- Email: [email protected]
Embryonic stroke volume and cardiac output in the chick
DEVELOPMENTAL
BIOLOGY
Embryonic
41, 14-21 (1974)
Stroke
Volume
and Cardiac
Output
in the Chick’
J. JOB FABER, THOMAS J. GREEN, AND KENT L. THORNBURG Department
of Physiology,
University
of Oregon Medical
Accepted
School, Portland,
Oregon 97201
July 8, I974
Cine recordings of the hearts of chick embryos of 3 days and 2 hr to 4 days and 21 hr incubation were projected and measured. The measurements were converted to volumes. Stroke volume was determined from the difference in end diastolic and end systolic volume and multiplied by heart rate to yield cardiac output. Mean stroke volume was 0.0058 (*0.00036 SEM) mm3 per mg body wt; mean cardiac output was 0.956 (+ 0.061 SEM) mm3/min per mg body wt. Stroke volume and cardiac output rose above their control values after intravascular injection of Ringer’s solution, and even more so after the injection of dextran solution. The increases in stroke volume were due to increases in end diastolic volume, in the case of dextran injected embryos they occurred in spite of a simultaneous increase in end systolic volume. It is concluded that the rise in cardiac output with growth of the embryo is in large part due to an increase in stroke volume, and that the increase in stroke volume depends in part on the known increase in embryonic blood volume. The experiments further suggest that a rapid hydrostatic and osmotic equilibrium exists between embryonic blood plasma and an extra vascular compartment. INTRODUCTION
Vertebrate embryos need a functional system for the circulation of blood as they begin to exceed the length of a few millimeters. The transparency of bird embryos and extraembryonic membranes allow the central circulation and much of the peripheral circulation to be seen after a few days of incubation. It is known, therefore, that the chick heart begins to beat, though not to pump, at about 30 hr of incubation. At this stage, the two cardiac primordia have not yet fused, except in the region that will later become the ventricle (Patten, 1949). Blood begins to circulate after about 40 hr of incubation when the connections between the dorsal aorta of the embryo and the small vessels of the yolk sac finally complete the circuit. The heart is thus one of the first permanent organs to become functional. It continues to function while undergoing the structural alterations that will separate atrium from ventricle, ventricle from aorta, and the left from the right chambers, alterations that take place in I Supported by a grant from the National Institute of Child Health and Human Development, HD 6313. 14 Copyright AU rights
Q 1974 by Academic Press, Inc. of reproduction in any form reserved.
the period between the third and the eighth days of incubation (see Patten, 1951). Cardiac output during development is believed to increase with increasing embryo size but cardiac outputs in embryos of less than 8 days incubation have been measured in only two single embryos (Hughes, 1949). At the time the heart begins to circulate blood, the myocardial cells contain fragments of myofilaments. The filaments appear almost randomly oriented in striking contrast to the severe parallel registration of myofilaments that one finds in the sarcomeres of the adult heart (Hibbs, 1956; Olivo et al., 1964; Manasek, 1968). The myofibrils often still lack alignment in the myocardium of chicks of 5 days incubation (Manasek, 1970). It is known that the heart of a chick embryo of 4 days of incubation possesses some inotropic mechanisms (Michal et al., 1967; Faber, 1968). Surprisingly, in view of the orientation of the myofilaments, heterometric changes in contractility can be evoked also (Faber, 1968). It seemed of interest, therefore, to investigate cardiac output during the first few days after the heart begins to beat to determine whether
FABER,GREEN AND THORNBURG an increase in cardiac output occurs and, if so, whether it is solely a reflection of the increase in myocardial mass or whether it is in part due to the increase in embryonic blood volume (Rychter et al., 1955) and ventricular filling. METHODS
Fertilized eggs were incubated for periods between 3 days and 2 hr to 4 days and 21 hr at 38°C and 50% relative humidity. This range of incubation ages yields embryos of Hamburger-Hamilton stages between 16 and 25 (New, 1966). We set an upper limit of 125 mg for embryonic weight in this study. The weights of the embryos used varied between 12.7 and 115.1 mg. The eggs were turned blunt end up, some time before the experiment, to allow the embryos to float to the position underneath the air chamber. Shell and both shell membranes were partially removed and the egg placed in a holder under a stereomicroscope. The surfaces of embryo and yolk sac were covered entirely with a thin layer of mineral oil to prevent evaporative loss of heat and water. In some preparations, a medium sized vitelline artery or vein was cannulated with a glass pipette with a beveled tip of about 50 micron diameter. Polyethylene tubing connected the pipette with a calibrated syringe which
Stroke Volume
15
