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226
Ventricular Pressure-Area Loop
Characteristics in the Stage 16 to 24
Chick Embryo
Bradley B. Keller, Norman Hu, Peter J. Serrino, and Edward B. Clark
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The accurate description of embryonic cardiovascular function requires the adaption of
standard measurement techniques to the small scale of the developing heart. In the mature
heart, the analysis of ventricular pressure and volume accurately defines function. Because in
vivo measures of volume are not feasible in the embryonic heart, we tested the hypothesis that
ventricular pressure-area loops accurately define ventricular function in the stage 16 to stage
24 white Leghorn chick embryo. We simultaneously measured ventricular pressure with a
servo-null pressure system and recorded video images at 60 Hz. The pressure waveform was
superimposed onto the video image in real time. Video fields were planimetered for epicardial
ventricular cross-sectional area and ventricular pressure. Pressure and area data were
smoothed using a fast Fourier transform filter and plotted. Data are reported as mean±SEM,
n.4, and were tested by regression analysis and analysis of variance (p<0.05). Heart rate
increased from 90±7 beats/min at stage 16 to 130±13 beats/min at stage 24. All pressure-area
loops displayed diastolic filling, isometric contraction, ejection, and isometric relaxation,
similar to pressure-volume loops of the mature heart. Isometric contraction time increased
from 42±5 to 62±4 msec (p<O.OS), while isometric relaxation time was 124±12 and 120±10
msec (p>0.05) between stages 16 and 24, respectively. The maximum ratio of instantaneous
ventricular pressure to area identified end systole better than peak ventricular pressure or
minimum ventricular area. Thus, pressure-area relations define ventricular function in the
embryonic chick heart. (Circultion Research 1991;68:226-231)
T he accurate measurement of ventricular function in the mature heart has been a primary
goal of clinicians and scientists for over a
century.' In the chick embryo, ventricular pressure,
ventricular cross-sectional area, and cardiac output
can be accurately measured shortly after the onset of
cardiac contraction.2,' Although the early embryonic
heart lacks formed valves or autonomic innervation,
ventricular blood pressure, stroke volume, and heart
rate are tightly regulated.4 Cardiac output increases
in proportion to embryo weight so that blood flow
indexed for embryo weight remains constant.5 In
addition, the structurally simple embryonic heart
accelerates ventricular growth in response to experimentally increased developed pressure.6
From the Cook Research Laboratory, Division of Pediatric
Cardiology, Department of Pediatrics, University of Rochester
School of Medicine, Rochester, N.Y.
Supported by National Institutes of Health National Research
Service Award HL-07824 (B.B.K.) and grant R01 HL-42151
(E.B.C.).
Address for correspondence: Bradley B. Keller, MD, Division of
Pediatric Cardiology, University of Rochester, 601 Elmwood Avenue, Box 631, Rochester, NY 14642.
Received April 12, 1990; accepted August 30, 1990.
In the mature heart, pressure-volume (PV) analysis allows the precise definition of ventricular filling
and ejection characteristics and the time of end
systole.78 Because the cardiovascular system operates as a closed system, the PV diagram also provides
a unique method to define the influence of the
vascular system on ventricular function.9 We therefore adapted the method of PV analysis to the
embryonic ventricle to more precisely measure ventricular function and to define ventricular/vascular
interactions during cardiovascular development.
We tested the hypothesis that ventricular pressurearea (PA) loops define cardiac function in the stage 16
to 24 chick embryo. Each PA loop displayed diastolic
filling, isometric contraction, ejection, and isometric
relaxation.'0'1 Isometric contraction time increased
from 42±5 to 62±4 msec (p<0.05), while isometric
relaxation time was 124±12 and 120±10 msec
(p>0.05) between stages 16 and 24, respectively.
PA loop analysis of the embryonic ventricle integrates dynamic morphological and functional measures of cardiovascular function and complements
our long-term investigation of the relation of form
and function in the developing heart.
