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THE ANATOMICAL RECORD 254:238–252 (1999)
Remodeling of Chick Embryonic
Ventricular Myoarchitecture
Under Experimentally Changed
Loading Conditions
DAVID SEDMERA,1,2* TOMAS PEXIEDER,2 VLASTA RYCHTEROVA,3
NORMAN HU,4 AND EDWARD B. CLARK4
1Institute of Physiology, Faculty of Medicine, University of Lausanne,
CH-1005 Lausanne, Switzerland
2Institute of Histology and Embryology, Faculty of Medicine, University of Lausanne,
CH-1005 Lausanne, Switzerland
3Department of Pathology, Third Medical Faculty, Charles University,
100 00 Prague 10, Czech Republic
4National 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 and Dentistry, Rochester, New York 14642
ABSTRACT
Adult myocardium adapts to changing functional demands by hyper- or
hypotrophy while the developing heart reacts by hyper- or hypoplasia. How
embryonic myocardial architecture adjusts to experimentally altered loading is not known. We subjected the chick embryonic hearts to mechanically
altered loading to study its influence upon ventricular myoarchitecture.
Chick embryonic hearts were subjected to conotruncal banding (increased afterload model), or left atrial ligation or clipping, creating a
combined model of increased preload in right ventricle and decreased
preload in left ventricle. Modifications of myocardial architecture were
studied by scanning electron microscopy and histology with morphometry.
In the conotruncal banded group, there was a mild to moderate
ventricular dilatation, thickening of the compact myocardium and trabeculae, and spiraling of trabecular course in the left ventricle. Right atrioventricular valve morphology was altered from normal muscular flap towards a
bicuspid structure. Left atrial ligation or clipping resulted in hypoplasia of
the left heart structures with compensatory overdevelopment on the right
side. Hypoplastic left ventricle had decreased myocardial volume and
showed accelerated trabecular compaction. Increased volume load in the
right ventricle was compensated primarily by chamber dilatation with
altered trabecular pattern, and by trabecular proliferation and thickening of
the compact myocardium at the later stages. A ventricular septal defect was
noted in all conotruncal banded, and 25% of left atrial ligated hearts.
Increasing pressure load is a main stimulus for embryonic myocardial
growth, while increased volume load is compensated primarily by dilatation.
Adequate loading is important for normal cardiac morphogenesis and the
Abbreviations used: HH, Hamburger-Hamilton; LAL, left atrial
ligation; LHH, left heart hypoplasia; LV, left ventricle; RV, right
ventricle; SEM, scanning electron microscopy; VSD, ventricular
septum defect.
r 1999 WILEY-LISS, INC.
Grant sponsor: Swiss National Science Foundation; Grant number: 3138889.92; Grant sponsor: National Institutes of Health
Specialized Center of Research; Grant number: P50-HL51489;
Grant sponsor: National Heart, Lung, and Blood Institute; Grant
number: HL42151.
*Correspondence to: David Sedmera, Department of Cell Biology and Anatomy, Medical University of South Carolina, 171
Ashley Avenue, Charleston, SC 29425. E-mail [email protected]
Received 10 March 1998; Accepted 10 September 1998
EMBRYONIC HEART REMODELING
239
development of typical myocardial patterns. Anat Rec 254:238–252,
1999. r 1999 Wiley-Liss, Inc.
Key words: conotruncal banding; left atrial ligation; compact myocardium;
trabecula; heart development; SEM
With increasing size of the organism, the heart architecture is modified accordingly to cope with increased demands. Examples of this relation between structural and
functional requirements can be found among species during ontogenesis. In fish, an increasing proportion of the
compact myocardium with emergence of coronary supply
correlates with the activity and life style of individual
species (reviewed by Agnisola and Tota, 1994). The proportion of compact myocardium increases during embryogenesis (Blausen et al., 1990; Pham, 1997; Sedmera et al.,
1998). Apart from increasing thickness, the architecture of
the myocardial wall undergoes extensive modifications
(Sedmera et al., 1997). Rychterova (1971) studied the
mechanism of thickening of the right ventricular wall in
the chick embryo, and showed that it is partly due to the
compaction of the basal parts of the trabeculae. These
results show that normal developmental adaptation to
physiologically increasing load is an augmentation of
proportion and thickness of the compact myocardium.
The influence of hemodynamics on heart morphogenesis
is well recognized. Recently, Hogers et al. (1997) demonstrated that there is a close correlation between modified
intracardiac streams and abnormal ventricular septation
after left vitelline vein clipping. Hemodynamic patterns
alter endocardial cells alignment (Icardo, 1989) possibly
due to increased shear stress. It is therefore reasonable to
expect that changed hemodynamic conditions may also
influence cardiomyocyte assembly at the organ level.