was driven by a micrometer and filled with Ringer’s or a solution of dextran in isotonic saline. The outline of the heart could usually be visualized with ease (see Fig. 1). A chick embryo is practically always lying on its left side (Romanoff, 1960) and the major axis of the combined ventricles is perpendicular to the optical axis of the microscope. The few embryos which were found to be in a bad position were discarded. It was possible, therefore, to determine the outline of the ventricles (Fig. 1). The stereomicroscope was equipped with a photography attachment and a “super-8” mm tine camera. Exposures were made at 50 frames/set on color reversal film. Only occasionally did a lack of light require the use of filming speeds of 25 or 36 frames/set. The developed films were projected frame by frame and their overall photographic magnification was measured by photographing a millimeter scale in place of the embryo and measuring its projected size on the screen. Measurements were made at either x 71 or x 142 linear magnification. Heart rates were counted visually by means of a stop watch at the time of the experiments in order not to be dependent on the exact speed of the tine camera. Cardiac outlines were retraced on paper. At least 50 consecutive frames were traced
FIG. 1 a. Cine recording of embryonic chick heart at 50 frames/set. in Fig. 1 b. Third frame shows end diastolic volume.
Copy from color film. Part of sequence 2
DEVELOPMENTALBIOLOGY
i
VOLUME 41, 1974
A Sequence I (control) 0 Sequence 2 0 Sequence 3
I
I
I
I
I
I
I
I
I
I
I
I
50
25
Frame Number FIG. 1 b. Computed cardiac volumes of embryo whose sequences 2 and 3 after injection of 6% and 16% of body wt after injection of 28% and 38% have been deleted for clarity. mms, respectively, but the last sequence had to be rejected 209/min to 18l/min (- 13%).
for each measurement of stroke volume. In most recordings, the boundary between the blood in the ventricle and the inside of the ventricular wall was not as clear as the boundary between the outside of the ventricular wall and the thoracic cavity of the embryo. This was due to the well documented presence of peculiar multiple pouches on the endocardial surface of the heart at this stage 9f development, which make the interior surface of the heart almost spongelike in appearance (Romanoff, 1960). The epicardial boundary was traced. The calculated cardiac volumes were, therefore, the combined volumes of myocardium and its contained blood. However, since the difference between the end
heart is shown in Fig. 1 a. Sequence 1 is control, respectively of dextran solution. Sequences 4 and 5 Stroke volumes were 0.43, 0.54, 0.77, 0.93, and 0.97 because heart rate dropped from its control value of
diastolic volume and the end systolic volume was due to the difference in the amount of contained blood, it represented true stroke volume. Two methods were used to convert a projected image of the heart to volume. At first, long axis (2a) and short axis (2b) of the ventricle were measured, suitably corrected for optical magnification, and converted to volume (V) according to the formula for a prolate spheroid V = (4/3) x ab2. Later, when an automatic planimeter (Hewlett Packard) became available the surface area (A) of the projection was measured, because this measurement was less sensitive to subjective error. Area was converted to volume by the formula for the
FABER. GREEN AND THORNBURG
sphere: V = (4/3) r-0.5 A’.5. The two methods yielded statistically indistinguishable results (see Results), and a series of ten sequences of cardiac contours analyzed for stroke volume by both methods showed a coefficient of correlation of 0.991 (P << 0.01) and a regression coefficient that did not significantly differ from its ideal value of one. Control tine recordings were made in 52 embryos. In 19 of these, known amounts of fluid not exceeding 50% of embryo body weight were then injected into the embryonic circulation and further tine recordings were made. No recordings were analyzed if there were any signs of bleeding from the site of cannulation. Such bleeding was easily detected against the light yellow background of the yolk. Usually, heart rate after injection remained between + 10% of its control value but nine recordings (out of a total of 49) were rejected because heart rates changed by more than 10%. In no case was the time between the control recording and the last recording more than 16 min and during this period the embryos cooled by only a few degrees centigrade as confirmed by thermocouple measurements. Finally, the embryos were dissected out, gently blotted and weighed. RESULTS
AND
DISCUSSION
The embryos selected for these experiments have an atrium, a ventricle and a truncus arteriosus that are clearly separated from each other by narrow junctions. All three chambers contract. There are as yet no valves and no separation of left and right hearts. The chambers contract in sequence. There is no reflux from ventricle to atrium or from truncus arteriosus to ventricle since the junctions close during the appropriate moments of the cardiac cycle. This is easily verified by observation or by slow motion analysis of the tine recordings of the (transparent) embryonic hearts. These observations are in full agreement with those of Patten et al.