Keller et al Embryonic Ventricular Pressure-Area Loops
227
D.C. AMPUFIER
FIGURE 1. Workstation diagram for pressure and area data acquisition and analysis.
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Materials and Methods
We studied white Leghorn chick embryos at stage
16 (2.3 days, n=4), stage 18 (2.8 days, n=8), stage 21
(3.5 days, n=9), and stage 24 (4 days, n=8) of a
46-stage (21-day) incubation period.12 Developmental landmarks including somite number, limb size,
and cardiac morphology identify each stage. Embryo
wet weight doubles between each selected stage.5
Fertile eggs were incubated blunt end up in a forced
draft incubator at 38.5°C. When incubated blunt end
up, the blastodisk floats to the top of the egg and the
embryo develops beneath the air cell. Access to the
embryo was gained by opening the shell and removing a small region of the inner and outer shell
membranes. The embryo was then positioned for
imaging on a photomacroscope stage.
Experiments were performed and analyzed at an
integrated physiology and morphometry workstation
(Figure 1). Video images were acquired using a
photomacroscope (model M400, Wild Leitz USA,
Inc., Rockleigh, N.J.), a video camera (model 70,
Dage-MTI, Michigan City, Ind.) with a grade 1
Newvicon tube, a fiber-optic light source (DolanJenner Industries, Woburn, Mass.), a VHS video
recorder (model VR9670, Magnavox North American Phillips, Jefferson City, Tenn.), and a time/date
generator (model VTG-33, FOR.A, West Newton,
Mass.). The Dage camera generated 60 sequential
video fields per second in the interlaced mode. The
image field was 1,000-2,000 ,gm in diameter, and the
effective raster spacing was 4-8 ,gm. One pixel in the
image equals 0.25% of the epicardial diameter. Real
time+0.005 second was recorded on each field. A
10-ptm scale scribed-glass standard was recorded in
the plane of each embryo after imaging.3
We simultaneously measured intraventricular
blood pressure with a servo-null pressure system
(model 900, WPI, New Haven, Conn.). A fluid-filled
7-,gm-tip glass capillary tube was positioned with a
micromanipulator (Leitz, Wetzlar, FRG) to puncture
the embryonic ventricle in the region of the primitive
right ventricle. Zero transtip pressure was measured
by immersing the electrode in the extraembryonic
fluid adjacent to the ventricle. The analog signal gain
was increased 10-fold by a direct-current amplifier
(Nelson Technical Services, South Old, N.Y.), sampled at 500 Hz by an analog-to-digital board (RC
Electronics, Santa Barbara, Calif.), and qualitatively
reviewed on the computer in digital oscilloscope
mode.
An analog device sampled the pressure waveform
at 15.75 kHz and placed a marker proportional to
instantaneous pressure onto each horizontal video
line in real time (model PONV, Ogden Scientific,
Spencerport, N.Y.). The device also placed zero, full
scale, and pressure baseline markers onto video fields
for calibration of the pressure scale. The composite
video fields were recorded on /2-in. VHS tape.
Individual video fields were analyzed at a workstation that included a minicomputer (Premium/286,
AST Research, Irvine, Calif.), a monitor (Multisync
II, NEC Information Systems, Boxborough, Mass.), a
frame grabbing board (model Targa M8, Jandel
Scientific, Corte Madera, Calif.), JAVA video analysis software and a mouse (Microsoft, Redmond,
Wash.), and a multipurpose video monitor (model
PVM1271Q, Sony Corp.).
