventricular wall composition are based on averages from
at least three hearts; they were corrected for slightly
different levels of slicing using linear regression. This
approach preserved the trends seen in individual
hearts, but, rendered some of the statistical analysis
invalid. The porosity of individual areas (layers) of
the trabeculated myocardium was determined as a percentage of the total layer area occupied by the intertrabecular spaces. By this definition, the porosity of
,
the compact layer is 0%.
To obtain an image for analysis, we oriented the
slices so they were viewed looking towards the apex,
with the dorsal side at the top of the image (Fig. 2). In
cases where they were viewed from the apical side
(e.g. no trabeculae visible on the non-apical side) lateral invertion of the image preserved the standard orientation. The first of two techniques used for the evaluation of trabecular orientation used a polar histogram
(adapted from Usson et al., 1994) of the lengths of the
intertrabecular spaces oriented in each direction. The
necessary data were obtained by measuring the length
and orientation of individual intertrabecular spaces
108
D. SEDMERA ET AL.
Fig. 2. Transversely dissected heart of HH stage 24 embryo. (a)
Frontal view showing levels of slicing and the direction of
observation (arrows). (b)-(e) Transversal slices from base
to apex. Note the differences between the patterns in the
left and right ventricles as well as bet ween the apex and
midportion slices. The atrioventricular cushions begin the
septkion of the atrioventricular canal. Scale bars, 100 pm. (f) Higher magnification of the lateral part of the right ventricle at HH
stage 24. Details of the myocardial architecture can be seen and the three main wall constituents (compact layer, trabeculae and
intertrabecular spaces) are easily distinguished. Arrows indicate the boundary between the basal and luminal layers in the trabeculated myocardium. Scale bar, 10 pm.
CHICK VENTRICULAR MYOARCHITECTURE
(Fig. 1), and then summing their lengths in 10” intervals. The expression of values as percentages enabled
a meaningful comparison of the different regions to
be made. This method is technically simple and the
results are essentially the same as direct measurements on individual skeletonized trabeculae (Sedmera
& Pexieder, 1995). To obtain data enabling the construction of ellipses representing orientation in polar
coordinates, we adapted the mean linear intercept
technique (Cowin, 1986) to use feature measurements
made on a Quantimet 970. In each direction (unit step
11.25”), the diameters (ferets, Fig. 1) of all intertrabecular spaces were measured and their average
calculated. These values were then plotted against the
angle of measurement in polar coordinates, resulting
in an ellipse-like curve representing the size and shape
of the “mean” intertrabecular space. This approach
gives the same results as the original method (Cowin,
1986), in which the dissector is rotated and the total
length of intercepts with intertrabecular spaces in each
direction is divided by the number of intercepts. For
further morphometrical analysis, the intertrabecular
spaces were classified according to their orientation,
area, and roundness. All these techniques were used
separately for different ventricular regions.
The thickness of the compact layer was measured
on SEM photographs using a digimatic caliper. For
consistency and comparison, the midportion slice,
containing both right and left ventricle, was selected
and standard points (at the intercepts of lines drawn at
O”, +45” and -45” from the centre of the interventricular septum with the lateral wall) were used. According to measurements of maximal transverse diameter performed on macrophotographs taken before
dissection (in isoosmotic buffer) and on SEM micrographs, between stages 2 l-29, the linear dimensions
shrink to approximately 52% of the wet values. To
approximate the in viva diastolic dimensions, the values of compact layer thickness can be multiplied by
factor 1.92. For all quantitative measurements a minimum of three hearts per stage were analysed. Statistical analysis was done using the Wilcoxon one-way
non-parametrical test (SAS) for evaluating the differences between stages and groups and a paired t-test
was used for comparison between the left and right
ventricle at any one stage. Results ~~0.05 were considered significant.
The experiments with chronic verapamil treatment
were performed as described by Clark et al. (199 1).
After opening of the membranes, one end of a piece
109
of PE-60 polyethylene tubing was positioned on the
surface of the extraembryonic vascular bed of HH
stage 21 (incubation day 3 l/2) chick, and the other
end attached to the flow modulator of an Alzet miniosmotic pump (Alza, Palo Alto, CA). The pump system was filled with 0.9% saline solution and immersed in normal saline. The pump had a constant
flow rate of 1 l.tl/h with reservoir capacity 200 fl. The
extraembryonic vascular bed was suffused with verapamil at 1 ng per hour, and the controls with 0.9%
saline alone. The eggs were then reincubated for 24,
48 and 72 hours up to (approximately) HH stages 24,
27 and 29, respectively. These stages were selected
because of approximately a two fold increase in embryo mass between them. Sampling, dissection, and
analysis was performed in the same way as described
earlier. As the values from embryos treated with 0.9%
saline did not differ from those of normal embryos,
measurements performed on normal embryos were
used for comparison.