The heart has a considerable functional plasticity enabling compensation when the functional demands are
changed abruptly. The adult heart responds by hypertrophy and/or dilatation according to the speed and magnitude of the increased load (Hutchins et al., 1978; Castaneda et al., 1994; Rockman et al., 1994). Pressure load is a
more powerful stimulus for cardiac growth than volume
load (Zak, 1974). Embryonic or fetal heart responds to
altered load conditions in a different manner. Increased
pressure load induced by outflow tract constriction in the
chick embryonic heart (Clark et al., 1989) or ascending
aorta banding in the fetal guinea pig (Saiki et al., 1997)
results in cardiomyocyte hyperplasia rather than hypertrophy. Similarly, decreased afterload by treating the embryo
chronically with verapamil leads to smaller hearts that
contain fewer cells of normal size (Clark et al., 1991).
Investigations of rearrangement of cardiomyocyte assembly in this model of decreased load conditions revealed
decreased compact layer thickness with relatively more
trabeculae (Sedmera et al., 1998).
Another approach to study structural adaptations to
altered hemodynamic conditions is left atrial clipping or
ligation (LAL), which redirects the blood streams from left
to right thus resulting in underdevelopment of the left
heart structures and compensatory overdevelopment of
the right ventricle (Rychter and Lemez, 1964; Rychter et
al., 1979). Unlike conotruncal banding in which the heart
works against an increased resistance (afterload), this
procedure changes the distribution of blood flow thus
creating a combined model of increased/decreased preload.
At later stages in severe cases of left heart hypoplasia
(LHH), the right ventricle has to act as a systemic pump.
This model, based on mechanical intervention with normal
hemodynamics, is one possible experimental approach to
study human hypoplastic left heart syndrome. Production
of chick LHH was also achieved by obstructing the left
atrioventricular ostium with an insertion of nylon thread
(Harh et al., 1973; Sweeney, 1981).
Hypoplastic left heart syndrome is relatively rare congenital pathology occurring in approximately 3.8% of
human congenital heart disease but contributing significantly to its mortality. Characterization and description of
this syndrome are attributed to Lev (1952), who called it
‘‘hypoplasia of the aortic tract complex.’’ The term hypoplastic left heart syndrome was introduced by Friedman et al.
(1951). It occurs sporadically in other species as well—pig
(Shaner, 1949), cattle and lamb (van der Linde-Sipman,
1978), minipig (van der Linde-Sipman and Wensing, 1978),
and domestic cat (van Nie, 1980)—which suggests that its
cause lies in perturbation of a generally operating developmental mechanism.
In order to investigate the reactions of embryonic heart
to altered hemodynamic conditions, we studied modifications of myocardial architecture in hearts which underwent conotruncal banding (model of increased afterload) or
left atrial clipping or ligation (redistribution of preload) by
histology and scanning electron microscopy. We hypothesized that 1) increased pressure load leads to accelerated
development in compact layer thickness and proportion,
and variable degree of dilatation; and 2) increased volume
load is compensated mainly by dilatation with only mild
and delayed ventricular wall thickening while the volumeunderloaded ventricle is thought to show delayed development. We found that increased pressure load accelerated
morphogenesis expressed as increased thickness of the
compact myocardium, and in addition a precocious trabecular spiraling. Altered volume load accelerated morphogenesis of the volume-underloaded left ventricle (LV), manifested by sooner trabecular compaction and changes of the
lumen. The increased volume load in right ventricle (RV)
produced dilatation, followed by trabecular proliferation
and increased thickness of the compact myocardium. Our
results indicate that cardiac growth and morphogenesis
are concomitant but distinct processes and that proper
pressure and volume load are prerequisites for normal
development of ventricular myoarchitecture.
MATERIALS AND METHODS
Conotruncal Banding
Fertilized White Leghorn chicken eggs were incubated
blunt end up in a 38°C forced-draft incubator to Hamburger-Hamilton (HH) stage 21 (3.5 days, Hamburger and
Hamilton, 1951). The embryo was exposed via a window in
the shell and an incision of the inner shell membrane. A
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10–0 nylon suture was passed around the mid-portion of
the conotruncus, and tied in an overhand knot snug
against its wall, but without any constriction to the blood
flow. The window in the shell was then sealed with
parafilm, and the eggs were reincubated to HH stages 24,
27, and 29 (n ⱖ 5 per stage). Sham controls had the suture
passed and removed. Normal embryos were unoperated.
These stages were selected because they represent approximately twofold increase in embryo mass. Additional sampling was done at HH stage 34 in order to evaluate
morphological anomalies after the completion of cardiac
organogenesis and the rearrangement of embryonic to
fetal ventricular wall structure.
Left Atrial Ligation
The eggs were incubated to HH stage 21 as described
above. The egg was positioned under the photomacroscope,
and the egg shell and its membrane were removed to
expose the embryo. The embryo was gently turned to the
left side up position, and a slit-like opening was made in its
thoracic wall using a pair of fine forceps. An overhand knot
of 10–0 nylon suture loop was placed across the left atrium
and tightened, constricting the left atrioventricular orifice
and decreasing the effective volume of the left atrium. The
embryo was then gently flipped to its original right side up
position. The opening in the egg shell was sealed with
parafilm, and the eggs were returned to the incubator for
reincubation until HH stages 29 (n ⫽ 18) and 34 (n ⫽ 34).
