Download Myocardial Volume and Organization Are Changed by Failure of

DEVELOPMENTAL DYNAMICS 228:152–160, 2003
ARTICLE
Myocardial Volume and Organization
Are Changed by Failure of Addition of
Secondary Heart Field Myocardium to the
Cardiac Outflow Tract
T. Mesud Yelbuz,1,2 Karen L. Waldo,1 Xiaowei Zhang,3 Marzena Zdanowicz,1 Jeremy Parker,1
Tony L. Creazzo,1 G. Allan Johnson,3 and Margaret L. Kirby1*
Cardiac neural crest ablation results in primary myocardial dysfunction and failure of the secondary heart field to add
the definitive myocardium to the cardiac outflow tract. The current study was undertaken to understand the changes
in myocardial characteristics in the heart tube, including volume, proliferation, and cell size when the myocardium
from the secondary heart field fails to be added to the primary heart tube. We used magnetic resonance and confocal
microscopy to determine that the volume of myocardium in the looped heart was dramatically reduced and the
compact layer of myocardium was thinner after neural crest ablation, especially in the outflow tract and ventricular
regions. Proliferation measured by 5-bromo-2ⴕ-deoxyuridine incorporation was elevated at only one stage during
looping, cell death was normal and myocardial cell size was increased. Taken together, these results indicate that
there are fewer myocytes in the heart. By incubation day 8 when the heart would have normally completed septation,
the anterior (ventral) wall of the right ventricle and right ventricular outflow tract was significantly thinner in the neural
crest-ablated embryos than normal, but the thickness of the compact myocardium was normal in all other regions of
the heart. The decreased volume and number of myocardial cells in the heart tube after neural crest ablation most
likely reflects the amount of myocardium added by the secondary heart field. Developmental Dynamics 228:152–160,
2003. © 2003 Wiley-Liss, Inc.
Key words: myocardium; neural crest ablation; secondary heart field; heart; development; MRI; myocardial volume
Received 21 April 2003; Accepted 6 June 2003
INTRODUCTION
Cardiac neural crest (CNC) ablation
in chick embryos results in the loss of
cardiac outflow tract septation and
malalignment of the outflow tract
(Kirby and Waldo, 1995). However,
long before these manifestations of
congenital heart defects can be
1
seen, the absence of CNC cells in
the pharynx of CNC-ablated chick
embryos results in primary myocardial dysfunction (Farrell et al., 1999).
This dysfunction is accompanied by
failure of the secondary heart field
to add the definitive myocardium to
the cardiac outflow tract (Yelbuz et
al., 2002). These results suggest that
the elongation of the outflow limb of
the cardiac tube by the secondary
myocardium is a critical step in the
normal process of looping insofar as
it provides myocardium that allows
the outflow tract to achieve a length
consistent with its role in attaining
Neonatal Perinatal Research Institute, Division of Neonatology, Duke University Medical Center, Durham, North Carolina
Department of Pediatric Cardiology, University Children’s Hospital, Münster, Germany
Center for In Vivo Microscopy, Duke University Medical Center, Durham, North Carolina
Grant sponsor: NIH; Grant numbers: HL36059; HD17063; HD39946; P41-05959; Grant sponsor: German Heart Foundation, German Research
Council; Grant sponsor: American Heart Association, Georgia Affiliate.
Dr. Yelbuz and Mrs. Waldo contributed equally to this work.
*Correspondence to: Margaret L. Kirby, Department of Pediatrics, Division of Neonatology, Box 3179, 157 Bell Building, Duke University
Medical Center, Durham, NC 27710. E-mail: [email protected]
2
3
DOI 10.1002/dvdy.10364
© 2003 Wiley-Liss, Inc.
MYOCARDIUM AFTER NEURAL CREST ABLATION 153
TABLE 1. Myocardial Volume (mm3) in Sham-Operated and CNC-Ablated
Embryos at Stage 22 Measured in MRM Imagesa
Sham
CNC-ablated
% change
P value
n
OFT
Ventricle
Atrium
5
5
191.8
74.0
261.4
0.001051
449.2
147.5
267.2
0.008714
318.6
160.7
249.1
0.040847
a
CNC, cardiac neural crest; MRM, magnetic resonance microscopy; OFT, outflow
tract.
normal alignment for wedging of the
aorta, which is critical to establish
continuity of the aorta with the left
ventricle, and alignment of the outflow septum with the ventricular and
atrioventricular septa (Yelbuz et al.,
2002). However, it is still unclear how
much myocardium is added to the
outflow tract by the secondary heart
field.