RESULTS
Qualitative description
At HH stage 21, the common ventricle had a considerable trabecula-free lumen and no distinguishable
interventricular septum. However, nascent trabecular
patterns were apparent. Thick, dorsoventrally aligned
ridges were found in the apex, and later in the central
(luminal) part of the left ventricle. In the right lateral
wall, the trabeculae were thinner, shorter, and oriented perpendicular to the outer compact myocardial layer.
Between HH stages 21 and 24 (Fig. 2), the trabeculation increased and the right ventricle was defined
by the interventricular septum. From stage 24 the
trabecular patterns of the prospective left and right
ventricles were dramatically different. In the left ventricle, the trabeculae were dorsoventrally oriented
with numerous interconnecting segments at the apex.
They formed sickle-like folds (trabecular sheets) with
no connections in the midportion of the ventricle and
the principal bundles were perpendicular to the outer
compact myocardium. In the right ventricle, there
were more interconnecting segments and, as a result,
the individual trabeculae were shorter, without a distinct pattern; they were also thinner than those of the
left ventricle. This arrangement was reflected by more
circular and smaller intertrabecular spaces and con-
110
D. SEDMERA ET AL.
Fig. 3 . Stage 24 heart treated by chronic verapamil infusion. (a)
The heart is distinctly smaller when compared with Fig. 2a
and the single ventricle shows no external signs of septation. (b) - (e) No internal interventricular partitioning is
visible; this delay is visible on the AV level where the
cushions are much less developed than in the control at
Figure 2c. The arrow indicates the entry into the conotruncus. Scale bars, 100 w.
firmed by mean linear intercept diagrams. In polar
histograms the patterns observed were narrow, vertical clusters in the left ventricle and wide almost semicircular ones in the right ventricle.
At the first sampling interval, the verapamil-treated embryos were growth retarded (as appreciated according to HH stages) and the hearts resembled those
of HH stage 21 (Fig. 3). The right ventricle could not
be distinguished and as such the common ventricle
was considered the left ventricle for measurement purposes. Its compact layer was significantly thinner than
normal for left ventricle on HH stage 24.
By HH stage 27, the pattern of trabeculation had
not changed significantly. A fine trabeculation net-
e
work almost entirely filled both ventricular cavities.
The interventricular septum as well as the compact
myocardial layer were about the same thickness as in
the previous stage. The hearts of verapamil-treated
embryos were visibly smaller. In the left ventricle,
there was a decreased contribution of the compact
layer to the ventricular cross section area accompanied by an increase of relative trabecular mass; the
compact layer thickness was markedly reduced. Similar thinning was observed in the right ventricle.
At HH stage 29 the trabeculae and the left ventricular compact layer were distinctly thickened (Fig.
4). A trabecula-free lumen reappeared in the basal
portions of both ventricles which were previously
CHICK VENTRICULAR MYOARCHITECTURE
Fig. 4. (a) Midportion slice from HH stage 29 heart. Both ventricles
are visible and individual wall components are labeled. The
asterisks in the right ventricle indicate points of measurement
of the compact layer thickness. The arrows indicate the boundary between the basal and luminal layers in the left ventricular
trabeculated myocardium. Scale bar, 100 /_tm. (b) The mean
linear intercept diagram quantifies the differences in shape,
size and orientation of the intertrabecular spaces in different
areas. Dorsoventral trabecular sheets enclosing oblong intertrabecular spaces are represented by a rather oblong ellipse oriented
almost vertically. The variable orientation of smaller and more rounded spaces in the right ventricle gives rise to a more circular
curve. The basal layers of both ventricles are represented by much smaller, almost circular curves. (c) and (d) The orientation
histograms for these areas reveal the similarity (almost semicircular pattern with no prevailing direction) between the basal layers
of both ventricles in contrast to differences in arrangement in the luminal ones. The direction of the main vertical cluster in the left
ventricle matches the orientation of the long axis of the ellipse in (b). The most prominent cluster at about 145” in the right
ventricle results from its sickle-like cross section being more than 180” wide so this direction is encountered twice. The diagram
can not distinguish between the two in this modality.