Sham-operated embryos (n ⫽ 9) had the suture tied
adjacent to the left atrial wall. Normal embryos (n ⫽ 9)
were unoperated.
Left Atrial Clipping
In the first experimental series (Rychter et al., 1979),
left heart hypoplasia was produced by clipping the left
atrial anlage at day 4 of incubation using a custom
designed microclips from electrolytically purified silver
wire. The aim of this study was to make an initial
description of the chick left heart hypoplasia (Rychter and
Rychterova, 1981). Sampling for histology was done in 48
hr intervals at incubation days 6, 8, 10, 12 and 14 (n ⫽ 6
per stage).
Histological Sections
The isolated hearts were fixed in 10% neutral formol,
embedded into paraffin, and serial sections of 7 µm thickness were cut in frontal plane. Measurements of compact
myocardium and trabecular thickness, ventricular cross
section area and diameters were performed using ocular
micrometer and planimetry on representative sections
(one per heart) running through the atrioventricular junction (‘‘four chamber view’’). Quantitative analysis was
based on four normal and four experimental (presenting
LHH of different severity but all having left atrium to right
atrium ratio 1:4 or less) hearts per stage.
(Wild, Switzerland) for the purposes of tissue shrinkage
estimation and ventricular length measurement. The specimens were cut transversely to the long axis with thinned
microdissection scissors into 0.25–0.50 mm thick slices.
Additional specimens were dissected in frontal plane. 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, United Kingdom), and
photographed in a JSM 630 OF scanning electron microscope (JEOL, Japan). At minimum, five embryos per stage
and group were transversely dissected, and four were
selected for quantitative evaluation performed on SEM
micrographs.
Quantitative Analysis
We analyzed the SEM micrographs of transverse slices
(n ⫽ 4 per group). Compact myocardium thickness was
measured using a digimatic calliper (Mitutoyo, Japan) on
the midportion slice which contained both ventricles at
standard points (at the intercepts of lines drawn at 0 deg.,
⫹45 deg. and ⫺45 deg. from the center of the interventricular septum with the lateral wall). To determine the ventricular wall composition, cross-sectional areas contributed by the compact myocardium, trabeculae and
intertrabecular spaces were measured using user-developed planimetric macroprograms on Quantimet 970 (Leica,
Cambridge, England) in various locations. Trabecular
orientation was evaluated visually and verified by measurements performed on intertrabecular spaces using techniques of polar histograms, mean linear intercept and
color classification according to orientation (Cowin, 1986;
Usson et al., 1994; Sedmera and Pexieder, 1995; Sedmera
et al., 1998) on Quantimet 970. For volume estimations
performed on HH stage 34, cross-sectional areas of the
ventricles and their different components (compact myocardium, trabeculae, intertrabecular spaces, trabecula-free
lumen) were estimated by unbiased point counting technique on digitized SEM photographs using Analyze 7.1
(CNSoftware, London, United Kingdom) image analysis
system run on HP UNIX platform. Volumes were calculated using a formula derived from Cavalieri’s principle
(Cavalieri, 1635), according to which the volume of a
feature is a product of its length perpendicular to slicing
direction and its mean cross sectional area. Typically, over
1,000 points per slice were counted. Theoretical discussion
and precision estimation of this methodology were given
by Gundersen and Jensen (1987). Lengths of ventricles
were measured on frontal macrophotographs of undissected specimens. The values were corrected for tissue
shrinkage during dehydration and critical point drying,
which caused diminution of linear dimensions to approximately 59% of the initial values. All volumes are expressed
in dry state; to approximate the in vivo diastolic values
they could be multiplied by a factor of 4.87.
Scanning Electron Microscopy (SEM)
After removal of the embryos from the shells, the hearts
were perfusion-fixed at high flow low pressure (Pexieder,
1981) with 2% glutaraldehyde–1% formaldehyde and 2 ⫻
10⫺5% verapamil in isotonic 0.1 M cacodylate buffer. The
hearts were then postfixed in 1% osmium tetroxide, and
frontal views were taken under a M400 photomacroscope
Statistical Analysis
Statistical analysis of all quantifiable data (compact
layer thickness, myocardial volumes, ventricular diameters, trabecular counts) was performed by non-parametric Mann-Whitney test. Results P ⬍ 0.05 were considered
significant.
EMBRYONIC HEART REMODELING
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RESULTS
Conotruncal Banding
Discrete changes of myocardial architecture were detected at HH stage 24 (12 hr after banding). The whole
heart was dilated, and the thickness of the compact
myocardium (Fig. 1) was increased. The trabeculae appeared thickened as well. The conotruncus was markedly
dilated downstream after constriction, and the conotruncal ridges were hypoplastic.