There are two ways to determine
the amount of myocardium added
from the secondary heart field. One
is to permanently mark the cells in
the secondary heart field, allow
them to incorporate into the outflow
tract, and measure the number or
volume of labeled myocardial cells.
Because we do not as yet have a
technique that could reliably mark
the cells of the secondary heart
field, this method is currently impractical. The second method is to inhibit
addition of cells from the secondary
heart field and measure the amount
of myocardium in the heart tube in
the absence of these cells. This
method is less accurate but should
provide an estimate of the amount
of myocardium added. Because
CNC ablation inhibits addition of
cells from the secondary heart field
to the heart, it provides the experimental disruption required.
CNC-ablated embryos are almost
always obvious because of the abnormal shape of the looped heart;
however, morphometric measurements in fixed hearts fail to show significant differences in the length of
the outflow tract or the ventricular
portion of the loop (Waldo et al.,
1999). On the other hand, close examination at Hamburger Hamilton
(HH, Hamburger and Hamilton, 1951)
stage 15/16 shows that the outflow
tract is straighter and the inner curvature smaller, which make the outflow appear shorter, whereas at HH
stage 22, the inner curvature and
outflow limb appear more normal
(Yelbuz et al., 2002). These apparent
discrepancies could be resolved by
determining the volume of myocardium rather than measuring outside
dimensions.
Magnetic resonance microscopy
(MRM) allows assessment of volume
without introduced artifacts that accompany reconstruction of histologic sections (Zhang et al., 2003).
We used MRM to perform three-dimensional (3D) morphovolumetric
analysis of chick hearts at HH stage
22 and compared myocardial volumes in normal hearts with hearts
from chick embryos after CNC ablation. This was coupled with confocal
microscopy to assess the organization
and thickness of the myocardial wall.
We determined that the volume of
myocardium in the heart was dramatically reduced and that the compact
layer was thinner. These changes
were not confined to the outflow
myocardium, indicating that myocardial changes occur throughout the
heart tube. At the same time, myocardial proliferation and cell size are
increased during the looping period
while myocardial cell death is absent in both sham-operated and
CNC-ablated embryos. On incubation day 8, when outflow septation
was nearing completion, the myocardial cell number and wall thickness recovered normal dimensions
with the exception that the anterior wall of the right ventricle and
the proximal outflow tract were significantly thinner in the CNC-ablated embryos. These results suggest that the secondary heart field
adds a significant volume of myocardium to the heart and that failure of this myocardium to be
added results in significant disorganization of the compact myocardium.
RESULTS
Myocardial Volume Is
Decreased After CNC Ablation
We used MRM to determine the volume of myocardium in the chick
heart at stage 22 in both sham-operated and CNC-ablated embryos.
Myocardial volume as described
TABLE 2. Number of Myocytes at Incubation Days 5 and 9a
Age
Treatment group
n
Body weight
(g)
Ventricle weight
(mg)
ID5
Sham
CNC-ablated
Sham
CNC-ablated
5
5
5
5
0.323 (0.053)
0.242 (0.028)b
1.89 (0.23)
1.78 (0.28)
2.20 (0.24)
1.57 (0.13)b
13.6 (4.2)
14.7 (2.4)
ID9
a
Data presented as mean (SD). ID, incubation day; CNC, cardiac neural crest.
P ⬍ 0.05 comparing Sham and CNC-ablated groups.
c
P ⫽ 0.075.
b
Cells/ventricle
(⫻105)
Cells/mg
ventricle
5.2 (0.9)
3.8 (0.2)b
28.6 (7.7)
34.0 (4.5)
2.4 (0.3)
2.5 (0.3)c
2.1 (0.2)
2.3 (0.2)
154 YELBUZ ET AL.
Fig. 1. Magnetic resonance microscopy (MRM) images from stage 22 sham-operated vs. Cardiac neural crest (CNC) -ablated embryos
in frontal section showing the cranial displacement of trabeculated myocardium (arrows) from the ventricle into the outflow tract (OFT)
as previously reported in histologic sections by Yelbuz et al. (2002). The white line indicates the level of the inner curvature.