The video measurement protocol for each embryo
included 1) calibration of measurement software for
length (millimeters) and area (square millimeters)
with the scribed standard; 2) planimetry of sequential
video fields for ventricular epicardial area (square
millimeters) and scaled ventricular pressure (millimeters); 3) planimetry of the zero, baseline, and full
scale pressure markers; and 4) selection of maximum
(end-diastolic) and minimum ventricular area images. The data analysis protocol included 1) calculation of heart rate from the cycle length of consecutive
end-diastolic images; 2) transformation of the scalar
pressure data into hemodynamic data (mm Hg);
228
Circulation Research Vol 68, No 1, January 1991
STAGE 18
STAGE 16
STAGE 24
STAGE 21
VENTRICULAR
PRESSURE
(mmHg)
FIGURE 2. Representative pressure and area curves for stage 16
to 24 chick embtyos. Pressure and
area curves are plotted on the
same time scale. Data are displayed as line drawings without
curve fitting.
1.50
VENTRICULAR
AREA
1.00.
(mm2)
0.500
o.oo
500 msec
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3) fast Fourier transform low-pass filter (three point)
of pressure and area data; 4) pressure and area data
plotted as PA loops; 5) calculation of diastolic filling
time as the time from minimum area to the onset of
ventricular contraction; 6) identification of end systole as the point of maximum instantaneous pressure
to area ratio for each cardiac cycle13; 7) calculation of
isometric contraction time as the number of video
fields from the onset of ventricular contraction to
95% of end-diastolic area, and isometric relaxation
as the number of video fields from end systole to the
first rise in ventricular pressure; and 8) determination of the point of maximum systolic pressure (tmax
Pm.), end-systolic pressure and area (tes, Pes, Aes),
and minimum ventricular area (tmmn, Amin) for each
cardiac cycle.
The mean values for each variable were compared
by analysis of variance and regression analysis with
significance assigned to p<0.05.
We previously documented that intraobserver and
interobserver error of area measurement by planimetry is not significant.3 Intraobserver and interob-
server error of pressure measurement by planimetry
was not significant (p>0.05).
Results
Heart rate increased from 90±7 beats/min at stage
16 to 130±13 beats/min at stage 24. Ventricular
pressure and area increased across the stage range
(Figure 2). A negative ventricular pressure occurred
at the onset of ventricular filling in all embryos.
Ventricular PA loops contained diastolic filling, isometric contraction, ejection, and isometric relaxation
(Figure 3).
From stage 16 to 24, isometric contraction time
calculated from the PA plots increased from 42±5 to
62±4 msec (p<O.05), while isometric relaxation time
remained similar at 124±12 to 120±10 msec
(p>O.OS, Figure 4).
Peak ventricular pressure, maximum pressure to
area ratio, and minimum area were identified in all
embryos (Figure 5). At the time of the maximum
pressure to area ratio (end systole), ventricular pressure was less than peak pressure (Figure 6), and
ventricular area was larger than minimum area (Fig-
STAGE 24
-> 2.25-
0.15-
* ISOMETRIC RELAXATION
STAGE 21
E 1.75E
1.25-
STAGE 16
o| 0.10U)
lLJ
D.
:D
o ISOMETRIC
0.75-
:2
F-
C/)
(A 0.25LL-0.250.00
0.25
1
1
0.50
0.75
1.00
1.25
AREA (mm2)
FIGURE 3. Representative pressure-area loops for stage 16
embryos. Data for two or three cardiac cycles are
included in each loop. Data are displayed as line drawings
without curve fitting.
to 24 chick
CONTRACTION
L
0.05-
n
nn
U.vv
(x + SEM)
&
-g
14
16
v.
.
18
20
22
24
26
STAGE
FIGURE 4. Isometric contraction and relaxation times for
are mean +SEM.
Linear regression curve of isometric contraction time was
plotted to raw data.
stage 16 to 24 chick embryos. Data plotted
Keller et al Embryonic Ventricular Pressure-Area Loops
2.001
_-] 1.75
E
aa
PRESSUREmnx
PRESSURE/AREAmax
'AREAmin
(N
E
1.25-
E
w
Ld
229
1* END-SYSTOUC
AREA
r = 0.865
y = 0.081x - 0.927
4O MINIMUM SYSTOUC AREA
1.50- y = 0.064x - 0.659 r = 0.863
SEE = 0.133
SEE = 0.107
1.00-
0.75-
0. 0.50-
m"
0.25-
0.00 -
LJL
14
ry -0.25^ _
Q
0.25
0.75
0.50
FIGURE 5. Representative pressure-area loops labeled for
peak ventricular pressure, maximum ventricular pressure to
area ratio, and minimum area.