By HH stage 27, the dilatation progressed, and the heart
shape became more spherical (Figs. 2, 3). The experimental heart was larger and distinctly elongated compared to
control. The trabeculae in the LV appeared coarser than
normal (Fig. 3), and the intertrabecular spaces in the RV
were smaller and more rounded. The trabeculae in the
supraapical part of the LV had started to spiral in some
cases counterclockwise when viewed from the base towards apex. The compact layer was thickened in both
ventricles, but its ratio to trabecular myocardium was not
significantly different from controls. The dilatation of the
conotruncus distally from the banded (stenotic) part was
even more distinct, as was the hypoplasia of the conotruncal ridges.
By HH stage 29, the differences between the sham
control and banded hearts were pronounced. In the banded
group, the LV lumen expanded, and extended as far as to
the apex (Fig. 4). The trabeculae in the supraapical portion
followed an evidently spiral course (Figs. 4, 5). Such
spiraling was never seen in control hearts at this stage,
though it was the characteristic of the definitive trabeculation pattern, and apparently started from HH stage 36
(Sedmera et al., 1997). The thickness of the compact
myocardium was increased (Fig. 1), but its ratio to the
trabeculated myocardium was augmented only in the
basal part of the LV, suggesting starting compaction. In the
RV, the external shape was markedly globular, consistent
with some degree of dilatation, and the interconnecting
segments between the trabeculae were more abundant.
The proportion of intertrabecular spaces was decreased,
and they appeared smaller than those of controls as a
result of trabecular coarsening and starting compaction.
Similarly to previous stages, the ratio of intertrabecular
spaces to trabeculae was slightly decreased but not substantially different from control in any ventricular region. As a
result of the increased number of cell layers (approximately 4–5 in control and 7–8 in banded), the contribution
of the compact layer augmented moderately in the apical
part, and its thickness was significantly increased. The
size of myocytes (as appreciated on high power view in
scanning electron microscope) was not different from controls. Coarsening of the atrial relief (pectinate muscles)
was noted, and the atrial wall appeared thicker than
control as well.
By HH stage 34, a ventricular septum defect (VSD) was
present (Fig. 6) with either double outlet right ventricle or
persistent truncus arteriosus, and trabecular compaction
was more advanced than in controls, resulting in increased
compact layer thickness (Fig. 1) and expansion of trabeculafree lumen. In some cases, morphology of right atrioventricular valve was changed from normal muscular flap-like
to bicuspid structure, similar to the mitral valve.
Fig. 1. Development of compact layer thickness between HH stages
24 and 34 in control and conotruncal banding groups. The values come
from measurements performed on critical point-dried material, so to
approximate the end-diastolic in vivo values these should be multiplied by
factor 1.92 for stages 24 to 29 and 1.69 for HH stage 34.
Left Atrial Ligation
In both series of experiments, qualitative and quantitative changes found at HH stage 29 (60 hr after ligation)
were generally mild. In the LV, the principal trabecular
sheets were more closely packed together (Fig. 5), while in
the RV, they were more widely spaced and thinner than
normal. In extreme cases, the normal predominantly
radial arrangement of RV trabeculae was changed to
parallel to the compact layer (Fig. 7) together with severe
dilatation and expansion of trabecula-free lumen. The
thickness of compact myocardium in either RV or LV was
similar to controls.
At HH stage 34, the changes in survivors (25–30% of the
operated embryos) were more pronounced. The trabeculae
in both ventricles were thinner than normal. In the LV,
they started to adhere to the lateral wall. The RV was
dilated and its trabeculae shifted toward the compact layer
(Fig. 8). Compact layer thickness was slightly decreased in
both ventricles in comparison with controls but not significantly due to considerable variability. In six of 20 examined hearts from the second series a VSD was observed in
subaortic position (Fig. 6), typically in moderate cases of
LHH.
Quantitative volume estimations performed at HH stage
34 revealed some significant differences between sham
and LAL hearts. Total volume of the LV was decreased
(0.691 ⫾ 0.143 vs. 1.107 ⫾ 0.185 mm3, P ⬍ 0.05) (mean ⫾
SD) while total volume of the RV was increased though not
significantly (1.488 ⫾ 0.517 vs. 1.075 ⫾ 0.418 mm3, P ⫽
0.16). This resulted into inversion of LV to RV ratio from
1.103 ⫾ 0.245 (sham) to 0.531 ⫾ 0.279 (LAL, P ⬍ 0.05)
while the volume of the entire heart remained similar
(sham 2.641 ⫾ 0.803, LAL 2.522 ⫾ 0.613 mm3, P ⫽ 1). The
volume changes differed by different ventricular compo-
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Fig. 2. Frontal view with indicated section planes and transverse slices obtained from sham HH stage 27
heart. Note the fine trabeculation filling most of the ventricular cavities and the extent of conotruncus division by
the conotruncal ridges. The entry to the conotruncus is marked by a star. Scale bars ⫽ 100 µm.