here is the volume occupied by cells
comprising the myocardial wall. We
found excellent correlation between images obtained from MRM
with those obtained by routine histology (Zhang et al., 2003). The 3D
MRM reconstructions of hearts from
sham-operated vs. CNC-ablated
embryos were made, and these
could be viewed as sections in any
plane (Fig. 1). In the frontal plane,
these images illustrate a significant
degree of cranial displacement of
trabeculated myocardium from
the right ventricle into the outflow
tract in CNC-ablated embryos as
reported previously (Yelbuz et al.,
2002). This finding suggests rearrangement of the myocardium in
the primary heart tube to compensate for the failure myocardium to
be added from the secondary
heart field. Average myocardial
volume in sham-operated and
CNC-ablated hearts is shown in Table 1. The reduction in myocardial
volume ranged from 50 to 60% with
the greatest reduction in the outflow and ventricular myocardium.
Fig. 2. Histogram showing the distribution of cell sizes in sham-operated vs. cardiac
neural crest-ablated (Exp) myocardial cells at stage 22.
MYOCARDIUM AFTER NEURAL CREST ABLATION 155
TABLE 3. Average Thickness of the Compact Myocardium in Various Regions of the Right and Left Ventricles at ID8a
Anterior
RV wall
n
Sham
CNC-ablated
P value
a
81 ⫾ 4
50 ⫾ 4
0.001
5
5
Lateral
RV wall
101 ⫾ 3
100 ⫾ 10
0.925
Posterior
RV wall
Lower
RVOF wall
Upper
RVOF wall
Ventricular
septum
69 ⫾ 4
78 ⫾ 11
0.490
128 ⫾ 6
84 ⫾ 4
0.001
131 ⫾ 9
78 ⫾ 7
0.002
475 ⫾ 55
440 ⫾ 31
0.570
Lateral
LV wall
140 ⫾ 7
143 ⫾ 16
0.800
Data presented as mean ⫾ SD. RV, right ventricle; RVOF, right ventricular outflow; LV, left ventricle; ID, incubation day.
TABLE 4. BrdU Incorporation in Stages 12–18 Sham and Cardiac Neural Crest-Ablated Embryosa
Number of BrdU-positive cells per 50 cells
Outflow
St 12
Sham
CNC-ablated
PNC-ablated
St 14
Sham
CNC-ablated
St 16
Sham
CNC-ablatedc
St 18
Sham
CNC-ablated
n
Inner
curvature
8
5
5
Ventricle
Inflow
Outer
curvatureb
Inner
curvature
Outer
curvatureb
7.5 (2.4)
5.0 (2.2)
2.3 (2.2)
26.5 (3.4)
13.3 (5.3)
9.0 (1.8)
10.3 (2.8)
5.3 (1.0)
5.3 (2.6)
21.5 (8.3)
14.3 (1.9)
11.8 (2.5)
7.8 (4.1)
6.3 (3.0)
6.3 (1.7)
17.0 (2.9)
12.3 (5.5)
10.0 (2.9)
5
5
10.5 (1.9)
11.5 (5.8)
30.0 (5.4)
42.0 (13.3)
22.3 (8.7)
21.3 (9.7)
34.3 (2.2)
42.3 (11.1)
21.5 (6.6)
23.5 (7.6)
26.3 (4.8)
33.8 (17.5)
5
5
9.2 (3.4)
14.5 (6.3)
17.8 (6.8)
31.3 (5.9)
17.0 (7.9)
21.5 (10.3)
34.8 (5.1)
41.5 (8.7)
13.8 (6.2)
20.0 (10.3)
21.5 (7.2)
37.0 (15.5)
5
5
16.3 (9.5)
16.3 (4.3)
23.0 (6.0)
34.8 (5.5)
19.3 (1.0)
23.3 (3.4)
35.8 (5.7)
39.8 (2.6)
22.8 (3.8)
18.8 (5.1)
38.5 (7.7)
33.0 (12.4)
Inner
curvature
Outer
curvatureb
a
Data presented as mean (SD). BrdU, bromodeoxyuridine; St, stage; CNC, cardiac neural crest; PNC, preotic neural crest.
