Download Volume on Cardiac Output and Arterial and Ventricular Blood

187
Effect of Changes in Circulating Blood
Volume on Cardiac Output and Arterial and
Ventricular Blood Pressure in the Stage 18,
24, and 29 Chick Embryo
Armin J. Wagnan, Norman Hu, and Edward B. Clark
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We studied the hemodynamic effects of changing volume loading in the chick embryo, before
autonomic innervation, to test the hypothesis that the Frank-Starling mechanism functions in
the embryonic myocardium. Dorsal aortic blood velocity was measured by pulsed Doppler.
Heart rate and aortic diameter were also measured to calculate cardiac output and stroke
volume index. Vitelline arterial and ventricular pressures were measured with a servo-null
micropressure system in stage 24 embryos. Infusing isotonic solution intravenously resulted in
linear increases in stroke volume index for stages 18 (y=388x+6.89), 24 (y=466x+7.86), and
29 (y=549x+4.96). The slopes and intercepts were statistically the same for all three stages.
Similar volume loading in stage 24 embryos initially increased mean arterial pressure linearly,
but at higher loading conditions, the rate of rise lessens. Thus, volume loading resulted in a
decrease in vascular resistance. Withdrawing blood from stage 24 embryos resulted in a
decrease in ventricular peak systolic and end-diastolic pressures. With reinfusion of the blood,
systolic and end-diastolic pressures initially rose above baseline levels and later returned to
normal. We conclude that a length-tension relation is present in the preinnervated embryonic
heart and that vascular resistance changes inversely with loading conditions. We speculate that
these mechanisms are the primary hemodynamic control mechanism in the early chick embryo.
(Circulation Research 1990;67:187-192)
In the mature animal, control of the cardiovascular system occurs through extrinsic and intrinsic
mechanisms. The extrinsic mechanisms operate
through the neurohumoral and autonomic nervous
systems. Both mechanisms play an important role in
hemodynamic regulation, but the embryonic cardiovascular system functions prior to autonomic innervation of the heart and before the adrenal gland
forms. Therefore, extrinsic mechanisms are probably
not present.
We hypothesized that intrinsic mechanisms, in
particular the Frank-Starling mechanism, control
cardiac function at early stages of development. We
sought to define the responses to changes in circulatFrom the Department of Pediatrics and Cardiovascular Center
at the University of Iowa Hospitals and Clinics, Iowa City, Iowa.
Supported by National Institutes of Health grants HD-14723
and HL-14388. A.J.W. was supported by National Institutes of
Health training grant HL-07413. E.B.C. is a recipient of National
Institutes of Health grant RCDA HD-00376.
Address for reprints: Armin J. Wagman, MD, Division of
Pediatric Cardiology, Department of Pediatrics, University of
Florida School of Medicine, J. Hillis Miller Health Center, Box
J-296, Gainesville, FL 32610.
Received September 26, 1988; accepted February 16, 1990.
ing blood volume to test this hypothesis. We studied
the embryo at a time when the heart undergoes rapid
growth and morphogenesis from a muscle-wrapped
tube to a nearly septated four-chambered mature
heart. These stages precede the development of
autonomic innervation of the heart and vascular
system.12 Our data demonstrated a length-tension
relation in the embryonic heart and embryonic regulation of hemodynamics by altering peripheral vascular resistance.
Materials and Methods
Fertile white leghorn chicken eggs were incubated
blunt end up in a forced-draft constant-humidity
incubator to Hamburger-Hamilton stage 18 (3 days),
stage 24 (4 days), or stage 29 (6 days).
The shell was carefully opened at the blunt end
where the air sac is located. With microdissecting
forceps, the outer shell membrane was gently
removed.
Cardiac output was calculated from mean dorsal
aortic blood velocity and dorsal aortic diameter.
Velocity was measured with a 20-MHz pulsed Doppler velocity meter (model 545C-4, University of Iowa
188
Circulation Research Vol 67, No 1, July 1990
PmHssue
2
-
-30
-60
-60I1
sec
FIGURE 1. Recordings showing ventricular pressure measured in a stage 24 embryo heart. Top tracing: Phasic
ventricular pressure. Bottom tracing: The first derivative of
pressure with respect to time (dPldt). End-diastolic pressure
was measured just before the rapid rise in pressure during
systole. This corresponded to dP/dt=0.
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during systole. This corresponded to dP/
dt=O (Figure 1). Vascular resistance was calculated
by dividing mean vitelline arterial pressure by the
mean dorsal aortic blood flow.