Stroke Volume
17
(1948). The ventricular stroke volumes are, therefore, “forward” stroke volumes. Stroke volume increased almost in proportion to the increase in embryonic weight. Figure 2 shows stroke volumes of the 52 embryos as a function of embryonic weight from 12.7 to 115.1 mg. The ventricular volumes of the first 29 embryos were measured with the ventricular shape approximated as a prolate spheroid. The ventricular volumes of the last 23 embryos were measured by conversion of planimetered ventricular areas. The regression coefficients relating stroke volumes to embryonic weight were not significantly different for the two groups (P r 0.3), and the mean stroke volumes normalized per mg body wt were 0.0055 (SE 0.00045) and 0.0062 (SE 0.00058) mm3/mg for the first and second group, respectively; these also were not significantly different. The results obtained with the two methods were therefore combined in Fig. 2 and Table 1. The increase of stroke volume with increasing embryo weight is substantial and statistically highly significant (Table 1). Within the restricted range of embryonic weights encountered in these studies, heart rate did not vary significantly with embryonic weight (r = 0.13, P z 0.35) although it is known to do so when large ranges are studied (Romanoff, 1960). The stroke volumes, even when normalized per mg embryo weight did not significantly correlate with heart rate (r = -0.08, P s 0.55). Cardiac output was calculated as the product of stroke volume and heart rate. Cardiac output increased with embryonic body weight as is shown in Fig. 3 and Table 1. In contrast to the normalized stroke volumes, which did not depend on heart rate, normalized cardiac output increased reliably with increasing embryonic heart rate (Table 1). We concluded from these relationships that in the ranges of heart rates that naturally occur in chick embryos, the diastolic period is sufficiently long to allow close to complete filling of the empty
18
DEVELOPMENTALBIOLOGY
0
25
FIG. 2. other lines (inner set) equivalent
75
50 Embryo
VOLUME 41, 1974
Wwght
150
100
(mg)
Stroke volume as a function of embryq body weight. Center line is least squares regression line. The are the 95% confidence limits for individual stroke volumes (outer set) and mean stroke volumes as functions of embryo weight (Snedecor and Cochran, 1967). One cubic millimeter is assumed to be to 1 mg.
ventricle. This is not to say that the filling of the ventricle cannot be increased, as will be shown below, but that it must be increased by increasing venous pressure rather than by increasing the duration of diastole. The variability in normalized cardiac output and in stroke volume exceeds the variability one finds in adults as is evident on inspection of Figs. 2 and 3. This variability must in part be due to unavoidable experimental inaccuracies in the present study. In the absence of another independent experimental method for determining cardiac output in embryos, one cannot determine the magnitude of this error. Figure lb shows that cardiac volumes determined by the present method are reproducible from cycle to cycle. Some of the observed variability is likely to be related to differences in the relative size of the extraembryonic circulations which are not
TABLE
1
STROKE VOLUME, HEART RATE, AND CARDIAC OUTPUT IN 52 EMBRYOSO Regressions as a function of embryo weight: S.V. = 0.120 + 0.00326 (SE 0.00067) wt. C.O. = 13.9 + 0.65 (SE 0.13) Wt. H.R. does not correlate significantly with weight S.V. and C.O. normalized per mg body wt; regressions as a function of heart rate. Mean (S.V./Wt.) ,100 = 0.58 (+ 0.036 SE) Mean (C.O./Wt.). 100 = 95.6 (+ 6.1 SE) (S.V./Wt.) does not correlate significantly with heart rate (C.O./Wt.). 100 = 28.1 + 0.41 (SE 0.206) H.R.