Significantly higher at all stages, P ⱕ 0.0002.
c
Significantly higher than shams in all regions, P ⫽ 0.003.
b
Myocardial Cell Size Is
Increased After CNC Ablation
The average area occupied by
sham-operated myocardial cells
was 14,785 (dimensionless units, n ⫽
663) compared with 29,711 (n ⫽ 636)
in the CNC-ablated embryos (P ⱕ
1 ⫻ 10⫺10). A histogram showing the
distribution of cell sizes can be seen
in Figure 2. Because of the reduced
volume and increased size of myocardial cells at stage 22 (day 4) after
CNC ablation, we surmise that there
are fewer cells.
Myocardial Cell Number Is
Reduced at Incubation Day 5
but Recovers by Day 9
Because the number of cells in the
CNC-ablated heart is less than normal
at day 4 of incubation, we examined
two additional days, one before car-
diac septation (day 5) and a second after cardiac septation is complete (day 9), to determine
whether recovery of cell number
occurs. By using a routine histologic
technique for assessing cell number, the number of myocardial
cells per ventricle was obtained at
two later times, incubation day (ID)
5 and ID9 (Table 2). Myocardial cell
number continued to be reduced
at ID5 but recovered by ID9 (Table
2), indicating a restitution of normal
cell number during the septation
phase of development.
Composition and Thickness of
the Myocardial Wall Is Altered
The organization of the myofibrils allowed identification of different layers across the myocardial wall. The
number of layers changed over time
and by region of the heart from two
at stage 12 to four at stage 22 (summarized in Fig. 3). At stage 12, a surface layer of the myocardium comprised the external layer of the heart
wall and this was underlain by an
inner layer of myocardial cells with
mostly disorganized myofibrils abutting the cardiac jelly. At stage 14,
the myocytes adjacent to the cardiac jelly became organized into a
third distinct layer containing wellaligned myofibrils in the ventricular
(midloop) portion of the loop. By
stage 16, both inflow and ventricular
portions of the myocardium had
three layers while the myocardium
of the outflow portion of the loop
continued to have only inner and
outer layers. In the CNC-ablated
embryos, no third layer appeared in
the outflow tract of stage 16 hearts.
At stage 18, the inflow and outflow
myocardium of sham and CNC-ab-
Fig. 3. A: Confocal images of whole-mount embryonic hearts from sham-operated embryos stained with rhodamine phalloidin at
stages (St) 12, 14, 16, 18, and 22 show sites where measurements of myocardial thickness were made. Rectangles demarcate the imaging
sites for each region of the heart. o, outflow tract; m, midloop (ventricle or prospective ventricular area); i, inflow region. B–D: The average
thickness of the myocardial wall, measured by focusing through the myocardial depth with a confocal microscope, was thinner in
cardiac neural crest (CNC) -ablated embryos. B: The outflow tract myocardium of CNC-ablated embryos was significantly thinner at all
stages. C,D: Although the myocardium was thinner in the ventricular and inflow regions, the reduction was not significant, except at the
earliest times.
lated hearts showed three layers,
whereas the ventricular portion of
the loop began to show a fourth,
trabeculated layer. A definitive
compact myocardium was present
at stage 22 that was composed of
the three outer layers. The fourth trabeculated layer had become much
thicker than the compact layer.
When focusing through the thickness of the myocardial wall, there was
a general impression in CNC-ablated
embryos that the depths of the myocardial wall and its individual layers
had decreased and that there were
fewer myofibrils. To confirm that the
myocardium was thinner in the CNCablated embryos, the width of the
myocardial wall was measured in the
inflow, midloop, and outflow portions
of the heart loop by using confocal
microscopy. In all stages examined,
there was a reduction in the thickness
of the myocardial wall in all regions of
the CNC-ablated hearts (Fig. 3). This
was most apparent in the outflow
tract in which the average width of
the myocardial wall decreased significantly over time (39%; Fig. 3B). The
ventricular (midloop) and inflow regions of the heart loop suffered a
much smaller decrease in myocardial
wall thickness (17% and 15%, respectively; Fig. 3C,D).