In the first group of experiments, circulating blood
volume was increased by injection of isotonic chick
Ringer's solution through a 10-,um-diameter glass
cannula inserted into a vitelline vein. After insertion
of the cannula, a baseline recording was made of the
heart rate and dorsal aortic blood velocity. Then a
specified volume of isotonic solution was injected, at
a constant rate, using a graduated syringe (Hamilton
Co., Reno, Nev.). Throughout the injection period,
there was a continuous recording of heart rate and
dorsal aortic blood velocity. At the end of the experiment, dorsal aortic diameter was measured. Dorsal
aortic diameter remained constant throughout the
cardiac cycle and with the injection of isotonic solution. From these measurements, we determined
baseline stroke volume index and maximum stroke
volume index after volume infusion. For control
studies there was insertion of an intravenous cannula
but without injection of fluid. Measurements were
made for 1 minute.
We calculated a percent increase in stroke volume
index as a function of the injected volumes. The
injected volumes used for each embryo stage are
listed in Table 1. A different group of embryos was
studied at each volume, so that each embryo received
one injection. For stage 18 embryos, these infusions
corresponded to 0.9-7.7% of circulating blood volume. For stage 24 embryos, these infusions corresponded to 1.0-14.9% of circulating blood volume.
For stage 29, these corresponded to 1.9-7.6% of
circulating blood volume.4 To compare the response
of the different developmental stages, the injection
volume was normalized for average embryo wet
weight.5 Percent change in stroke volume index was
plotted against normalized injection volumes.
Volume infusion experiments were also done in
stage 24 embryos while measuring arterial blood
pressure. The same injection volumes were used as
listed in Table 1 for the stage 24 embryos. Control
studies were done in the same fashion as described
above. Five embryos were studied at each injection
volume and in the control experiments.
In other experiments, we examined the effect of
altering circulating blood volume on ventricular prespressure
3
Ventricular
Bioengineering, Iowa City.) that is linear over the
of 0-16 m/sec (y=0.85x+1.15, r2=0.99,
SEE=0.49 m/sec).3 An 850-,um piezoelectric crystal
was positioned over the dorsal aorta at a 450 angle to
record phasic and mean velocities. Heart rate was
determined by counting pulsations between time
lines. Dorsal aortic diameter was measured with a
filar micrometer eyepiece, calibrated with a 10-gm
scribed-glass standard. At these stages the embryo is
transparent, which allows direct visualization of the
heart and dorsal aorta. Mean dorsal aortic blood flow
was calculated from the product of the mean velocity
and the cross-sectional area of the aorta (area= rd2/
4, where d is the dorsal aortic diameter). Stroke
volume index was calculated by dividing mean dorsal
aortic blood flow by the heart rate.
Blood pressure was measured with a servo-null
micropressure system (model 900, World Precision
Instruments, New Haven, Conn.) that is linear over a
range of 0-30 mm Hg (y=0.995x-0.23, r2=0.99,
SEE=0.11 mm Hg).3 The frequency response is 1090% in 20 msec and linear to 10 Hz. A 5,gm-diameter drawn-glass micropipette cannula was
inserted into a vitelline artery or the ventricular
cavity to measure arterial or ventricular pressures,
respectively. dP/dt was electronically derived from
the pressure tracing. Ventricular peak systolic and
end-diastolic pressures were analyzed. End-diastolic
pressure was measured just before the rapid rise in
range
TABLE 1.
Injection Volumes Used in Stroke Volume Index Experiments in Stages 18, 24, and 29 Chick Embryos
Stage 29
Stage 24
Stage 18
n
Injection volume (gl)
n
n
Injection volume (,ul)
Injection volume (,ld)
0.12
6
5
5
1.0
10
0.25
5
5
9
10
2.5
0.50
5
9
15
5
5.0
1.00
5
9
20
7.5
5
10.0
13
... ...
9
12.5
... ...
...
...
n, Number of embryos studied at each injection volume.
Wagman et al Hemodynamic Control Mechanisms in the Chick Embryo
40o
Velocity 201mm/sec
O
0
Injection
l
Mean
10
VelocitY
5
mm/sec
0
1
sec
FIGURE 2. Recordings showing an example of the response
of dorsal aortic blood velocity in a stage 24 embryo to a 5-,il
volume load (6% of circulating blood volume). Stroke volume
index is derived from the velocity, aortic diameter, and heart
rate (see text). Top tracing: Phasic velocity. Bottom tracing:
Mean velocity. Baseline stroke volume index is 0.25 mm3/
cardiac cycle. After volume infusion, there is a 52% increase in
stroke volume index.
