Download Evidence that blood pressure controls heart rate in

/ . Embryol exp. Morph. Vol. 36, 3, pp. 685-695, 1976
Printed in Great Britain
685
Evidence that blood pressure controls heart rate
in the chick embryo prior to neural control
By G. M. RAJALA, 1 J. H. KALBFLEISCH 2 AND S. KAPLAN 3
From the Department of Anatomy, The Medical College of Wisconsin,
Milwaukee, Wisconsin
SUMMARY
Blood pressure increases will increase heart rate in intact chick embryos, prior to tne
development of neural control. Similarly, in surgically isolated hearts, increases in intraventricular fluid pressure will increase the rate of beat. However, fluid pressure applied
equally to both interior and exterior surfaces of the isolated heart does not result in increased
heart rate. Therefore, we conclude that the increased pressure stretches the heart muscle
and that this stretch stimulates the increased heart rate.
While heart rate is clearly influenced by blood pressure, the reverse is not true. Propranolol
reduces the heart rate to about half normal in intact embryos but does not significantly
alter the blood pressure.
INTRODUCTION
The embryonic chick heart begins to beat early on the 2nd day of incubation
(Patten & Kramer, 1933), but vagal control of heart rate does not become
functionally established until the 5th day (LeGrande, Paff & Boucek, 1966),
and sympathomimetic receptors are not functionally established until the
4th day (Paff & Glander, 1968). Although numerous reports exist describing
temporal changes in embryonic heart rate, there have been no suggestions
made concerning how the heart rate is regulated during the intervening period
between initiation of heart beat and functional neural influence. Indeed, it
appears that it is not known whether heart rate is regulated at all during this
period.
During the course of teratological experiments in which ventricular blood
pressure and heart rate were being measured in 3-day-old chick embryos, it
was observed that a correlation existed between these two parameters; that
is, when pressure was high, heart rate was high, and the reverse. Experiments
were then designed to determine if the degree of stretch of the heart tube wall,
1
Author's address: Department of Anatomy, Hahnemann Medical College, 203 North
Broad Street, Philadelphia, Pennsylvania 19102, U.S.A.
2
Author's address: Department of Preventive Medicine (Biostatics), The Medical College
of Wisconsin, 1725 West Wisconsin Avenue, Milwaukee, Wisconsin 53233, U.S.A.
3
Author's address: Department cf Anatomy, Medical College of Wisconsin, 561 North
15th Street, Milwaukee, Wisconsin 53233. Send reprint requests here.
686
G. M. RAJALA, J. H. KALBFLEISCH AND S. KAPLAN
induced by introducing additional fluid into the bloodstream and measured
in units of pressure, is a factor in the control of heart rate in the preneural
embryonic heart. The results, reported here, indicate that pressure indeed
does control heart rate at these early stages.
MATERIALS AND METHODS
General. Fertile White Leghorn chicken eggs were stored at 10 °C for no
longer than one week after receipt from the supplier. The eggs were incubated
in a forced draft incubator at 38 °C and 55-75 % relative humidity (standard
conditions, Landauer, 1967) until the embryos had reached HamburgerHamilton (1951) stages 18, 19 and 20 (3-3£ days).
The experiments required the use of a blood pressure apparatus sensitive
enough to record mean ventricular blood pressure (MVBP) and versatile
enough to artificially alter intraventricular pressure in these small embryos.
A water manometer system, used by Landis (1926) to measure capillary
pressure in the frog mesentery and developed by Paff, Boucek & Gutten (1965)
and Grabowski, Tsai & Toben (1969) to measure MVBP in chick embryos,
was adapted for use in this study. The system consisted of a micrometer
controlled syringe connected to a saline (0-85 % NaCl, w/v) reservoir, manometer, and cannula by means of two three-way stopcocks. The cannula
and manometer were connected with polyethylene tubing. The entire
apparatus was mounted on a plastic stand supported with a thick plastic base
(Fig. 1).
The procedure used to measure mean ventricular blood pressure was as
follows. An egg was placed under a dissecting microscope and a window was
made in the shell with a motor driven microsaw and forceps to expose the
embryo. An air-curtain incubator was used to keep the embryo at 38 °C (this
was monitored with a thermistor probe). The height of the saline in the
manometer (Fig. 1) was then adjusted to heart level and this was defined as
the zero pressure level. The chorionic, amniotic, and pericardial membranes
of the embryo were moved aside with fine tipped forceps. The cannula, flamedrawn from capillary tubing to a diameter of approximately 50 jam, was
positioned in the lumen of the ventricle with the aid of a mechanical manipulator. Successful cannulation was signaled when erythrocytes entered the
cannula tip. At that time the manometer and cannula were opened to the
syringe. The micrometer screw was then turned until the small stream of
erythrocytes, located just in the tip of the cannula, was seen to oscillate with
systole and diastole of the heart. At this time the MVBP was recorded from
the manometer and consisted of the height above heart level of the saline
column.