Ventral Wall of the Right
Ventricle and Outflow Is
Thinner after CNC Ablation
To determine whether the reduction
in the myocardial wall during looping impacted the postseptation
heart, we compared the compact
myocardial layer of incubation day
8 CNC-ablated and sham-operated
hearts. Measurements were made
of the ventral, dorsal, and lateral
right ventricular walls, the left ventricular free wall, and the ventricular septum (Fig. 4). Most of the
measurements from the myocardium of the CNC-ablated embryo
were similar to those from the
sham-operated embryo. However,
the ventral wall of the right ventricle and the proximal outflow tract
were significantly thinner in the
CNC-ablated hearts (Table 3).
Myocardial Proliferation Is
Altered After CNC Ablation
Because proliferation of cardiomyocytes in the primary heart tube
would have a significant impact on
our assessment of myocardium
added from the secondary heart
field, we examined 5-bromo-2⬘-deoxyuridine (BrdU) incorporation as a
measure of cell division. Significantly,
more BrdU-labeled cells were seen
in the myocardium of the outer curvature than the inner curvature in all
regions of the heart loop in both
CNC-ablated and sham-operated
embryos between stages 12 to 18.
Within the outer curvature itself, differences were apparent in BrdU labeling in the CNC-ablated vs. shamoperated embryos. At stage 16,
proliferation in the outer curvature of
the CNC-ablated hearts was significantly higher than that in sham
hearts (Table 4). No differences were
seen in the BrdU incorporation in endocardium or epicardium between
the CNC-ablated and sham-operated embryos. As noted previously
(Waldo et al., 1999), the thickness of
the cardiac jelly was variable in the
experimental embryos, whereas it
was uniform in sham-operated embryos.
While myocardial proliferation was
increased in CNC-ablated hearts at
stage 16, cell death was normal at
all stages, i.e., in both the sham and
CNC-ablated hearts, there was a
complete absence of dying cells
from the outflow and ventricular regions of the myocardium. In the inflow myocardium, a few dying cells
could be seen in the atrioventricular
canal and sinus venosus (data not
shown). These results are consistent
with those of Watanabe et al.
(1998), who found that there was no
cell death in the looping chick heart
before stage 22.
DISCUSSION
To understand how the heart is affected when myocardium fails to be
added from the secondary heart
field, we have examined myocardial development from several perspectives by performing tests to determine the volume of myocardium,
myocardial proliferation, cell death,
and organization. By using 3D highresolution MRM, we were able to determine that the myocardium derived from the primary heart fields
and comprising the heart tube until
looping, was significantly reduced in
all portions of the tube but most severely in the ventricle and outflow
regions where myocardium should
be added from the secondary heart
field. We have shown previously that
the length of the tube is shorter and
straighter in living embryos (Leatherbury et al., 1990) after CNC ablation
but appears to have a normal
length in postmortem specimens
even though the configuration of
the outflow tract is abnormal (Waldo
et al., 1999; Yelbuz et al., 2002). This
finding suggests a rearrangement of
myocardial cells in the looping heart
tube to compensate for the lack of
myocardial cells that should have
been added from the secondary
heart field. This rearrangement is further substantiated by the presence
of trabeculae at the base of the outflow tract seen in this and a previous
study (Yelbuz et al., 2002).
Assessment of the myocardial wall
confirmed that the compact myocardium is thinner in the CNC-ablated hearts during the looping period with the greatest effect in the
ventricular and outflow myocardium. At the same time, myocardial
cell size is larger, indicating that
there are significantly fewer cells in
the CNC-ablated hearts, as might
be predicted by the failure of myocardial cells to be added from the
secondary heart field. This finding is
confirmed by actual myocardial cell
counts at incubation day 5, showing
that the myocardial cell population
is still below normal. Thus, we believe
that the myocardial cells in the primary cardiac tube, i.e., those derived from the primary heart fields,
respond to the lack of addition of
myocardium from the secondary
heart field by rearranging a smaller
number of larger cells in an attempt
to compensate for the missing cells.