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in stage 24 embryos first by removing blood and
then by reinfusing it. After insertion of a glass
cannula into a vitelline vein, the pressure cannula
was inserted into the ventricular cavity. First, to
decrease intravascular volume, 10 gl blood was withdrawn intravenously from the embryo at 0.5 ,ll/sec.
After 15 seconds, the same 10 ,ul blood was reinfused
at the same rate. Five embryos were studied. Ventricular pressure and heart rate were recorded continuously. In some experiments, dorsal aortic blood
velocity was simultaneously recorded.
Data are expressed as mean±SEM and analyzed
by analysis of variance and analysis of covariance.
Vascular resistance calculations are expressed as
mean±+SEE and analyzed by linear regression and
Kendall's Statistical significance was defined by a
value of p<0.05.
Results
Dorsal aortic blood velocity increased in response
to volume infusion (Figure 2). Stroke volume index
increased since it is proportional to dorsal aortic
blood velocity. For all experiments and control studies, there was no significant change in heart rate
(Table 2). Therefore, at stages 18, 24, and 29, stroke
volume index increased linearly as a function of
injection volumes (Figure 3). Injection volume was
normalized for wet embryo weight in Figure 3D.
There were no statistical differences in the slope or
sure
r.
189
intercept of the response among the different stages.
Stroke volume index did not change during control
studies (Table 2).
Phasic and mean arterial pressure increased in
response to volume infusions in stage 24 embryos
(Figure 4). At the lower injection volumes, the percent
change in mean arterial pressure increased linearly. But
with volumes greater than 5 ,ul, the rate of rise of mean
arterial pressure begins to level off (Figure 5). The rise
in stroke volume index was linear at all injection
volumes. Thus, vascular resistance decreased with
increased volume loading: y= -0.027x+ 1.11, r= -0.84,
p=0.001 (Table 3). Mean arterial pressure did not
change during control studies (Table 2).
During the withdrawal of blood, ventricular peak
systolic pressure, ventricular end-diastolic pressure,
and mean dorsal aortic blood flow (i.e., stroke volume index) all decreased. With reinfusion of the
blood volume, these measurements increased (Figure
6). An example of the effect of withdrawal and reinfusion of 10 ,ul blood on peak ventricular systolic and
end-diastolic pressures is shown in Figure 7. During
blood withdrawal, systolic and end-diastolic pressures
decreased. When the volume was reinfused, peak systolic and end-diastolic pressures increased above the
baseline pressures, but after a short time, these
returned to the baseline measurements.
Discussion
The Frank-Starling mechanism has been studied
extensively in fetal and neonatal animals. Numerous
studies6-10 have shown that it functions in fetal and
neonatal lamb hearts, but its importance as a control
mechanism has been debated.
Hemodynamic regulation has not been well studied in the embryo. Faber et al"l studied the FrankStarling mechanism in the embryo in vivo. They
showed that an intravenous bolus of either isotonic
chick Ringer's solution or a hypertonic dextran solution increased end-diastolic volume as measured by
the area on a cinephotomicrograph.
Our results support the hypothesis that the FrankStarling mechanism is functional in the chick embryo
heart. The increase in stroke volume index is partly
related to increase in preload (resting tension). This
is consistent with our previous experiments in which
an injection of 5 1,u chick Ringer's solution in a stage
TABLE 2. Changes in Stroke Volume Index and Mean Arterial Pressure During Control Studies and Effect of Volume
Loading on Heart Rate in Stages 18, 24, and 29 Chick Embryos
Change in heart rate (%)
Control
Experimental*
Change in control MAP (%)
Stage
Change in control SVI (%)
-0.6±0.8
-1.4+1.5 (5)
1.3±1.3 (5)
(5)
*
18
-5.5+1.0 (9)
-2.0
-2.3±0.4
±2.0
-1.8±1.5
24
(9)
(5)
(9)
-1.7±0.5 (5)
-0.5±0.5 (5)
-2.7±0.8 (5)
29
Values are mean±SEM. Numbers in parentheses represent the number of studies performed for each group of
experiments. SVI, stroke volume index; MAP, mean arterial pressure.
*Values indicate maximum change in heart rate during volume loading.