Preliminary MVBP measurements were recorded from stage 18 through
20 embryos to test the apparatus. The average blood pressure was 12-13 mm
Control of heart rate in chick embryo
687
Calibration
scale (mm)
Screw-opcratcd syringe
From cannula
Polyethylene
Uibiim
3-\vav valves
Glass cannula
To manometer
Polyethylene tubing
Fig. 1. Apparatus used to measure blood pressure, and to alter blood pressure by
introducing fluid into the caidiovascular system.
of water, which was comparable to the results of Paff et ah (1965) and
Grabowski et ah (1969) for similarly staged embryos.
Intact embryo experiments. The blood pressure and heart rate in beats per
minute (bpm) were recorded from 19 intact embryos. The MVBP measurement
was considered as the baseline blood pressure for each particular embryo. The
pressure was then increased in increments of 10 mm of water by turning the
syringe screw (Figs. 1 and 2 A) and the heart rate was measured at each
increment. After the pressure had been increased by several increments of
10 mm (with heart rate recorded at each increment), the ventricular pressure
was decreased in increments of 10 mm. Heart rate was again recorded at each
increment. In this way, data were obtained and analyzed to determine whether
a correlation existed between ventricular pressure and heart rate in the intact,
preneural embryo.
Isolated heart experiments. The hearts of 19 stage-18 to -20 embryos were
688
G. M. RAJALA, J. H. KALBFLEISCH AND S. KAPLAN
SI'
S\
-
""
c
c
A. Intact
B. Isolated
1 mm
C. Isolated
D. Intact
Chamber
Propranolol
Fig. 2. Summary of the four types of experiments. Single arrows indicate pressure;
double arrows indicate oscillation of blood cells as described in the text. (A)
Cannula tip inserted in the heart of an intact embryo. (B) Isolated heart, occluded
at the bulbus cordis with a 7-0 silk ligature. (C) Isolated heart in a closed chamber
in which pressure could be raised or lowered. (D) Heart in an intact embryo in
which propranolol solution was used to reduce the rate of beat. Abbreviations:
C = cannula, BC = bulbus cordis, SV = sinus venosus.
excised using small iris scissors to cut at the junction of the vitelline veins,
duct of Cuvier and truncus arteriosus. Once excised, the heart was placed in
a small dish of Locke's solution kept at 38 °C by a temperature-controlled
warming stage mounted on a dissecting microscope. The venous end of the
heart tube was slipped on a cannula with fine-tipped forceps and was tied
in place with 7-0 silk ligature. The bulbus cordis was also ligated with 7-0 silk,
leaving the heart tube occluded at one end and connected directly to the
pressure apparatus at the other end (Fig. 2B). The cannula and manometer
were opened directly to the syringe (Fig. 1). Pressure was controlled by
manipulating the micrometer screw and heart rates were recorded at pressure
levels of zero (manometer fluid level at heart level) and at increments of 10 mm
above zero. Heart rates were also recorded as the pressure was reduced to
the initial level. The aim here was to vary intraventricular pressure in the
isolated heart (clearly, this heart was free of any possible central neural connexions) and to observe whether or not heart rate was directly correlated
with these changes in ventricular pressure.
The next series of experiments was designed to test the effects of pressure
Control of heart rate in chick embryo
689
applied to all surfaces of the heart. Hearts were isolated as outlined previously
and were placed in a sealed chamber containing Locke's solution kept at
38 °C. The chamber consisted of two round cover glasses separated by a
neoprene O-ring and clamped in a metal holder. A port in the side of the
holder admitted a 32-gauge needle which was connected to the blood pressure
apparatus. In this way, fluid pressure was monitored and altered in the entire
chamber, which contained an isolated but unligated heart. Again, pressure
was increased in increments while monitoring heart rate to determine whether
pressure applied uniformly to all surfaces of the heart can control the preneural
heart rate (Fig. 2C).
Propranolol experiments. In the previous experiments, ventricular pressure
was artificially controlled and was the independent variable while heart rate was
the dependent variable. This last series of experiments was designed to reverse
the variables to determine whether artificial changes in heart rate (induced
by treatment with propranolol) would affect ventricular pressure. Propranolol
was chosen because it has been found to cause an immediate and significant
decrease in heart rate in the chick embryo heart after its application (Jaffee,
1972; Kolesari, 1975).
