Download Ventricular function and morphology in chick embryo from stages 18

Ventricular function and morphology
in chick embryo from stages 18 to 29
EDWARD
B. CLARK,
NORMAN
HU, JAMES
GREGG K. VANDEKIEFT,
CHERYL
OLSON,
L. DUMMETT,
AND ROBERT
TOMANEK
Cardiology, Department of Pediatrics, and Department uf Anatomy and
The Cardiovascular Center, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242
Division
of Pediatric
null pressure system to measure cardiovascular function
in the chick embryo (5). We found a marked increase in
hemodynamic
function with development. We hypothesized that developmental changes in morphological
char19): H407-H413,1986.- We evaluated wet and dry ventricular
acteristics of the embryonic heart parallel the increase
and embryo weights, hemodynamic parameters of ventricular
in hemodynamic function. In this paper, we describe the
function, and myocardial myocyte organellecomposition in the
changes
in ventricular and embryo weight during develdeveloping chick embryo. Phasic and dP/dt ventricular pressure were measuredwith a servo null pressure system, and opment (from stages 18 to 43). We also measured the
volume of mitochondria
and myofibrils in
phasic, mean,and dV/dt dorsalaortic velocities were measured proportional
myocytes, ventricular
pressure, and dorsal
with a 20-MHz pulsed-Dopplermeter. Ventricular and embryo myocardial
weight increasedgeometrically with development,but at differaortic blood velocity from stages 18 to 29, the period of
ent rates, sothat the ventricle-to-embryo weight ratio decreased rapid cardiac morphogenesis.
These results define the
from 0.02 to 0.001just prior to hatching. Ventricular systolic changes in ventricular
function that occur with develand end-diastolic pressureincreasedfrom 1.31 t 0.05/0.33 t
opment in the chick embryo.
CLARK, EDWARD B., NORMAN Hu, JAMES L. DUMMETT,
GREGG K. VANDEKIEFT, CHERYL
OLSON, AND ROBERT ToMANEK. Ventricular
function. md morphology in chick embryo
from stages18 to 29. Am. J, Physiol. 250 (Heart Circ. Physiol.
0.03 mmHg at stage 18 to 3.45 t O.lO/O.SZ& 0.03 mmHg at
stage 29, while dP/dt increasedfrom 23.04 -f 1.32 to 79.55 t
3.69 mmHg/s over the sameperiod. Dorsal aortic dV/dt increasedfrom 878 t 17 to 2,076 k 65 mm/s2 from stuge 18 to
29. Myocyte percent volume of myofibrils increasedfrom 16.7
t 0.9% at stage18 to 23.6 t 1.1% at stage 27 and diminished
to 18.4 t 0.8% at stage 29. Mitochondrial percent myocyte
volume remainedconstant at about 11%. Thesedata define the
parameters of normal ventricular function and morphology
during embryonic development in the chick.
ventricular weight; ventricular blood pressure;myocardial myocyte; dP/dt; dV/dt
THE HEART
IS THE FIRST functioning
organ in the em-
bryo. Myoblasts derived from the embryonic splanchnic
mesoderm are organized in a homogenous layer of cells
surrounding the cardiac tube. In the chick embryo, myofilaments begin to form within the myoblasts at stage
9’ (29-33 h of incubation), and the heart tube starts to
pulsate at stage 10 (33-38 h). Blood flow to the embryonic
and extraembryonic
circulation starts at stage 12 (45-49
h of incubation)
(11). During the X-day incubation
period, the heart provides circulatory support for the
embryo while undergoing morphogenesis from the cardiac tube to the complex, four-chambered heart.
Embryologists have long hypothesized that there is an
interrelationship
between form and function in the developing cardiovascular system. The difficulty in defining
the relationship
has rested on the lack of techniques to
quantify the hemodynamics of the early circulation. Recently, we applied pulsed-Doppler
technology and servo
0363-6135/86
$1.50
Copyright
METHODS
Fertile white Leghorn eggs were incubated in a forceddraft, con stant-humidity
incubator and staged by the
system of Hamburger and Hamilton
(9)
Embryonic and ventricular weights. Wet and dry embryo and ventricular weights were measured from stage
18 (3 days) to stage 43 (19 days). An embryo was removed
from the shell after the extraembryonic
membranes were
stripped away. It was then gently blotted to remove
excess water and weighed on a Mettler balance accurate
to +lO
pg. A ventricle including the conotruncus was
weighed after the vessels distal to the aortic sac and atria
were trimmed off. For dry weight, the embryo or heart
was heated in an oven at 80°C for 3 h and transferred to
a desiccator containing silica gel for 12-24 h. At stages
18-29, 9 or 10 hearts were pooled for each measure of
wet and dry weight, while from stage 31to 43 hearts were
weighed individually.