Circulation Research Vol 67, No 1, July 1990
190
90
x
W
a
z
W
W
0
cc X
I-LW
40
B
70 ~
A
Stage 18
Stage 24
80
W
0
2 80 F
30
W
n
50
0
A
20
UI
I-
y= 21.92x + 6.89
a: 40
r = 0.88
10
Gu
y= 5.86x
r=0.82
30
0. 1
0a
0.2
zz
0.4 0.5 0.6 0.7 0.8 0.9
VOLUME OF INJECTION (pA)
0.3
1.0
ot
z
20
10
WU
0>
50
C
Stage 29
4
3
2
1
40
5
8
7
6
9
10
11
12
13
(g I)
VOLUME OF INJECTION
30
C0Z
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CDae
20
z
10
~~~
cr0
00
90
80
70
60
Wl W
40
0>
30
20
/ ~~~~~Y = 2.05x + 4.96
~~~~~~~r =0.91
W
be
0
a:x
I-Lu
2
4
6
8
10
12
14
16
18
20
VOLUME OF INJECTION (.L 1)
D
50
-Stage 18
Stage 24
----Stage 29
-
101
A
A
A
.01 .02.03.04.05.06 .07.08 .09.10.11 .12.13.14.15.16
VOLUME OF INJECTION
(gllmg)
FIGURE 3. Graphs showing response of stroke volume index to volume loading in stage 18 (panel A), stage 24 (panel B), and
stage 29 (panel C) embryos. Data points are mean +SEM. In panel D, the change in stroke volume index is compared among stages
by plotting against injection volumes normalized for average wet embryo weight at each stage of development. Data points and
standard error bars are omitted for clarity. For panel D, linear regression equations are as follows: y=388x+6.89, r=0.88 for stage
18; y=466x+ 7.86, r=0.82for stage 24; and y=549x+4.96, r=0.91 for stage 29. Total number of experimental embryos studied at
each stage was 20 for stage 18, 59 for stage 24, and 21 for stage 29.
24 chick embryo resulted in a 22% increase in the
atrial pressure.'2
The similarity of responses among the different
stages was not expected. There are marked differences in development. The stage 18 embryo is a
muscle-wrapped bent tube with a single atria and
ventricle. By stage 29, the heart has assumed a more
mature shape, and there are four distinct chambers
with septation nearly complete. There is also a
15-fold increase in wet embryo weight from stage 18
to 29.5 Although the slopes in Figure 3D suggest a
slight trend toward an increased response in the
LU
a:
30F
i
0.
tL
Arterial 1 r
Pressure 0.51mmHg 0L
.i
20
W
cc
z
:
zc
1F
Mean
Pressure 0.5
Lu
-
mmHg
0
10
0
z
-
U
1 sec
-
1
a:
MC
W
0
Injection
1
-
FIGURE 4. Recordings showing an example of the response
of vitelline arterial pressure in a stage 24 embryo to a 2.5- pi
volume load (3% of circulating blood volume). Top panel:
Phasic pressure. Bottom panel: Mean pressure. Baseline
mean pressure is 0.9 mm Hg. After volume infusion, there is a
17% increase in mean pressure.
1
2
3
4
5
a
7
a
8
-
1
A
9
10
11
t
A
12
13
VOLUME OF INJECTION (j. I)
FIGURE 5. Graph showing response of mean arterial pressure in the stage 24 embryo to volume loading. Data points are
mean +SEM. Five embryos were studied at each of six injection volumes for a total of 30.
Wagmnan et al Hemodynamic Control Mechanisms in the Chick Embryo
191
TABLE 3. Comparison of Total Vascular Resistance Before and After Volume Loading in the Stage 24 Chick Embryo
Vascular resistance (mm Hg/mm3/sec)
Injection volume (,ul)
Baseline
After volume loading*
Change in vascular resistance (%)
0.0 (control)
1.11±0.16
+3.6
1.15±0.16
1.0
1.28±0.15
-6.3
1.20±0.13
2.5
1.04±0.10
0.97±0.09
-6.7
5.0
0.96±0.11
0.88±0.08
-8.3
7.5
1.06±0.12
0.87±0.09
-17.9
10.0
1.01±0.08
0.83±0.07
-17.8
12.5
1.15±0.11
0.86±0.09
-25.2
Values are mean±SEE. Vascular resistance is calculated from the ratio of mean vitelline arterial pressure and mean
dorsal aortic blood flow.
*Values were calculated using peak mean pressure and peak blood flow.
Downloaded from http://circres.ahajournals.org/ by guest on April 30, 2017
older embryos, there appears to be a consistency in
how these different developmental stages respond to
hemodynamic changes. The large increases in stroke
volume index occur despite the immaturity of the
embryonic myocardium. Electron microscopy examination of the myocardial cells has shown marked
myofibril disarray at stages 18, 24, and 29.5 The
myofibrils become more organized by stage 29, but
there is still not a significant difference in the rise in
stroke volume index.