The MVBP and heart rate for 15 intact embryos were recorded. The cannula
was left in position in each embryo while administering 10/d of a 0-13 % (w/v,
dissolved in Locke's solution) propranolol solution directly on the ventricle
(Fig. 2D). The MVBP was recorded for each heart rate measured and these
measurements were made while heart rate was decreasing. The collected data
were analyzed to determine if the direct relationship between ventricular
pressure and heart rate was observed with heart rate as the independent
variable, in contrast to the previously described experiments with ventricular
pressure as the independent variable.
RESULTS
Intact embryos. A blood pressure-heart rate linear relationship was determined
for each individual embryo as:
HR = a + b(BF),
where HR = heart rate (dependent variable), a = intercept, b = slope, and
BP = ventricular blood pressure (independent variable). Strong and significant
blood pressure-heart rate linear correlations were observed in 15 of the 19
embryos in the study. The overall average regression equation (Table 1) yielded
a high correlation and strong significance, indicating that heart rate will
increase or decrease as ventricular blood pressure increases or decreases.
Fig. 3 shows the plotted data points and average equation from the 19 intact
embryos, again showing the direct relationship.
Isolated hearts. The same blood pressure-heart rate linear relationship was
observed in the 19 surgically isolated, ligated hearts. Strong and significant
44
EMB 36
690
G. M. RAJALA, J. H. KALBFLEISCH AND S. KAPLAN
240 -i
210 -
180 -
150 -
«
120 -
90 -
60 -
30 -
0 -
0
20
40
60
80
100
Ventricular blood pressure (mm H : O)
Fig. 3. Intact embryo hearts, ventricular blood pressure versus heart rate.
correlations were seen in all 19 individual hearts, as well as in the overall
average regression equation (Table 1). The average equation and data points
(Fig. 4) clearly indicate that ventricular pressure changes result in concomitant
heart rate changes in the absence of central neural connexions.
The six isolated, unligated hearts subjected to pressure changes in a closed
chamber yielded a negative slope in the overall average regression equation
and an overall negative correlation (Table 1). This indicates that pressure
increases surrounding and compressing the heart will decrease heart rate some-
691
Control of heart rate in chick embryo
240 -,
210 -
180 -
150 -
•~
120 -
9 0 •-
60 -
30-
0-
1:
l
l
I
20
40
60
I
80
100
Ventricular fluid pressure (mm H 2 O)
Fig. 4. Isolated, ligated embryo hearts, ventricular fluid pressuie versus heart late.
what (Fig. 5). This is in contrast to the previous experiments in which intraventricular pressure increases (stretching the ventricle) increase the heart
rate.
Propranolol experiments. This experiment was designed to reverse the variables
described in the previous experiments. A heart rate-blood pressure linear
relationship was determined for each embryo as:
BP = c+b(HR),
44-2
692
G. M. RAJALA, J. H. KALBFLEISCH AND S. KAPLAN
Table 1. Summary of the correlation analysis
for all experimental groups
N
Overall regression
equation*
Correlation
Significance
level
19
19
6
HR == 110+1-643 (BP)
HR == 43 +2-050 (BP)
HR == 84-0-115 (CP)
+ 0-95
+ 0-95
-0-40
P < 0001
P < 0001
P < 001
15
BP =: 11-4 + 0015 (HR)
+ 0-21
N.S.f
Group
Intact embryos
Isolated ligated hearts
Isolated unligated
hearts (chamber)
Propranolol treated
intact embryos
* Abbreviations: HR = heart rate, BP = blood pressure , CP = chamber pressure.
t Not significant, i.e. P > 005.
150 -i
120 -
90 -
60 -
30 -
0 -
20
40
60
80
100
Chamber pressure (mm H 2 O)
Fig. 5. Isolated, unligated embryo hearts, chamber pressure versus heart rate.
where BP = mean ventricular blood pressure (dependent variable), a = intercept, b = slope, and HR = heart rate (independent variable). Non-significant
heart rate-blood pressure linear correlations were determined in 14 of the
15 embryos. The overall average regression equation (Table 1) was also not
statistically significant, indicating that propranolol treatment, which decreases
693
Control of heart rate in chick embryo
20
10 -
0 -
40
r
80
i
120
r
160
l
200
Heart rate (bpm)
Fig. 6. Intact embryo hearts, heart iate versus mean ventricular blood pressure
after propranolol treatment.
heart rate, does not result in concomitant decrease in MVBP. Indeed, 8 of
15 embryos yielded individual slopes of zero. The average regression equation
and data points are plotted in Fig. 6.