Myocyte proportion measurements. Fractional myocardial cell volume was determined for mitochondria,
myofibrils, glycogen, and sarcoplasm at stages 18,21,24,27,
and 29. We harvested a minimum
of six ventricles at
each stage, which were immediately
fixed in isotonic
2.5% glutaraldehyde
with buffered 0.1 M sodium cacodylate and subsequently embedded in Spurrs plastic.
Random thin sections cut from the ventricle were stained
with uranyl acetate and lead citrate and photographed
on a Hitachi H600 electron microscope. The micrographs
were printed at a final magnification
of ~24,000.
We used a point counting technique to analyze the
@ 1986 the American
l
Physiological
Society
H407
CHICK
H408
EMBRYO
VENTRICULAR
FUNCTION
AND MORPHOLOGY
FIG. 1. Stage 21 chick embryo in ovo
with 7-pm glass electrode cannula inserted into ventricular chamber for p ressure measurement (X14).
micrographs
after
Fifty micrographs
cell vacuoles and nuclei were excluded.
from five embryos
were counted
for
each stage with an overlay grid of 1,092 intercepts per
micrograph. The number of intercepts (points) falling on
a given structure (Pi) and the total number of points
(P,,lJ are related to the relative volume fraction (Vi) of
the structure per total cell volume (V,,,,) as follows: Vi/
Vcell
=
Pi/Pcell~
In this study we determined
the relative fractions
(volume densities) of mitochondria,
myofibrils, and glycogen. All other cytoplasmic organelles (e.g., ground substance, sarcoplasmic reticulum, lipid) were categorized
as sarcoplasm.
The individuals counting the micrographs (Dummett
and Vandekieft)
were unaware of the embryo stage.
Intra- and interobserver errors were determined by t test
on repeat counts of 10 micrographs. The intraobserver
correlation was high, with the lowest P value of 0.25 for
an individual
and 0.17 for the two observers. Interobserver correlation was also high as the lowest P value
was 0.62.
Hemodynamic measurements. For in ovo physiological
measurements, an egg was removed from the incubator
and positioned on a dissecting microscope stage. We
opened the shell and removed the outer and inner shell
membranes to expose the embryo.
We measured ventricular pressure with a servo null
pressure system that is accurate over the range of O-30
CHICK
EMBRYO
VENTRICULAR
mmHg ( y = 0.995x: - 0.23, ? = 0.99; SEE = 0.11 mmHg)
and has a rapid response from 10 to 90% in 20 ms (5).
dP/dt was electronically
derived from the pressure curve
by a differentiating
channel that was calibrated with a
ramp voltage generator. The 5- to 7-pm-diameter
tip of
the glass pressure cannula was inserted through the
ventricular wall to measure the ventricular pressure (Fig.
1). The phasic and differentiated
ventricular pressures
were recorded (Fig. 2A). The zero pressure was measured
bc
V
FUNCTION
AND
H409
MORPHOLOGY
by positioning
the micropipette
tip in the extraembryonic fluid at the level of the ventricle. An individual value
for dP/dt was determined by averaging the peak value of
40 consecutive pressure curvesWe measured dorsal aortic blood velocity with a 20mHz pulsed-Doppler
velocimeter that is accurate over a
range of O-16 mm/s (y = 0.85~ - 1.15, r2 = 0.99, SEE =
0.49 mm/s) (5). dV/dt was electronically
derived from
the velocity curve by a differentiating
channel that was
calibrated with a ramp voltage generator. The I-mm
piezoelectric crystal was positioned at a 45” angle over
the dorsal aorta at the level of the sinus venosus. Phasic
and differentiated
dorsal aortic blood velocities were
recorded (Fig. ZB). An individual
value for dV/dt was
determined by averaging the peak value of 40 consecutive
velocity curves.