nents (Fig. 9). Most pronounced was the absolute volume
decrease of LV trabeculae compensated by increase of RV
trabeculae. In relative numbers, a significant decrease of
LV trabecular volume (P ⬍ 0.05) was accompanied by
relatively increased proportions of compact myocardium
(P ⫽ 0.16) and lumen (P ⬍ 0.05). Local porosity (percentage of intertrabecular spaces in the trabeculated myocardium) was slightly higher in LAL hearts but not significantly different between sham and LAL embryos in neither
ventricle. In the RV, the proportion of the compact myocar-
EMBRYONIC HEART REMODELING
Fig. 3. Transverse dissection of banded heart at HH stage 27. Note ventricular elongation and shape
change, most spectacular in the RV. The LV trabeculae are remarkably thicker when compared with control
(Fig. 2). The dilatation of conotruncus (entry marked by a star) is remarkable as well as the hypoplasia of the
conotruncal ridges. Scale bars ⫽ 100 µm.
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SEDMERA ET AL.
17.99 vs. 168.01 ⫾ 9.2 mm, P ⬍ 0.05) in the RV and
continued to be higher on day 14, but the process of
trabecular compaction was delayed, and dilatation persisted.
DISCUSSION
Fig. 4. Slices from normal (a) and banded (b) HH stage 29 hearts at
the apex level. Note the honeycomb arrangement of the LV trabeculae in
this area and presence of the RV on the cross section in the normal heart.
In the banded one, only LV with more circular cross section is present in
this location, its trabeculae show spiral course and there is a distinct
trabecula-free lumen. Scale bars ⫽ 100 µm.
dium was decreased as well as the lumen (P ⫽ 0.16 and
0.29, respectively).
The observations performed at later developmental
stages in the first experimental series showed the continuing trend of inversion of LV to RV cross-section ratio while
the total cross-section area of the ventricular compartment
did not differ significantly from controls. Similar information was obtained by measurements of the ventricular
length (Fig. 10). The phenotype of left heart hypoplasia
became gradually more pronounced (Fig. 11) with variable
penetrance. Right atrioventricular valve remodeling towards fibrous bicuspid structure was related in its extent
to degree of left ventricular hypoplasia. In extreme cases,
left atrioventricular valve was also dysplastic (Fig. 11) or
even atretic. There were more trabeculae than normal in
the hyperplastic RV on days 8 and 10, respectively (30.4 ⫾
0.54 vs. 25.0 ⫾ 0.35 and 29.8 ⫾ 0.46 vs. 27.4 ⫾ 0.21, P ⬍
0.05). They continued to be significantly thinner than
normal in both ventricles (data not shown). Compact layer
thickness which was not different from normal before
increased suddenly above control values at day 12 (82.65 ⫾
The selection of suitable quantitative parameters to
describe the complex patterns of embryonic myocardial
architecture is not easy. Quite straightforward solution is
the measurement of compact myocardium thickness, which
is altered under changed pressure loading (Sedmera et al.,
1998), and in some mutant mice with cardiac phenotype
(‘‘thin myocardium syndrome’’) where it is usually associated with poor survival in later fetal development (e.g.,
Sucov et al., 1994; Kastner et al., 1994; Jaber et al., 1996).
Estimation of myocardial volumes in different compartments is informative and reliable, but requires timeconsuming point counting (Bouman et al., 1997). The value
of simple length and area measurements giving a general
idea about heart shape and dimensions should not be
underestimated. All these techniques provide us with
statistically comparable values, but do not tell much about
trabeculation patterns.
The proportion of compact to trabeculated myocardium
and ventricular wall porosity give us some idea about the
myocardial fabric, but the planimetric approach employed
on SEM slices needs correcting for different slicing level
(Sedmera et al., 1998) thus preventing a rigorous statistical comparison. It provided two interesting results. First,
there is a gradient of increasing proportion of compact
myocardium from LV apex towards the base, which is
accentuated in the conotruncal banding group. Second, the
local porosity of the trabeculated myocardium did not
show substantial modifications under altered loading conditions, suggesting that this ratio must be maintained in
relatively narrow limits to ensure an efficient blood pumping in the trabeculated heart without coronary circulation.
The computer-based classification of intertrabecular spaces
does clearly demonstrate the anisotropy of the trabeculated myocardium, but is not of much help for quantification of differences between different groups and does not
add new information to visual appreciation of trabecular
patterns in SEM. There is probably no simple mathematical formula that could describe the complex pattern of
ventricular trabeculation, and careful dissection of wellperfused matched specimens stays the method of choice.
A topic deserving discussion is the exchange of material
between the compact layer and trabeculae. Initially, the
early compact layer is the source of cells for the trabeculae,
as it presents two to three times higher proliferative
activity and less cardiomyocyte differentiation (Jeter and
Cameron, 1971; Tokuyasu, 1990; Franco et al., 1997). On
the other hand, the compacting trabeculae start to add
volume to the free wall from approximately HH stage 31.