112
D. SEDMERA ET AL.
Fig. 5. Midportion slice from stage 29 heart treated with verapamil. In comparison with normal heart at this stage (Fig. 4a) the matched
slice is smaller, and the compact myocardium, trabeculae and interventricular septum are thinner. Despite this, the left and right
trabecular patterns are normal. Scale bar, 100 pm.
filled by trabeculations. There were regional differences in the orientation of the trabeculae. In the apical
part of the left ventricle, they maintained almost isotropic arrangement as seen in Figure 2. In the midportion, the trabecular sheets preserved the dorsoventral
alignment observed in earlier stages. They assume’d
an alignment parallel to the lateral wall, or the interventricular septum when they were close to these
structures. In the right ventricle the trabeculae were
arranged in a fan-like pattern, radiating from the interventricular septum perpendicular to the outer compact layer. Unlike the fenestrated trabecular sheets in
the left ventricle, they were more rod-like entities.
In the non-compact (trabeculated) part of the ventricular wall two different layers can be distinguished
from stage 24: basal parts of the trabecular sheets
adjacent to the compact myocardium, and central portions located luminally. In the basal area the trabeculae were fine, short, and oriented perpendicular to the
outer compact layer. This layer was quite thin (on
average 50 ~_un), the intertrabecular spaces were small,
rounded (Figs. 2, 4), and there was no difference in
pattern between the left and right ventricle. These
features were best demonstrated by the mean linear
COMPACT LAYER THICKNESS
80
70
60
* pco.05
LV normal
A RV normal
0 LV verapamil
A RV verapamil
l
S.D.
I
Ofi
21
24
27
29
HH STAGE
Fig. 6. Increase in compact layer thickness (mean&SD) during development. Note the much smaller slope for the right ventricle, as well as virtually no difference between stages 24
and 27. The values for verapamil-treated hearts are decreased.
CHICK VENTRICULAR MYOARCHITECTURE
Fig. 7. The histograms of ventricular wall composition at different developmental stages. The decrease in the proportion of trabeculae in
the left ventricle is accompanied by an increasing proportion of compact myocardium. The percentage of compact layer shows an
apicobasal gradient. The proportion of trabeculae is relatively stable in the right ventricle and regional differences are less
pronounced. Trabeculae form most of the ventricular myocardial mass during these stages.
114
D. SEDMERA ET AL.
intercept diagram (Fig. 4). The differences between
the different layers and ventricles were verified by
the classification of the intertrabecular spaces according to their area, length, roundness, and orientation
(Sedmera & Pexieder, 1995; and unpublished data).
The verapamil-treated hearts were markedly smaller than normal by stage 29 (Fig. 5). The compact
layers of both ventricles were thinner, but not significantly (Fig. 6). The proportion of trabeculae relative
to the other components was augmented in both ventricles (Fig. 8). The pattern of trabecular orientation
was normal.
Quantitative measurements
At all stages examined, the compact layer in the left
ventricle was significantly thicker than in the right
ventricle (Fig. 6). The left to right ratio increased from
2: 1 at HH 24 to 2.5: 1 at HH 29. The increase in thickness between the individual stages was not significant
Q~0.05) between HH stages 24 and 27 in the left, and
24 and 27, and 27 and 29 in the right ventricle.
The composition of the ventricular wall showed
spatial and temporal gradients (Fig. 7). At HH stage
2 1, the single non-septated ventricle was quite homogeneous with the compact myocardium forming about
20% of the wall cross-section and the trabeculae about
57%. An apicobasal gradient in the proportion of the
compact myocardium soon became established in the
left ventricle accompanied by a decreased contribution from the intertrabecular spaces. This gradient
became more pronounced during subsequent development; at HH 29 the compact myocardium formed
almost 80% of the cross section of the basal region of
the left ventricle. The wall composition was more homogenous in the right ventricle: the compact myocardium comprising 15 to 25% and trabeculae 40 to 60%.
This did not change much during the period of study.
The highest proportion of intertrabecular spaces was
repeatedly seen in the middle slice. From the data
collected it was possible to determine the ratio between compact and trabeculated myocardium. With
the exception of the left ventricle’s basal part of HH
stage 29, the trabeculated component was the greater.