The data are shown as means t SE of the mean for
embryo and ventricular weight, ventricular pressure, dorsal aortic blood velocity, and proportional measurements
of cell volume. The relationship of ventricular to embryo
weight was determined by ratio analysis as well as by
log-log plot.
RESULTS
Ventricular and embryonic weights from stages 18 to 43
(Table 1). Both wet and dry ventricular weights increased
IO
5
0I
V
t2
during development. However, the ratio of heart to embryo weight decreased from 0.02 to 0.001 just prior to
hatching. When expressed as log ventricular wet weight
versus log embryo wet weight, there was a linear correlation (Fig. 3). The dry weight log-log relationship was
similar to the wet weight relationship (logdrY y = 0.8295
logdw x - 1.634, r = 0.995).
Myocyte proportion measurements from stages 18 to 29
(Table 2, Fig. 4). The percent volume of mitochondria
in
the myocytes remained constant from stuges 18 to 29 at
dV/dt
FIG. 2. A: ventricuIar
phasic and dP/dt pressure tracing from stage
21 chick embryo.
B: dorsal aortic phasic, v; mean, ir; and dV/dt veIocity
tracing from stage 24 chick embryo.
about 11%. However, the percent volume of myofibrils
increased gradually from 16.7% at stage 18 to 23.6% at
stage 27, resulting in a gradual decrease in mltochondrialto-myofibril
ratio. Between stages27 and 29, the percent
myofibril volume decreased from 23.6 to 18.4%. Glycogen
volume percent was variable. Sarcoplasm comprised approximately
60% of the myocyte volume during this
1. Ventricular and embryo weights of chick embryo
TABLE
Weights,
n
Stwe
Wet
ventricle
18
21
.
24
27
29
10
10
10
9
-26
-37
34
36
38
40
43
Values
10
are means
25
24
24
24
20
& SEM;
0.35,tO.Ol
0.42~0.03
0.97kO.08
1.54t,O.l1
3.18k0.18
AXkkO~l5
6.76kO.14
16.31t0.54
39.44t1.06
65.7721.65
102.65k3.56
n, no. of preparations.
n
10
IO
10
10
10
10
10
10
10
10
IO
Solid
ventricle
0.03MI.002
0.04kO.002
0.05~0.003
0.10~0.004
0.14-1-0.006
.0>54%.01
0.8fdO.02
1.69M.10
4.27kO.08
8.50k0.13
14.66t0.45
n
30
30
30
30
9
-9
9
9
9
9
9
mg
Wet
embryo
17.7t0.9
34.4Ik1.9
79.5k3.6
149.2k4.8
267.5t9.7
516>3&29,7
1016.5t15.1
282O.Ok44.0
423O.Ot,187.0
9590.02 110.0
19123.0t275.0
Solid
embryo
25
25
25
25
25
-25
25
25
25
25
23
1.02k0.05
1.66~0.11
3.06kO.09
6.61kO.19
15.36t,O.30
-46Jwa94
71.38k1.68
162.43k2.69
369.62t8.65
1230.7Ok34.46
3198.26H06.56
H410
CHICK
EMBRYO
VENTRICULAR
FUNCTION
AND MORPHOLOGY
m,#
= -8498 bq,# - I.6W
SEES a631
r=.9%
-l.ooj
T
a
m
v
1.4
1.0
11
11
m
1.6
2.0
2.4
T
J
2.6
a
1’
lDGwETEmRmwEGHr)
FIG.
TABLE
Slap
3. Log-log plot of heart vs. embryo wet weight. Note linear and significant
2. Percent of myocardial cell volume
n
Mitochondria
18 50 lL'2kO.6
21 52 10.0~0.5
24 37 12.1-t0.8
27 52 11.0&0.5
29 55 1 l.O-tO.4
Values are means k
Myofibril
Mitochondrialto-Myofibril
Ratio
16.7kO.9
17.5kl.l
21.6k1.3
23.6d.l
18.4k0.8
SEM; n, no,
Glycogen
0.82~0.08
2.3k0.3
0.7620.08
6.2kO.9
0.68kO.07
2.9kO.4
0.56kO.05
4.9k0.7
0.67~0.05
6.5kO.6
of preparations.
Sarco plasm
69.8k0.9
66.451.0
63.5tl.l
6Oa5&1.1
64.1-cl.O
period of development.