This process should be kept in mind when comparing the
values between different experimental groups. The increased thickness of the compact myocardium can come
either from accelerated or more abundant compaction
(probably the case for HH stage 34 banded hearts, and RV
at later stages of LHH), or increased proliferative activity
in stages preceding compaction. The importance of compaction for force generation by the definitive multilayered
compact myocardium is suggested by the lethality of
mutant mice where it is impaired (e.g., Sucov et al., 1994;
Kastner et al., 1994; Jaber et al., 1996). Delay of this
EMBRYONIC HEART REMODELING
245
Fig. 5. Ventral halves of frontally dissected control (a), banded (b) and LAL (c) HH stage 29 hearts. Note
spiraling of LV trabecular course in the banded heart as well as elongation of whole heart. The changes in LAL
heart are subtle, but slightly closer packing of the LV trabeculae can be noted. Stars mark the entry to the
conotruncus. Scale bar ⫽ 100 µm.
process might be responsible for impaired survival of LAL
embryos, as the trabeculated RV might not be able to
generate enough pressure to meet the increasing circulatory demands of the growing embryo at later stages. On
the other hand, acceleration of this process in the conotruncal banding group might not be too advantageous in the
absence of properly established coronary circulation (despite the normal capillary density, Tomanek et al., 1996),
and could account, together with increasing severity of
obstruction, for some later lethality in this group.
Conotruncal banding has, apart from increasing pressure load, also pronounced effects on morphogenesis, producing cardiovascular defects in survivors (89% lethality
until hatching; Clark et al., 1984, 1989). These can be
characterized by alignment problems of the outflow tract,
expressed as increased aortic-mitral separation with invariable occurrence of VSD, with either double outlet right
ventricle or persistent truncus arteriosus. This might well
be due, apart from mechanical intervention of the suture,
to changes in blood flow patterns (Hogers et al., 1997), and
hypoplasia of the conotruncal ridges. The influences of this
procedure on myocardial architecture were rapid and
pronounced. Initially, the heart dilated and became more
spherical, similar to changes in the adult heart (Hutchins
et al., 1978). Trabeculae normally fill the ventricular
cavities almost entirely between HH stages 21 and 31
(Ben-Shachar et al., 1985; Sedmera et al., 1997), and the
precocious reappearance of a trabecula-free lumen is a
sign of dilatation and accelerated trabecular compaction.
Consistent with this is slightly decreased proportion of
intertrabecular spaces. Together with relatively small
magnitude of this change (similar to insignificantly in-
creased porosity in LHH model), it suggests that there are
probably certain limits necessary for adequate myocardial
nutrition and efficient blood pumping that cannot be
overcome and define survival.
Increased pressure load is a powerful stimulus for
cardiomyocyte proliferation in the developing heart (Zak,
1974; Saiki et al., 1997). Indeed, purely hyperplastic
response was reported in this model by Clark et al. (1989).
This response occurs both in the compact myocardium and
trabeculae, as indicated by their increased thickness and
relatively minor changes in their ratio and supported by
our preliminary data on the proliferative structure. The
patterns of trabeculation are modified: the intertrabecular
spaces are smaller and more circular in the RV, and
trabecular spiraling occurs in the LV. This is an interesting
phenomenon, since it mimics the orientation of definitive
trabeculation in this location (Sedmera et al., 1997), and is
similar to the course of muscle fibers in the compact layer
(Streeter, 1979; Jouk et al., 1995). These are normal
features of development which likely reflect adaptation to
gradually increasing functional demands. We speculate
that spiraling is a mechanism of increasing the pumping
efficiency of the pressure-overloaded embryonic heart. In
unperfused contracted hearts from HH stages 24 to 31,
slight spiraling can be observed in the apical trabeculation
(Harh and Paul, 1974; our unpublished observations),
suggesting that the roughly orthogonal alignment is the
relaxed state, and the contraction is effected by an anticlockwise twist similar to the adult heart (Gibbons Kroeker
et al., 1993; Fig. 12). Accelerated trabecular compaction
with increased compact myocardium proportion and thickness between HH stages 29 and 34 is another phylogeneti-
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Fig. 6. Frontal dissection of HH stage 34 sham (a), banded (b) and
LAL (c) hearts. Note distorted atria, changed proportions of the ventricles
and defect of interventricular septum (star) in the LAL one. The trabeculae
in the LV show precocious adherence to the lateral wall and changed
orientation with circumferential alignment in the right. The muscular
flap-like morphology of the right atrioventricular valve is changed to a
bicuspid, mitral-like structure. Left atrioventricular valve is behind the
section plane because of the dorsal shift of the whole ventricle (compare
with Figure 8). Ventricular septum defect is seen also in banded heart,
where the trabecular compaction is distinctly advanced in both ventricles
resulting in thickening of the compact myocardium and interventricular
septum and similar modification of right atrioventricular valve morphology.
Dorsal halves, scale bar ⫽ 1 mm.
cally well established mechanism that increases heart
performance (Tota et al., 1983; Agnisola and Tota, 1994).