P < 0.01 P < 0.01 PxO.35
P= 0.55 P5 0.05
“S.V., stroke volume (mm”); C.O., cardiac output (mmJ.min-I); H.R., heart rate (min-I); Wt., embryo body weight (mg).
Stroke Volume
FABER. GREEN AND THORNBURG
accurately reflected in body weight. Upon inspection, the extraembryonic circulation appears to us to accommodate more than half of the cardiac output of the early chick embryo. Figure 4 shows the changes in stroke volumes that were observed in 19 embryos after the injection of Ringer’s or dextran solution into a vitelline blood vessel. (All of these stroke volumes were analyzed by planimetry.) The stroke volumes were expressed as percent of the control (preinjection) values and injected volumes were expressed as percent of embryo weight. (Rychter et al. (1955) estimated that blood volume of 3-5 day old embryos is about 40% of body wt.) Thirteen measurements of stroke volume in embryos injected with Ringer’s solution showed that the stroke volume obeyed the least-squares regression: Stroke volume (%) = 106 + 2.21 (SE 0.41) injected volume (%) (r = 0.85, P < 0.01). Forty-six measurements of stroke
0
25
FIG. 3. Cardiac output as a function those in Fig. 2.
volume in embryos injected with dextran solution obeyed the least-squares regression: Stroke volume (%) = 104 + 3.86 (SE 0.32) injected volume (%) (r = 0.88, P < 0.01). Since nine measurements were discarded, because heart rates changed by more than 10% from control value, changes in cardiac output closely paralleled the changes in stroke volume. End diastolic volumes increased significantly after the injection of Ringer’s solution (r = 0.75, P < 0.01) or dextran solution (r = 0.92, P < 0.01). The increase in stroke volume is, therefore, a form of heterometric autoregulation. End systolic volumes did not significantly correlate with injected volume in the series injected with Ringer’s solution, but correlated reliably in the larger series injected with dextran solution (r = 0.57, P < 0.01). We conclude, therefore, that the heart emptied less completely after the injection of dextran solu-
50 Embryo
19
75 Weight
100
125
(mg)
of embryo body weight. The indicated
lines have the same functions
as
DEVELOPMENTAL
BIOLOGY
Infused Solution
VOLUME 41. 1974
(mg/mg)
FIG. 4. Increases in stroke volumes as a function of infused volumes. Stroke volumes are expressed as % of control stroke volumes and infused volumes are expressed as % of embryo body weight. The lines are the 95% confidence limits of the mean stroke volumes as functions of injected volume (1 mm3 = 1 mg).
tion. Ejection fractions could not be calculated as the measured cardiac volume included myocardial volume. The regression coefficients 2.21 and 3.86 relating stroke volume to injected volume in the Ringer’s and dextran injected series respectively were statistically highly significantly different from each other (P < 0.01). The experiments showed, therefore, that the increase in stroke volume produced by injection of dextran exceeded the increase produced by the injection of an equal volume of Ringer’s solution. This was most likely due to rapid loss of Ringer’s solution from the vascular compartment to another, unidentified, embryonic or extraembryonic compartment. These compartments are too numerous to permit identification (interstitial fluid of the embryonic soma, seroamniotic fluid, amniotic fluid, allantoic fluid, subgerminal yolk or albumin). To our knowledge, no measurements of the colloid osmotic pressure of embryonic chick plasma are available. It cannot be ruled out, therefore, that the
colloid osmotic pressure of the injected dextran solution was relatively hypertonic. If so, it may have attracted extra vascular water to the vascular compartment of the embryo causing an increase in blood volume in excess of the volume of water already injected. The injected volumes shown in Fig. 4 do not, therefore, either in the case of Ringer’s solution or in the case of dextran solution, necessarily reflect the actual change in circulating embryonic blood volume. We conclude that embryonic stroke volume depends on embryonic blood volume and increases with embryonic growth. This increase must be in part dependent on the known increase in blood volume. The increase in embryonic cardiac output with growth is due to the known increase in heart rate and the increase in stroke volume. A continuous and rapid equilibrium of the colloid osmotic and hydrostatic pressures in the blood plasma and an unidentified extravascular compartment would account for the observed differences between
FABER. GREEN AND THORNBURG
the effects of injected Ringer’s and dextran solutions. The authors thank Mrs. Alice Fitzgerald, Robt. P. Roth and Dr. E. Louise Kremkau.