We have noted previously some
other changes in the myocardium after CNC ablation. At the microscopic
level,
myofibrillar
disorganization
could be seen in the outflow myocardium at stages 14 –18 (Waldo et al.,
1999). Of interest, although a prolifera-
158 YELBUZ ET AL.
Fig. 4. Transverse sections of hearts from sham-operated and cardiac neural crest-ablated embryos at incubation day 8. Sections have
been matched at mid-ventricular (top row), proximal right ventricular outflow (RVOF; middle row), and a slightly more cranial RVOF level
(bottom row). The magnification bars (100 ␮m) are on the dorsal surface of the heart in each panel. Black bars mark the region and
thickness of the compact layer sampled. Five measurements were made in the midventricular sections, but only one measurement was
made in the two levels of the right ventricular outflow to determine whether the thin compact layer of the ventral right ventricular wall was
extended into the outflow tract myocardium. Numeric results are presented in Table 3. LV, left ventricle; RV, right ventricle.
tive response might have been predicted as a compensatory response,
proliferation was abnormally enhanced at only one stage during looping. The distribution of the cardiac jelly
was irregular; causing changes in the
shape of the heart loop (Waldo et al.,
1999). Here, we show that myocardial
proliferation is normal, except at stage
16 when it is up-regulated in the entire
heart tube. Our data are consistent
with that of Sissman (1966) and Stalsberg (1969), who showed that the primary heart field and looping heart in
avian embryos undergo waves of
stage-correlated synchronous regional
proliferation. Although up-regulation of myocardial proliferation after CNC ablation for a few hours
might serve to restore some of the
myocardial cell numbers, it obviously is not enough to reconstitute
a normal population of myocardial
cells. This up-regulation results in a
dramatic decrease in myocardial
volume at stage 22. Because myocardial cell death is absent, the
missing volume of myocardium is
due to the failure of myocardial
cells to be added from the secondary heart field.
Even though the number of myocardial cells is still below normal at ID5, by
ID9 the number of myocardial cells is
normal. Because the ventricular wall
thickness at ID8 was normal except for
the anterior wall of the right ventricle
and the cell number is normal at ID9,
the inference can be made that the
cell size is restored to normal. How does
the normal myocardial cell number relate to the thin anterior right ventricular
MYOCARDIUM AFTER NEURAL CREST ABLATION 159
wall at day 8? It is most likely due to the
finding that the anterior wall of the right
ventricle is a small proportion of the entire myocardium and the margin of error in estimating the total number of
myocytes in the ventricles is too large to
distinguish this regionally localized thinning of the myocardial wall.
A remaining question to be answered is why the myocardium of
the secondary heart field is not
added to the heart after CNC ablation because the CNC would not be
in proximity to the secondary heart
field in an intact embryo. Although
more experiments will be needed to
determine the answer, we have
shown previously that CNC alters the
availability of FGF8 in the pharynx
(Farrell et al., 2001). Presumably, the
commitment and/or differentiation
of myocardial cells from the secondary heart field is influenced by a variety of signals that emanate from
the pharynx (Waldo et al., 2001).
CNC could alter the availability of
such signals, which would be in superabundance in its absence. The
identity of these hypothetical factors
is still to be determined.
EXPERIMENTAL PROCEDURES
Embryo Preparation
CNC-ablated and sham-operated
embryos were prepared as described previously (Waldo et al.,
1999). Embryos were collected at
stages 12, 14, 16 18, 22, and ID8. Embryos used for myocardial proliferation studies were treated in ovo 1 hr
before collection with 20 ␮l of a 40
mM BrdU solution according to the
method used by Brand-Saberi et al.
(1995). Embryos collected for confocal microscopy were placed in 1.8%
phosphate buffered saline (PBS)-buffered potassium chloride solution (243
mM) until the hearts ceased beating
in diastole. The embryos were staged
by counting somites or examining external morphology after the extraembryonic membranes were removed
(Hamburger and Hamilton, 1951).
They were then rinsed in saline and
fixed overnight in 2% paraformaldehyde in 0.1 M Hepes buffer. ID8 embryos were fixed overnight in 4% paraformaldehyde in PBS.
MRM
Sham-operated and CNC-ablated
embryos were harvested at stage
22–23 (day 4). The fixative preparation, perfusion, and fixation of embryos as well as the technique for
MRM and imaging volume rendering and histology have been described in detail (Zhang et al., 2003).
Myocardial Proliferation
Five CNC-ablated and sham-operated embryos were examined at
each of the stages. The number of
BrdU-positive cells per 50 myocardial
cells was counted from digital photomicrographs of transverse sections
stained with anti/BrdU and hematoxylin.