If only one ventricle has to function against increased
resistance, changes in the interventricular topography
take place (e.g., shift of the interventricular septum to the
left in an isolated RV pressure overload), as was demonstrated both mathematically and experimentally by Beyar
et al. (1993). However, both ventricles in conotruncal
banding have to act against increased resistance, so appropriate changes in shape occur in both, approaching to the
‘‘ideal’’ shape (spherical; Hutchins et al., 1978), and the
relative position of the interventricular septum remains
the same. On the other hand, shifting of the interventricular septum was observed in LHH or right ventricular
hypoplasia by Rychter et al. (1979), where uneven volume
loading occurs.
The phenotype of LAL hearts shows considerable variability from almost normal to extreme involution of the LV.
The results from different techniques (clipping or ligation)
are comparable. The selection of only distinct phenotypic
cases for quantitative analysis was essential, because
otherwise the already considerable variability of measured
parameters would completely obscure any significant differences. Relatively higher variability of LAL data reflects
the different penetrance of the phenotype. This continuous
spectrum of cases allows qualitative perception of RV
response to a graded overload. Generally small differences
between sham and LAL hearts at HH stage 29 could be
explained by persistent interventricular communication
which allows some blood to pass to the LV. The occurrence
of VSD (ca. 25%) is similar to results obtained recently by
Hogers et al. in left vitelline vein clipping model (Rychter
et al., 1979; Hogers et al., 1997), and might be likewise
attributed to changes of intracardiac blood stream patterns. Other associated abnormalities were described in
chick embryo LHH model (Rychter et al., 1979; Rychter
and Rychterova, 1981) and resemble features reported
from human pathology (e.g., Noonan and Nadas, 1958).
Not surprisingly, there is generally altered atrial morphology with an abnormal development of the interatrial
septum. In extreme cases, right atrioventricular valve
loses its typical muscular flap-like morphology and looks
more like a bicuspid fibrous valve, similar to the left
atrioventricular (mitral) valve. This resembles changes
observed under increased pressure load conditions, suggesting that loading is an important determinant of morphology of the developing valvular structures. The structure of
atrioventricular valves is often abnormal in congenital
heart disease. The myocardialization of the right atrioventricular valve is specific for birds, and does not normally
occur in mammals; but abnormal muscularization of the
antero-superior leaflet is a relatively frequent finding in
Ebstein’s malformation (e.g., Zuberbuhler et al., 1979),
indicating that this potential is existing as well. The
ascending aorta is hypoplastic, and the blood is conducted
by largely dilated ductus arteriosi. This led Rychter and
Lemez (1964) to propose it as a model of aortic coarctation.
The development of coronary circulation is delayed by
approximately 2 days at day 14 (Dbaly and Rychter, 1967).
Lymphatics are also abnormal, being absent in the hypoplastic LV and showing dysplastic valves in the basal
EMBRYONIC HEART REMODELING
Fig. 7. Transverse midportion slices of sham (a) and LAL (b) HH stage 29 hearts. Note marked dilatation,
and changed proportion of left to right ventricle. In this rather extreme case of LHH, RV trabeculae changed
their normal radial arrangement to parallel with the outer compact layer. Scale bar ⫽ 100 µm
247
248
SEDMERA ET AL.
Fig. 8. Midportion slices of HH stage 34 sham (a) and LAL (b) hearts. Note shrinkage and dorsalwards shift
of the LV and dilatation of the RV. Scale bar ⫽ 100 µm
EMBRYONIC HEART REMODELING
Fig. 9. Dry myocardial volumes of different compartments at HH stage
34 show decrease of LV muscular mass, mostly due to trabecular loss and
compensatory increase of the RV mass, again predominantly in the
trabecular component.
Fig. 10. Development of ventricular length, measured on frontal
histological sections in control and LHH hearts. Note the inversion of the
LV to RV proportion in the experimental group, resulting in switching of
their positions in the graph at HH 40.
portion of the hyperplastic RV (Rychter and Rychterova,
1981).
Adaptation of the RV to gradually increasing volume
load occurs in three steps. First, dilatation becomes evident as slight thinning and decreased proportion of the
compact myocardium. In extreme cases the trabeculae
249
even change their radial orientation (Rychter and Rychterova, 1981; Ben-Shachar et al., 1985; Sedmera et al.,
1997) to parallel with the outer compact layer, indicating
increased circumferential strain (Cowin, 1986). Second,
proliferation of trabeculae (starting from HH stage 34) is
suggested by their increased number and relative and
absolute volumes, but they are finer and their orientation
in some cases remains altered. Also diminished compact
layer thickness and increased local porosity (proportion of
the intertrabecular spaces within the non-compact myocardium) give evidence of persisting dilatation. Third, the
compact myocardium appears thicker, a finding we interpret as an acceleration of normal course of development
(Rychterova, 1971) and probably necessary for more efficient force generation (e.g., Greer Walker et al., 1985).