Hemodynamic measurements from stages 18 to 29 (Table 3). At stage 18, the ventricular early diastolic pressure
was near zero, and the end-diastolic pressure was accentuated presumably by the contributed volume of atria1
systole (Fig. 2A). With development, systolic, end-diastolic, and ventricular dP/dt pressures increased. Among
embryos of the same stage, ventricular dP/dt was unrelated to heart rate.
The dorsal aortic blood velocity tracing showed only
antegrade blood movement, suggesting that the conotruncal cushions act as valves in the outflow tract of the
embryonic heart. (Fig. ZB) Aortic blood velocity increased with development as did dV/dt. However, among
embryos of the same stage, we observed no relationship
between dV/dt and heart rate.
DISCUSSION
Understanding
the relationships between ventricular
morphology and hemodynamic
function in the chick is
important in the eventual understanding of mechanisms
of cardiac development.
J
3.2
3.6
’
r
4.0
J
J
4.0
1
f
5.2
nq
correlation.
Ventricular
weight changes rapidly during development in the chick. We found that the relationship
between ventricular
and embryo weight was not linear
as the ventricular-to-embryo
weight ratio declined during
development. Our measurements of embryo weight agree
with those reported by Romanoff (16) and Van Mierop
and Bertuch (18).
The linear log-log, ventricular-to-embryo
weight ratio
is similar to the heart-to-body weight ratio noted among
animals. Relative heart weight is greatest in newborn
animals and declines as the animal matures (15). Among
mature animals of different sizes, the larger the animal
the relatively smaller its heart weight (8). The greater
relative heart weight early in development may relate to
the circulatory requirements of the embryo. In the chick,
vascular resistance is initially high and decreases as new
resistance units are added to the circulation (5).
Intracellular
organization
of the myocytes is also
changing rapidly with development. Myofibrils first appear in the chick cardiac myocytes at stage 9+ but account
for only a small proportion
of the cell volume (11).
Between stages 18 and 29 we noted an increase and
subsequent fall in the proportion
of myofibrils.
The
myofibrils
became more organized with development,
and assumed a more orderly arrangement, presumably
parallel to lines of stress. However, we could not quantitate the alignment process, since there is no reference
point for comparison.
In the lamb and rat, mitochondrial
and myofibril volume increase during development. Brook et al. (2) noted
a progressive increase in the number and organization of
intracellular
organelles beginning in the 29-day-old fetal
lamb. Hirakow and Gotch (10) noted that in the rat
CHICK
EMBRYO
VENTRICULAR
FUNCTION
AND
H411
MORPHOLOGY
FIG. 4. Electron
micrograph
of chick ventricular
myocytes. A: stage 18; B: stage 27. Major
difference
between
these stages is increased
volume
density
of myofibrils
at
stage 27. mi, Mitochondria;
gl, glycogen;
my, myofibril.
3. Hemodynamic
TABLE
measurements
Ventricular
stage
18
21
24
27
29
Values
n
Systole,
mmHg
20
20
20
20
21
1.31-to.05
1.61+0.04
1.96kO.05
2.35-cO.08
3.45LO.10
are means
+ SEM;
Pressure
End-diastole,
mmHg
0.33kO.03
0.34kO.02
0.40~0.03
0.56kO.03
0.82+0.03
Dorsal Aortic Blood Velocity
dP/dt,
mmHg/s
n
Mean velocity,
mm/s
23.04e1.32
31.98k1.28
44.64f1.81
52.9M2.31
79.55+3.69
30
30
30
30
30
2.99kO.12
4.22f0.08
6.40k0.15
11.45~kO.22
15.33+0.62
dV/dt,
mm/s2
878+17
931*14
1,199&23
1,740+35
2,076+63
Heart rate,
beats/min
146f2
155+2
172t2
183-c3
208f5
n, no. of preparations.
cardiac myocyte, myofibrils increased from 22% at day
16 to 35% at day 25 (birth) while mitochondrial
percent
volume increased from 17 to 35%. On the basis of these
data, we hypothesize that there is a further increase in
the percent volume of mitochondria
and myofibrils during later development in the chick embryo.
The relationship
of myofibril
organization
to heart
function is unclear. At the electron microscopic level, the
H412
CHICK
EMBRYO
VENTRICULAR
randomly arranged myofibrils seem incapable of coordinated myocardial
contraction.