Downloaded from http://circres.ahajournals.org/ by guest on May 7, 2017
ure 7). The time of peak ventricular pressure occurred 5±4 to 29±6 msec sooner than end systole
(p<0.05), and the time of minimum area occurred
52±13 to 124±12 msec after end systole (p<0.05)
from stage 16 to 24, respectively (Figure 8).
Discussion
The precise definition of terms is crucial in quantifying cardiac function. At the simplest level, systole
is the period of active ventricular contraction and
ejection, and diastole is the period of ventricular
relaxation and filling. Preload is the force exerted on
the ventricle while in a passive state, and afterload is
the force against which the ventricle contracts. However, the measurement of any one of these parameters of cardiac function and the study of their regulation during cardiovascular development are
complicated by their complex interdependence. We
therefore adapted the PV method to the embryonic
heart to define ventricular function and ventricular/
vascular interactions.
Diastolic filling in the mature heart is determined
by the interaction of the passive ventricle and the
STAGE
FIGURE 7. Area at time of maximum pressure to area ratio
versus minimum area for stage 16 to 24 chick embryos. Data
plotted are mean +SEM. Linear regression curves are plotted
to raw data.
venoatrial preload system. Similar to data in the
mature ventricle,'1415 we found that intraventricular
pressure was negative at the termination of isometric
relaxation in all embryos. While the exact atrioventricular pressure differential is not known for the
embryonic heart, a negative ventricular pressure at
the onset of ventricular filling is consistent with the
presence of ventricular diastolic suction.16
The mechanism by which negative pressures are
generated in the thin-walled embryonic heart is unknown. Radially oriented fibers extend from the
endocardium to the myocardium in the extracellular
matrix of the early embryonic heart.'7 These fibers
are stretched as the wall thickens during cardiac
contraction and may provide a restoring force during
diastole.18 Because the contents of the extracellular
matrix can be selectively altered by proteases19 and
hyaluronidase,20 measuring ventricular function after
these perturbations will further define the role of the
matrix in generating diastolic suction in the embryonic heart.
In the mature heart, ventricular systole begins with
the rise in pressure that occurs after the completion
0.151 Otmin area
,- 3.00
*PEAK SYSTOUC PRESSURE
y = 1.900 - 0.160x + 0.007x2 r = 0.897 SEE = 0.154
2.50 oEND-SYSTOLIC PRESSURE
y = 2.430 - 0.209x + 0.008x2 r = 0.871 SEE = 0.164
2.00-
22
20
18
1.00
AREA (mm2)
E
E
"--
16
(x i SEM)
24
26
-
tes area
Otes pressure tmax
-
pressure
o 0.10
(D
U)
LLJ
L .J
0.05.
D 1.50-
:2
U) 1.oo
L0
CL
0-5041 4
(x i SEM)
.
16
18
20
22
24
26
versus
maximum pressure
to area ratio for stage 16 to 24 chick embryos. Data plotted are
mean+±SEM. Second-order regression curves are plotted to
raw
data.
14
9
16
(x- ± SEM)
t
18
.
.
20
22
24
26
STAGE
STAGE
FIGURE 6. Peak systolic pressure
0.00X
FIGURE 8. Time difference between time of end systole
defined as the maximum instantaneous pressure to area ratio
and time of minimum area or time of maximum ventricular
pressure. Data plotted are mean +SEM.
230
Circulation Research Vol 68, No 1, January 1991
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of atrial contraction. This sequence depends on
electrical isolation between the chambers and the
time delay of atrioventricular node depolarization.