Delayed trabecular compaction underscores the more important role of RV trabeculae in contractile function than
in the LV, which starts to rely on its compact myocardium
sooner (Sedmera et al., 1997). Significant decrease in the
volume of RV free wall (compact myocardium) was reported from the most severely affected hearts (double
outlet right ventricle) of HH stage 34 chick embryos after
treatment with retinoic acid (Bouman et al., 1997) and
seemed to correlate with decreased heart performance.
Although volume estimates were made from serial histological sections, their data correspond quite well with our
results.
In pediatric pathology, coarsening of the trabecular
relief and thickening of the RV compact myocardium is a
common finding in cases where the RV functions as a
systemic pump (e.g., Oren et al., 1994). This thickening
thus seems to be a secondary effect of increase in the
pressure load. The relatively slow nature of these changes
contrasts with fairly rapid response (increased cell number, compact layer and trabecular thickening) observed as
soon as 24 hr after increased pressure loading (Clark et al.,
1989). The proliferative structure of LAL hearts is altered
(Rychter et al., 1979; Rychter and Rychterova, 1981) with
overall decrease in mitotic counts. It shows that increased
pressure load in the chick embryonic heart is a powerful
growth stimulus for hyperplastic cardiomyocyte growth
while increased volume load is compensated preferentially
by cardiac dilatation.
The changes in myocardial architecture of the underloaded LV are characterized by precocious adherence of
trabeculae to the lateral wall and their compaction, perhaps resulting from their closer packing in diminished
space. This is illustrated by shift of LV composition at HH
stage 34, where proportion of trabeculae is decreased in
favor of compact myocardium and lumen. This indicates
an acceleration of the normal developmental processes
which shows that growth and morphogenesis are two
concomitant but distinct processes. On the other hand,
decreased pressure loading achieved by chronic verapamil
suffusion results in hypoplastic hearts containing fewer
cells of normal size (Clark et al., 1991) with decreased
proportion and thickness of the compact myocardium
(Sedmera et al., 1998). It would be interesting to extend
the pilot experiments of Rychter et al. (1979) of inverted
procedure (right atrial clipping), which results in hypoplasia of the RV and hyperplasia of the LV. The authors
reported changes in inner heart relief and altered heart
form but detailed analysis was prevented by poor survival
of operated embryos, perhaps due to problems with outflow
tract alignment. In addition, the higher occurrence of VSD
250
SEDMERA ET AL.
Fig. 11. Frontal sections of control (a,c) and LHH (b,d) hearts at HH
stage 38 (a,b) and 40 (c,d). Note remarkably thickened RV compact
myocardium and inversion of the interventricular septum in the LHH at HH
stage 38. Moderate changes in the right atrioventricular valve morphology
are present in this case, while much more remarkable remodeling towards
mitral-like morphology can be seen in severe case at HH stage 40 as well
as dysplastic changes (thickened appearance, less condensed fibrous
tissue) in the left atrioventricular valve. Scale bars ⫽ 1 mm.
(60% of cases) made the search for consistent rearrangements of ventricular myoarchitecture more difficult.
We have shown differential response of the embryonic
heart to changes in pressure and volume loading (summa-
rized in Fig. 12). Increased pressure load induced first
dilatation, compensated rapidly by cardiomyocyte proliferation, trabecular and compact myocardium thickening, and
precocious spiraling and compaction of the trabeculae, all
EMBRYONIC HEART REMODELING
Fig. 12. A diagram summarizing the mechanics of trabeculated heart
contraction at HH stage 29 in normal and experimental hearts. In banded
heart, there is a thickening of the compact myocardium and trabeculae,
and spiralled trabecular course even in diastole. LAL heart presents
dilatation of the right ventricle with alteration of preferential orientation of
its trabeculae from radial to circumferential. The left ventricle is shifted
dorsally.
of which can be regarded as an acceleration of normal
developmental processes. Decreased volume load resulted
in shrinkage and accelerated morphogenesis of the LV.
Increased volume load to the RV led to its dilatation, only
later followed by trabecular proliferation and thickening of
the compact myocardium. The slow rate of compensatory
processes with high mortality of operated embryos may
reflect the poor ability of the RV to function as a systemic
pump. Its morphologic (Hirokawa, 1972; Sedmera et al.,
1997) and molecular (Zimmer, 1994) differences seem to be
the factors responsible for the invariably fatal outcome of
unoperated LHH in humans. Our study indicates the
importance of analysis of myocardial architecture in the
abnormal hearts, as it is of considerable importance for
their performance. Such data, supplemented by functional
evaluation obtainable for example by recent technique of
magnetic resonance tagging (Young et al., 1996), could
influence the considerations of suitability for operation in
some congenital cardiovascular anomalies such as transposition of great arteries (Castaneda et al., 1994).
ACKNOWLEDGMENTS
We thank Mrs. Ariane Gerber and Mr. Claude Verdan for
excellent technical assistance, and Mr. Franco Ardizzoni
for technical support with scanning electron microscopy.
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