Cardiac Morphometry
Confocal microscopy (Bio-Rad Laboratories, Inc., Hercules, CA) was
used to measure variations in the
thickness of the myocardium. The
myocardial myofibrillar organization
was visualized by using rhodamine
phalloidin. Measurements of the
myocardial thickness were made on
five CNC-ablated and sham-operated embryos from each stage.
Stacked images consisted of 2–10
scans, each made with a Z-step of
0.25 microns. The myocardial thickness was determined by multiplying
the number of Z-steps needed to focus through the myocardium by the
depth of the Z-step.
Myocardial volume was measured
in MRM images by using ImageJ software available from NIH. The myocardium was outlined manually by two
independent observers to obtain the
area in each visual section.
To compare the myocardial thickness of five ID8 sham-operated and
CNC-ablated embryos, transverse
sections at the ventricular level (7
␮m thick) were stained with myosin
heavy chain antibody MF20 (Developmental Studies Hybridoma Bank,
Iowa City, IA). SPOT software was
used to measure and document the
thickness of the myocardium.
Immunohistochemistry
BrdU was visualized by using the
ZYMED Staining Kit (ZYMED Laborato-
ries, Inc., San Francisco, CA). Cell
death was assessed by using the
APOPTAG Apoptosis Detection Kit
(Oncor, Intergen). The monoclonal
antibody for myosin heavy chain
MF20 was applied to ensure that
only myocardial cells were assessed
(Yelbuz et al., 2002). For confocal microscopy, fixed whole-mount embryos were stained with rhodamine
phalloidin as described previously
(Price et al., 1996).
Myocardial Cell Size
Myocardial cell size was measured
at stage 22 by using myocardial cells
that had been freshly dissociated using trypsin and DNase as reported in
detail previously by Creazzo (1990).
At stage 22, the heart contains few
other cell types and these can be
recognized in dissociated cell preparations as spherical, nonpigmented, cells with obvious inclusions. Cell size was measured on 636
cells from three CNC-ablated hearts
and 667 cells from four sham-operated hearts by using ImageJ software.
Number of myocardial cells per
ventricle was obtained at incubation days 5 and 9 by using the
method of Anversa and colleagues
(1990) as previously described
(Creazzo et al., 1994) and summarized here. The hearts were trimmed
of the atria and great vessels, and
the ventricles weighed and fixed in
0.1 M Sorenson’s phosphate buffer
(pH 7.2) containing 2% glutaraldehyde and 2% formaldehyde and
embedded in Araldite. These blocks
were sectioned at either 0.75 ␮m for
cross-sections or 2 ␮m for longitudinal sections, and the sections were
stained with toluidine blue. The number of myocytes per ventricle and
the number of myocytes per unit volume of myocardium were determined by counting nuclei and measuring nuclear length in tissue
sections (Anversa et al., 1990). Seventy-five fields were measured from
the ventricles of each heart. Because no difference was found in
comparing apical, middle, and
basal regions of the ventricles, the
measurements reported here were
made from approximately the middle region. The 0.75-␮m sections
160 YELBUZ ET AL.
were visualized at ⫻1,000 and the
number of cross-sectional myocyte
nuclear profiles (N) were counted in
a defined area (A), overlaid by an
ocular grid covering an area of
5,900 ␮m2. The values for N and A
were averaged to determine the
density of nuclear profiles per unit
area of myocytes, N(A). The average
nuclear length (D) was determined
from 85 to 100 measurements of
each tissue sample in the 2-␮m-thick
sections. The number of myocyte nuclei per unit volume of ventricle, N(V),
was determined by using the equation N(V) ⫽ N(A)/D. Assuming a tissue
density of 1.05, the volumes of the
ventricles were calculated from their
measured wet weights. The number
of myocytes per ventricle was calculated from N(V).
Statistical Analysis
Two-tailed, unpaired t-tests were
used to compare values for shamoperated vs. experimental hearts.
ACKNOWLEDGMENTS
We thank Michael A. Choma for
help in reconstruction techniques.
All MRM was performed at the Duke
Center for In Vivo Microscopy, an
NIH/NCRR National Resource (P41
05959). M.L.K. and G.A.J. received
funding from the NIH and T.M.Y. received fellowship grants from the
German Heart Foundation (Deutsche Herzstiftung), German Research Council (DFG), and the
American Heart Association, Georgia Affiliate.
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