The stroke volume index and arterial pressure
experiments in the stage 24 embryo also demonstrate
that vascular resistance falls as circulating blood volume rises with volume loading. This change is most
likely more dependent on the extraembryonic vascular
bed since it is more extensive than the intraembryonic
blood vessels. The mechanisms for changes in vascular
resistance are unknown. There is no innervation of the
extraembryonic vascular system. Mechanical forces,
due to the rise in circulating blood volume, may open
additional vascular beds, resulting in decreased vascular resistance. ,-Receptors that can alter vascular
resistance have been demonstrated in the extraembryonic vasculature.13 Thus, vascular changes could be
mediated by local receptors responsive to stretch.
In the mature animal, autoregulation usually
results in vasoconstriction in the face of increasing
pressure and flow. In the embryo, we theorize that
the reverse would occur in the extraembryonic vascular bed. This would result in maintaining a more
constant hemodynamic state within the embryo itself.
We also theorized that a decrease in circulating
blood volume would result in an increase in vascular
resistance. Again, this process would maintain a
more constant hemodynamic state for the embryo at
the expense of the extraembryonic vascular bed,
which is less critical to the embryo at least for short
periods of time.
The results of the experiment that measured ventricular pressure while decreasing and then increasing circulating blood volume also support both of our
hypotheses. The end-diastolic pressure, an index of
preload, changed directly with changes in circulating
blood volume. This is not a pure response, since the
peripheral vascular resistance could not be controlled, but the immediate association between
changes in end-diastolic pressure and peak ventricular pressure demonstrate a length-tension relation of
the embryonic ventricular myocardium. Stroke vol-
3
Ventricular 2
Pressure 1 L
mmHg
-
-
0L
8
6
Velocity 4
mm/sec 2 F
Mean
30
Velocity 20
-
m m/sec 10
m dil
mr
bq,;,!
'i
h
wnicr
l v Iallt s
O_
i
Withdrawal
U
2 sec
Reinfusion
I
FIGURE 6. Slow recording of the response of ventricular pressure and dorsal aortic blood velocity to withdrawal and reinfusion
of 10 Md blood in a stage 24 embryo. Top tracing: Ventricular pressure. Middle tracing: Mean dorsal aortic blood velocity, which
is directly proportional to stroke volume index Bottom tracing: Phasic aortic blood velocity.
192
Circulation Research Vol 67, No 1, July 1990
may also be critical for normal cardiac development
because this may be related to flow. The ability of the
myocardial muscle cell to respond with a lengthtension relation is a fundamental property of the
myocyte and is present even in the early stages of
heart morphogenesis.
3.0
2.8
E
E
2.6~
co
LU
CO)
W
2.4
k
00
2.2
cc
CL
CO
Acknowledgments
I would like to thank Dr. Alan Cantor for his help
in the statistical analysis. I would also like to thank
Deborah Floyd for preparing the manuscript.
2.0
1.8
0
-
Withdrawal
No Intervention
- Reinfusion
1.6
0.1
0.2
0.3
0.4
0.5
0.6
END-DIASTOLIC PRESSURE (mmHg)
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FIGURE 7. Graph showing ventricular pressure changes
during the withdrawal and reinsion of 10 /1 blood in a stage
24 embryo. Baseline peak ventricular systolic and end-diastolic
pressures are shown at point A. With withdrawal of blood, the
solid line plots the fall in peak systolic and end-diastolic
pressures to point B. After a short time to allow parameters to
stabilize (dash-dot line between B and C), the blood is
reinfused starting at C and ending at D (dashed line). After a
short period of time after the end of reinfusion, pressures return
to E (dash-dot line) and are nearly equal to the baseline
pressures.
ume index was also altered as a function of enddiastolic pressure.
We favor the following interpretation of the ventricular blood pressure experiments as support for
the hypothesis that vascular resistance is related to
circulating blood volume. Withdrawal of blood
decreases circulating blood volume and elevates vascular resistance. When the blood is reinfused, systolic
and end-diastolic pressures rise to levels above baseline because of a higher resistance of the vascular
bed. With time, systolic and end-diastolic pressures
return to baseline as vascular resistance returns to
normal.
We speculate that control of hemodynamics is
crucial to the embryo. Circulating blood volume and
vascular resistance may be markedly altered by environmental changes. A length-tension relation is
important for maintaining normal hemodynamics in
the chick embryo. Precise control of hemodynamics
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KEY WORDS * cardiac development * hemodynamics Frank-Starling mechanism * vascular resistance * chick embryo
Effect of changes in circulating blood volume on cardiac output and arterial and
ventricular blood pressure in the stage 18, 24, and 29 chick embryo.
A J Wagman, N Hu and E B Clark
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Circ Res. 1990;67:187-192
doi: 10.1161/01.RES.67.1.187
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