DISCUSSION
The experiments presented have provided evidence for an intrinsic autoregulatory mechanism for heart rate control in the preneural heart. In the
fitst two series of experiments, the ventricular muscle was artificially stretched
by increased filling (measured in units of ventricular pressure) and this, in
turn, resulted in increased heart rate in intact embryos as well as surgically
isolated, ligated hearts.
Pressure was applied to all surfaces of isolated, unligated hearts in the third
series of experiments. Such compression resulted in slightly decreased heart
rate, the effect being the opposite of the effect observed by intraventricular
pressure (which stretched the heart tube). These results suggest that before
functional central neural connexions to the heart develop, heart rate can be
regulated by the degree of stretch of the heart tube wall.
The final series of experiments was done to reverse the blood pressure-heart
rate variables. Propranolol, a known beta-adrenergic blocking agent, was used
to decrease heart rate following baseline heart rate and MVBP measurements.
The hearts were probably slowed through the local anaesthetic action of the
94
G. M. RAJALA, J. H. KALBFLEISCH AND S. KAPLAN
drug, since functional sympathomimetic receptors are thought not to exist
in the chick heart at 3 days of development. Following treatment with propranolol to decrease heart rate, a significant concomitant decrease in ventricular
blood pressure was not observed. This is perhaps due to two observations
made during the experiments. The ejection fraction of blood from the ventricle
during systole appeared to be altered after drug treatment decreased the heart
rate. A distinct stream of blood cells was seen distending the outflow tract
from ventricle to bulbus cordis during the entire cardiac cycle. This suggests
that regurgitation through an incompetent outflow valve elevated enddiastolic pressure. Another striking observation was that blood flow in
the anterior cardinal vein was seen to reverse diiections and result in
hemorrhages in the small vessels leading to the vein. This suggests that
the filling pressure generated by the atrium was elevated when the heart
rate decreased. It is perhaps for these reasons that MVBP was maintained
at near normal levels following propranolol treatment to decrease heart
rate.
The conclusion is that heart rate of the preneural embryonic heart is
intrinsically autoregulated by pressure within the heart tube. This control is
apparently mediated through the stretch of the heart tube wall by changing
blood volume. Thus, ventricular blood pressure changes result in concomitant
heart rate changes. The reverse of this relationship does not appear to be
true.
Finally, we speculate that the build up of intraventricular pressure, which
proceeds as the cardiovascular system develops in the very early embryo, may
play a role in the stimulation and maintenance of the first heartbeats.
We thank F. D. Anderson and H. Klitgaard for their constructive criticism of the manuscript. Supported by a Wisconsin Heart Association Grant and PHS GRS Grant 5 SOI
FR-5434.
REFERENCES
C. T., TSAI, E. N. C. & TOBEN, H. R. (1969). The effects of teratogenic doses
of hypoxia on the blood pressure of chick embryos. Teratology 2, 67-76.
HAMBURGER, V. & HAMILTON, H. L. C1951). A series of normal stages in the development
of the chick embryo. J. Morph. 88, 49-92.
JAFFEE, O. C. (1972). Effects of propranolol on the chick embryo heart. Teratology 5,
153-158.
KOLESARI, G. L. (1975). Amphetamines, insulin and trypan blue: their ability to cause caudal
hematomas in the chick and their mechanisms of teratogenic action. Ph.D. dissertation,
Anatomy Department, Medical College of Wisconsin.
LANDAUER, W. (1967). The Hatchability of Chicken Eggs as Influenced by Environment and
Hereditary. Rev. ed. Monograph 1, University of Connecticut, Agricultural Experiment
Station, Storrs.
LANDIS, E. M. (1926). The capillary pressure in frog mesentery as determined by microinjection methods. Am. J. Physiol. 75, 548-571.
LEGRANDE, M. C , PAFF, G. H. & BOUCEK, R. J. (1966). Initiation of vagal control of heart
rate in the embryonic chick. Anat. Rec. 155, 163-166.
GRABOWSKI,
Control of heart rate in chick embryo
695
G. H., BOUCEK, R. J. & GUTTEN, G. S. (1965). Ventricular blood pressures and
competency of valves in the early embryonic chick heart. Anat. Rec. 151, 119-124.
PAFF, G. H. & GLANDER, T. P. (1968). The time appearance of sympathcmimetic receptors
in the embryonic chick heart. Anat. Rec. 160, 405 (abstr.).
PATTEN, B. M. & KRAMER, T. C. (1933). The initiation of contraction in the embryonic
chick heart. Am. J. Anat. 53, 349-375.
PAFF,
{Received 1 July 1976)