Mr.
REFERENCES FABER, J. J. (1968). Mechanical function of the septating embryonic heart. Amer. J. Physiol. 214, 475481. HIBBS, R. G. (1956). Electron microscopy of developing cardiac muscle in chick embryos. Amer. J.
Anat. 99, 17-52. HUGHES, A. F. W. (1949). The heart output of the chick embryo. J. Roy. Microsc. Sot. 69, 145-152. MANASEK, F. J. (1968). Embryonic development of the heart. J. Morph. 125, 329-366. MANASEK, F. J. (1970). Histogenesis of the embryonic myocardium. Amer. J. Card. 25, 149-168. MICHAL, F., EMMETT, F., and THORP, R. H. (1967). A study of drug action on the developing avian cardiac muscle. Comp. Biochem. Physiol. 22, 563-570.
Stroke Volume
21
NEW, D. A. T. (1966). “The Culture of Vertebrate Embryos.” Academic Press, London. OLIVO, M. O., LASCHI, R., and LUCCHI, M. L. (1964). Genesi delle miofibrille de1 cuore embrionale di’ poll0 osservate al microscopio elettronico e inizio dell’ attivita contratille. Lo Sperimentale 114, 69-78. PAITEN, B. M., KRAMER, T. C. and BARRY, A. (1948). Valvular action in the embryonic chick heart by localized apposition of endocardial masses. Anat. Rec. 102, 299-311. PAITEN, B. M. (1949). Initiation and early changes in the character of the heart beat in vertebrate embryos. Physiol. Reu. 29, 31-47. PAT~EN, B. M. (1951). “Early Embryology of the Chick.” McGraw-Hill, New York. ROMANOFF, A. L. (1960). “The Avian Embryo.” Macmillan, New York. RYCHTER, Z., KOPECK?, M., and LEMEZ, L. (1955). A micromethod for determination of the circulating blood volume in chick embryos. Nature 175, 1126-1127. SNEDECOR, G. W., and COCHRAN, W. G. (1967). “Statistical Methods,” 6th ed. Iowa State University Press, Ames, IA.