The values of the local porosity of different regions reflected the changes in ventricular wall composition. To make a developmental comparison, the
values measured in the midportion slice were selected; the measurements of the compact layer thickness
were also performed in this region. The small sample
size and relatively high variability prevented any ex-
tensive statistical analysis of this quantitative data. In
the midportion of the left ventricle the porosity of the
luminal layer increased from 30%, during stages examined, to almost 60% by HH stage 29. The porosity
of the basal layer was around 20% with a small decline from HH 24 to HH 29. Unlike that of the left
ventricle, the porosity of the right ventricle luminal
layer did not change during the examined period; it
remained at around 38%. The porosity of the basal
layer increased from 18 to 36%, converging with values for the luminal layer. As with the values of compact layer thickness, there were no obvious differences between HH stages 24 and 27.
I.
_.. l._“_ _.. . .
. . . . _ . ..._ ..
..I I.^.
t
/
-40
-20
ml compact
5%
80
trabec 0 spaces
_. ._. . ._. ,.,.
, .~” ^_.,.__” . . “” ,_”
9 normal
7%
....,,~_X-
80
.v+._____I1-x
,,.lxx-““<^“--““-“--“-
-----j
*
compact
trabecm
spaces +?+ normal
Fig. 8. Ventricular wall composition of stage 29 hearts treated with
verapamil. Note the increased proportion of trabeculae
(normal values are shown by markers).
CHICK VENTRICULAR MYOARCHITECTURE
Verapamil-treated hearts showed a decreased proportion of compact myocardium and an increased proportion of trabeculae; heart development was retarded by about one day in comparison to non-treated
embryos (Fig. 8). However, the local porosity was
within normal limits. A statistically significant reduction of the compact layer’s thickness was observed in
stages 24 and 27 in both ventricles (Fig. 6).
DISCUSSION
Development of trabecular architecture
The first signs of trabeculation, of the initially smooth
inner ventricular wall, can be seen by HH stage 15 or
16 for the chick (Steding et al., 1982; Icardo & Fernandez-Teran, 1987) and between 8 and 9 dpc for the
mouse (Challice & Viragh, 1973). The trabeculae increase in number rapidly, filling most of the ventricular cavity within 24 hours (HH stage 21). Transformation to the definitive pattern, where trabeculation
becomes limited to the wall as a result of ventricular
expansion and a free lumen is re-established, occurs
at approximately HH stage 34 (Sedmera et al., 1997),
when the cardiac septation is completed. A qualitative description of the trabecular arrangement of the
chick embryonic heart has been made in earlier studies (Rychter & Rychterova, 198 1; Ben-Shachar et al.,
1985; Icardo & Fernandez-Teran, 1987). Our data refine the description of the non-compact (porous) part
of the ventricular wall (called “spongy” by Icardo &
Fernandez-Teran, 1987, or “trabeculated” by Rychterova, 197 1) by dividing it into two sublayers: basal
and luminal, as suggested by Rychterova (1982),
which are characterised by precise morphological criteria. The finer basal parts of the trabeculae were
sometimes overlooked in previous investigations due
to the use of suboptimal fixation. The presence of the
basal trabecular layer is temporary; it regresses during the development of coronary circulation (Rychter
& Ostadal, 1971; Vrancken Peeters et al., 1997). Its
function is probably mainly nutritive as suggested by
Minot (1901). It is consistent in its high degree of
isotropy and homogeneity; i.e. uniform size and the
absence of a preferred orientation of the intertrabecular spaces. These characteristics are clearly demonstrated by the mean linear intercept diagram (Fig. 4).
Using such a diagram, all the parameters of directional strength obtained from measurements on confocal
images using Denslow’s algorithms (Denslow et al.,
1993) can be determined which makes the technique
115
used in this paper available for people without a strong
mathematical background.
Rychterova (1971) made a quantitative study of
the further development of the right ventricular
trabeculae between incubation days 6 and 14 and described the process of the gradual disappearance of
the basal layer. There was an absolute reduction in
the number of trabeculae as established from frontal
sections from 24-26 on day 6 (HH stage 29) to 19 on
day 14 (HH stage 40) and a gradual thickening of the
remaining trabeculae as well as the compact layer. As
an underlying mechanism it was suggested that there
were variations in the orientation of mitoses in the
different regions of the trabeculae: random in the basal portion adjacent to the compact layer, and parallel
to trabecular axis in the luminal part. Our measurements of the local porosity ratio agree with these observations. The decrease of porosity in the basal layer
preceeds the factual compaction in the left ventricle.