These structures are not present in the embryonic
heart, and no electrical isolation exists between the
atria and ventricles.21
Simultaneous atrial and ventricular myograms of
the embryonic heart confirm that ventricular contraction begins before the completion of atrial contraction.22 This overlap is likely responsible for the
increase in ventricular area noted after the onset of
systole (Figure 3). In addition, the mature ventricle
changes shape during isovolumic contraction,23 and a
similar mechanism would change the amount of the
embryonic ventricle imaged in a single plane during
isovolumic contraction. Despite this overlap of atrial
and ventricular contraction, we defined the onset of
systole as the beginning of isometric contraction
displayed in the PA loop.
The presence of isometric contraction and relaxation periods in each PA loop confirms previous
observations that the atrioventricular and conotruncal cushions function as valves during the cardiac
cycle.24 Retrograde flow through the atrioventricular
cushions does not occur during ventricular systole.
Forward flow through the conotruncal cushions occurs shortly after the onset of active ventricular
contraction.3 The increase in isometric contraction
time noted from stage 16 to stage 24 is consistent with
the generation of a higher preejection ventricular
pressure. The increased isometric contraction time
may relate to ventricular-vascular coupling of the
ventricle to progressively higher aortic sac pressures.
Systolic function is greatly influenced by the interaction of the ventricle with the arterial system. Arterial impedance is a measure of the property of the
arterial system to oppose ventricular ejection.25 Because blood is ejected in a pulsatile manner, resistance must also include components of arterial compliance and blood inertia.26 In the mature ventricle,
changes in arterial impedance characteristics predictably alter the shape of the ejection trajectory defined
by the upper right corner of the PV loop.27 In the
chick embryo, hydraulic load decreases from stage 18
to 29 because of a progressive decline in vascular
resistance.28 We found that from stage 16 to 24 the
ejection curve of the PA loop became progressively
curvilinear and peak ventricular pressure occurred
earlier in systole. These changes in ventricular ejection characteristics defined by PA loops are consistent with the increased vascular compliance calculated by Fourier analysis of simultaneous aortic blood
pressure and flow.28
Systole ends with the completion of active ventricular contraction. In the mature heart, end systole is
best defined as the time of maximum ventricular
elastance7 and can be approximated as the time of
maximum pressure to volume ratio.'3 The time of end
ejection is greatly influenced by vascular impedance
and occurs after end systole in the ejecting heart. The
right ventricular PV loop has a rounded upper left
corner because of the low resistance of the pulmonary vascular bed relative to the inertance and
elastance components of pulmonary arterial impedance.29 This low-resistance vascular impedance increases the difference between the time of end
systole defined by maximum ventricular elastance
and end ejection.
We found that in the embryonic heart, the time of
end systole differed significantly from the time of
end-ejection area or peak systolic pressure. Following ventricular ejection, the contractile conotruncus
provides additional energy for the ejection of blood
into the aortic sac. Thus, end ejection is not a valid
index for the end of systole in the developing cardiovascular system. The time difference between peak
ventricular pressure and maximum pressure to area
ratio increased across the stage range. Because of the
rapid change in arterial impedance characteristics
that occurs during vascular development, instantaneous maximum pressure to area ratio, rather than
peak ventricular pressure or minimum ventricular
area, best defines the time of end systole.
In the mature heart, pressure and volume data
have been used to test mathematical models of
mechanics and energetics.7 The analysis and modeling of ventricular mechanics by PA loops in the chick
embryo will provide a framework for defining mechanisms of normal and experimentally altered cardiovascular development.
Acknowledgments
This work would not have been possible without
the inspiration and supervision of the late Dr. Kiichi
Sagawa.
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pressure-volume relations * cardiovascular
KEY WORDS
development * ventricular function * chick embryo
Ventricular pressure-area loop characteristics in the stage 16 to 24 chick embryo.
B B Keller, N Hu, P J Serrino and E B Clark
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Circ Res. 1991;68:226-231
doi: 10.1161/01.RES.68.1.226
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1991 American Heart Association, Inc. All rights reserved.
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