Only a portion of the
myofibrils, those with sarcolemmal attachment, may be
functional while others are in various phases of assembly
(11). However, when the whole heart is viewed under
polarized light, there are hoop-like bands of myofibrils
that encircle the heart. (13) Circumferential
contraction
of the tubular heart would be very efficient. With increasing geometric complexity of the septating heart, the lines
of stress and strain would change. We hypothesize that
the final organization
of the myocardium
is in part
determined by the hemodynamic
forces interacting with
the developing ventricular walls.
With the changes in cardiac morphology, there are
remarkable changes in hemodynamic
function. The ventricular systolic pressure rapidly increased nearly threefold from stages 18 to 29. The systolic pressure noted in
our studies were similar to those reported by Paff et al.
(14), who used a modified Landis apparatus, and Faber
(6), who used an electromechanical
transducer. We were
not able to make exact comparisons with the other
workers’ data, because they reported embryo development by incubation day, which is more variable than
morphological
stage.
We noted a gradual increase in ventricular diastolic
pressure, which we hypothesize is due to a decrease in
ventricular compliance as the heart walls thicken. The
decline in early diastolic pressure to near zero implies
that wall stress changes markedly during the cardiac
cycle. Changing wall stress is likely an important extrinsic factor in the regulation of myocardial formation (19).
The contour of the ventricular pressure curve in the
embryo is markedly similar to that recorded from a
mature heart. The distinct early diastolic pressure suggests that the endocardial and bulbar cushions have a
valve-like function in the early heart. We speculate that
the accentuation in the end-diastolic pressure is due to
atria1 systolic augmentation
of ventricular filling.
Pump performance
of the embryonic ventricle increased with development. dP/dt and dV/dt, preejection
and ejection phase indexes of cardiac performance, respectively, are directly related to preload, myocardial
contractility,
and heart rate and are inversely related to
afterload. With expansion of the vascular system, we
expect that preload increases with development.
The
progressive increase and alignment of myofibrils would
increase myocardial contractility.
Afterload, as measured
by vascular resistance, decreases markedly from stages
18to 29(4).The relationship
between ventricular
function and
heart rate is less clear. Between stages, dP/dt and heart
rate increased. However, among embryos at each stage,
we did not find a relationship between dP/dt and heart
rate. Investigators who found a relationship between dP/
dt and heart rate studied the same fetal animal at different paced heart rates (7, 17). We measured the dP/dt at
naturally occurring, rather than at art&ally
paced heart
rates.
At each stage, ventricular systolic pressure was higher
than the vitelline arterial pressure (5). At stage 18 the
difference was 0.49 mmHg and increased progressively
to 1.45 mmHg at &ge 29. The aortic arches and dorsal
FUNCTION
AND MORPHOLOGY
aorta are the vascular channels between these two points
in the cireu2ation. We hypothesize that this pressure
drop may be important in the mechanism of aortic arch
selection. Cardiac embryologists have long suggested that
the aortic arches are selected by vectorial distribution of
aortic arch blood flow (3). Such a pressure difference
could provide the energy necessary for the orientation of
vecterial blood flow.
From these studies, an intriguing question arises. Does
functional load affect ventricular
development?
In the
earliest stages of heart development, cardiac looping can
proceed in the absence of pump function of the heart
(12). However, at what point in development does the
embryonic ventricle respond to increased work load?
There is indirect evidence from human congenital heart
defects to suggest that growth can be accelerated in the
developing heart (1). The existence of a feedback mechanism by which ventricular
function, in part, controls
ventricular
growth may be the early manifestation
of
growth processes responsible for ventricular hypertrophy
in the mature animal.
The authors thank Sue Kucera and Trudi Fawcett for skillful work
with the manuscript and Horst Jordan for the graphs.
This study was supported by National Heart, Lung, and Blood
Institute Grants HL-14388 and HL-18629.
E. B. Clark is the recipient
of Research Career Development Award HD-000376 from the National
Institute of Child Health and Human Development. J. L. Dummett
and G. K. Vandekieft received Summer Research Fellowship H-107485.
Address for reprint requests: E. B. Clark, M.D., Div. of Pediatric
Cardiology, Dept. of Pediatrics, Brady 516, Johns Hopkins Hospital,
600 N. Wolfe St., Baltimore, MD 21205.
Received 12 March 1985; accepted in final form 17 October 1985.
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