In the right ventricle, where the process of compaction is delayed, we observed a diminution of the outside-to-inside porosity gradient. Later it was impossible to separate these two regions as the smallest
intertrabecular spaces disappeared during the process
of compaction and the remaining trabeculae thickened (Sedmera et al., 1997).
Methodological aspects
As making conclusions about three dimensional myocardial properties relying solely on a single dissection
plane would be tricky, we make reference to dissections performed in frontal (Steding et al., 1982; BenShachar et al., 1985) and sagittal (Pexieder, 1978)
planes as well as some additional dissections. From
this data it can be concluded that the three-dimensional orientation is radial, i.e. perpendicular to the
compact layer at any point. Differences between the
left and right trabecular patterns are in part caused by
different shapes of the embryonic ventricles, however, the left ventricular luminal trabeculae are coarser
and less branched, and the intertrabecular spaces larger and more oblong. This aspect is visible in both
diagrams used for the evaluation of orientation. Polar
histograms are useful for samples with several different directions, while the mean linear intercept technique is suitable for samples with one preferential
orientation; and can provide additional strength information. However, neither technique can distinguish
between polarized features oriented in multiple directions and unpolarized (circular) shapes giving both
116
D. SEDMERA ET AL.
cases an air of random distribution. Visual evaluation
of a colour feature classification by orientation is an
useful complement.
Another problem is the possible distortion of the
proportion of different layers in sections which are
not always perpendicular. This was sometimes viewed
in the apical area. Supplementary information was
obtained from frontal dissections which showed the
radial arrangement of the luminal trabeculae, as well
as the extent of the basal trabecular layer and the
circularity of its intertrabecular spaces. The shape of
the embryonic chick heart is more cylindrical than
spherical, unlike in mammals, so use of the dissection
plane perpendicular to its long axis should lead to
minimal error in comparison with other planes. Our
histological series, not included in this study, confirm
the above, and the error in the estimation of compact
layer thickness is minor. Furthermore, for comparative measurements we have chosen the midportion
area where the section plane is perpendicular to the
outer heart curvature.
The reported porosity values should be corrected
for section thickness (approximately 50 pm). This error might be eliminated using semithin sections (ca. 1
c,1111 thick) which can be considered to have zero thickness for this purpose. Such problem, which is still one
of the unresolved questions of stereology, does not
outweigh the advantages of using the method of “thick
slices” (Jaygasinghe et al., 1994). It was shown by
Zhu et al. (1994), that the variations in the directional
properties of porous biological materials such as cancellous bone from one section plane to another are
enormous, so the use of relatively thick “optical sections” (or, more precisely, profiles) gives a more realistic idea of existing patterns and properties. It is
sometimes difficult to define individual layers and
intertrabecular spaces on histological sections, and as
such time-consuming three-dimensional reconstructions (Blausen et al., 1990) and/or confocal microscopy (Usson et al., 1994) are required. We have shown
that consistent results can be achieved with the techniques used in this paper. These methods were used in
different stages and locations, giving data that were
consistent and comparable allowing observed gradients to be reproduced. It should be realized, however,
that trabeculations are like fingerprints: there is a
common building plan, but each heart has its own
unique, precise pattern of trabeculation similar to the
situation described in human mitral valves by Solomon and Nayak (1994).
Functional and comparative aspects
We agree with Challice and Viragh (1973), and
Wenink et al. (1996) that the extensive trabeculation
with a radial three-dimensional arrangement is important for the pumping function of the embryonic
heart. According to our results, the trabeculae form
most of the myocardial mass during early stages. Values between 64 and 84% were reported for early human embryos using three dimensional reconstructions
from histological series by Blausen et al. (1990). The
differences between left and right trabecular patterns
in humans were studied quantitatively by Wenink
(1992), and it was found that early trabeculae in the
right ventricle are finer than those of the left ventricle; this is in agreement with our observations of embryonic chicks. The cells in the trabeculae are more
differentiated, containing more myofibrils that are
better aligned than the cells in the compact zone
(Markwald, 1969; Challice & Viragh, 1973). The
myocytes in the conotruncus show a high degree of
differentiation and circumferential alignment which
is probably important for its sphincter-like function
(Tokuyasu, 1990).
The recently produced mice mutants with a disruption of genes for neuregulin or its receptors (reviewed
by Marchionni, 1995) lack most or all ventricular
trabeculation and die in utero at gestation day 10.5.
The probable cause of death was heart failure as an
irregular heartbeat and atria1 dilatation were observed.
These results give further evidence for the importance
of trabeculation in the proper pumping function of the
embryonic heart; these mice provide a model in which
the effects of the absence of trabeculation on contractile efficiency can be studied.
During development the proportion of the compact
layer becomes more substantial, especially in the left
ventricle. This suggests that from HH stage 29 onwards, the left ventricle relies mostly on this layer as
implied by its increasing thickness. This seems to correspond with the results of a comparative study performed by Ostadal and Schiebler (197 1) on fish hearts
of different species. Rather than finding a correlation
with the phylogenetic position, the proportion of compact myocardium increased parallel to body size (and
blood pressure) suggesting that the increased proportion of the compact layer is a way to improve heart
performance. Greer Walker et al. (1985) showed that
proportion of the compact myocardium is dependent
on the animal’s life style; more active fish have a
higher proportion of the compact myocardium. The
CHICK VENTRICULAR MYOARCHITECTURE
augmented proportion of the compact layer coincides
with the development of coronary circulation, which
starts in the chick embryo after a certain thickening of
the compact layer at HH stage 3 1. Details of this process and epicardial origin of the coronary endothelium
were demonstrated by Poelmann et al. (1993) using
chick-quail chimeras. This thickening seems to be
crucial for the proper pumping function of the heart at
later stages as shown by embryonic lethality at gestation day 12.5 of neuropilin-overexpressing mouse
embryos which have a very fine compact myocardial
layer and problems with ventricular septation (Kitsukawa et al., 1995). Embryonic lethality was reported
in VCAM-1 knockout mice which display a reduction
of the compact layer of the ventricular myocardium
and interventricular septum (Kwee et al., 1995).
Importance for perturbation studies
Measurements of the compact layer serve as markers
for experimental procedures that alter the load conditions of the embryonic heart. From the decreased proportion of compact myocardium relative to trabeculae
in verapamil-treated hearts one can draw the conclusion that a developmental increase of compact layer
thickness is a mechanism for increasing pumping efficiency. So the smaller number of cardiomyocytes of
normal size and composition found in verapamil-induced decreased afterload (Clark et al., 1991) is also
abnormally arranged. Less remarkable differences
seen at HH stage 29 could be caused by higher mortality of embryos with more severely affected hearts
or to insufficiency of dose due to the rapid embryonic
growth. However, the treated hearts showed no specific malformations. Compact layer thickness measurements could be useful in interspecies comparisons.
The developmental trends of compact layer thickness
are similar to those seen in the mouse heart between
gestation days 12 and 17 (Pham, 1997), but the increase in thickness of the compact layer of the left
ventricle is not as marked as in the chick. This resembles the adult state, where the left/right ventricular
wall thickness ratio is 51 in birds, but about 3:l in
human or other mammals (Komarek et al., 1985; our
unpublished observations).
In conclusion, we have devised a simple, reproducible methodology to analyse embryonic ventricular myocardial architecture. We use the above mentioned methods to describe the chick embryonic
ventricle during organogenesis and in the experimental model of decreased pressure load. It was shown
117
that left and right ventricle exhibit distinct morphogenesis from the time of their appearance. The developmental trends of parametres such as proportion of
the compact myocardium reflect the adaptation of the
heart to the gradually increasing demands of the growing embryo. Myocardial growth and architecture are
influenced by epigenetic factors. Thus, the in utero
environment is likely an important determinant of the
fabric of the mature myocardium influencing long
term survival.
ACKNOWLEDGEMENTS
We would like to express our thanks to Mrs. Ariane Gerber
for her excellent technical assistance, Dr. Richard Hayden
(Leica, England) for his advice about image analysis and
Dr. Penny Thomas (NHLI, London) for inspiring discussions and helpful criticism. Dr. Si Minh Pham is acknowledged for helpful discussions on mouse trabeculation development. Our further thanks belong to Mr. Didier Sevin
for his assistance in computing, to Ms. Tina Hernandez for
helping with the statistical analysis and Mr. Jay Legue for
final language revision. This work was supported by grants
from the Swiss National Science Foundation 31-33889.92
(T. Pexieder) and the National Heart, Lung and Blood Institute (NHLBI) Specialized Center of Research in Pediatric
Cardiovascular Diseases p50-HL51498 (E.B. Clark and N.
Hu).
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Received: May 1997
Accepted after revision: December 24, 1997