BIOLOGY
Embryonic
41, 14-21 (1974)
Stroke
Volume
and Cardiac
Output
in the Chick’
J. JOB FABER, THOMAS J. GREEN, AND KENT L. THORNBURG Department
of Physiology,
University
of Oregon Medical
Accepted
School, Portland,
Oregon 97201
July 8, I974
Cine recordings of the hearts of chick embryos of 3 days and 2 hr to 4 days and 21 hr incubation were projected and measured. The measurements were converted to volumes. Stroke volume was determined from the difference in end diastolic and end systolic volume and multiplied by heart rate to yield cardiac output. Mean stroke volume was 0.0058 (*0.00036 SEM) mm3 per mg body wt; mean cardiac output was 0.956 (+ 0.061 SEM) mm3/min per mg body wt. Stroke volume and cardiac output rose above their control values after intravascular injection of Ringer’s solution, and even more so after the injection of dextran solution. The increases in stroke volume were due to increases in end diastolic volume, in the case of dextran injected embryos they occurred in spite of a simultaneous increase in end systolic volume. It is concluded that the rise in cardiac output with growth of the embryo is in large part due to an increase in stroke volume, and that the increase in stroke volume depends in part on the known increase in embryonic blood volume. The experiments further suggest that a rapid hydrostatic and osmotic equilibrium exists between embryonic blood plasma and an extra vascular compartment. INTRODUCTION
Vertebrate embryos need a functional system for the circulation of blood as they begin to exceed the length of a few millimeters. The transparency of bird embryos and extraembryonic membranes allow the central circulation and much of the peripheral circulation to be seen after a few days of incubation. It is known, therefore, that the chick heart begins to beat, though not to pump, at about 30 hr of incubation. At this stage, the two cardiac primordia have not yet fused, except in the region that will later become the ventricle (Patten, 1949). Blood begins to circulate after about 40 hr of incubation when the connections between the dorsal aorta of the embryo and the small vessels of the yolk sac finally complete the circuit. The heart is thus one of the first permanent organs to become functional. It continues to function while undergoing the structural alterations that will separate atrium from ventricle, ventricle from aorta, and the left from the right chambers, alterations that take place in I Supported by a grant from the National Institute of Child Health and Human Development, HD 6313. 14 Copyright AU rights
Q 1974 by Academic Press, Inc. of reproduction in any form reserved.
the period between the third and the eighth days of incubation (see Patten, 1951). Cardiac output during development is believed to increase with increasing embryo size but cardiac outputs in embryos of less than 8 days incubation have been measured in only two single embryos (Hughes, 1949). At the time the heart begins to circulate blood, the myocardial cells contain fragments of myofilaments. The filaments appear almost randomly oriented in striking contrast to the severe parallel registration of myofilaments that one finds in the sarcomeres of the adult heart (Hibbs, 1956; Olivo et al., 1964; Manasek, 1968). The myofibrils often still lack alignment in the myocardium of chicks of 5 days incubation (Manasek, 1970). It is known that the heart of a chick embryo of 4 days of incubation possesses some inotropic mechanisms (Michal et al., 1967; Faber, 1968). Surprisingly, in view of the orientation of the myofilaments, heterometric changes in contractility can be evoked also (Faber, 1968). It seemed of interest, therefore, to investigate cardiac output during the first few days after the heart begins to beat to determine whether
FABER,GREEN AND THORNBURG an increase in cardiac output occurs and, if so, whether it is solely a reflection of the increase in myocardial mass or whether it is in part due to the increase in embryonic blood volume (Rychter et al., 1955) and ventricular filling. METHODS
Fertilized eggs were incubated for periods between 3 days and 2 hr to 4 days and 21 hr at 38°C and 50% relative humidity. This range of incubation ages yields embryos of Hamburger-Hamilton stages between 16 and 25 (New, 1966). We set an upper limit of 125 mg for embryonic weight in this study. The weights of the embryos used varied between 12.7 and 115.1 mg. The eggs were turned blunt end up, some time before the experiment, to allow the embryos to float to the position underneath the air chamber. Shell and both shell membranes were partially removed and the egg placed in a holder under a stereomicroscope. The surfaces of embryo and yolk sac were covered entirely with a thin layer of mineral oil to prevent evaporative loss of heat and water. In some preparations, a medium sized vitelline artery or vein was cannulated with a glass pipette with a beveled tip of about 50 micron diameter. Polyethylene tubing connected the pipette with a calibrated syringe which
Stroke Volume
15
was driven by a micrometer and filled with Ringer’s or a solution of dextran in isotonic saline. The outline of the heart could usually be visualized with ease (see Fig. 1). A chick embryo is practically always lying on its left side (Romanoff, 1960) and the major axis of the combined ventricles is perpendicular to the optical axis of the microscope. The few embryos which were found to be in a bad position were discarded. It was possible, therefore, to determine the outline of the ventricles (Fig. 1). The stereomicroscope was equipped with a photography attachment and a “super-8” mm tine camera. Exposures were made at 50 frames/set on color reversal film. Only occasionally did a lack of light require the use of filming speeds of 25 or 36 frames/set. The developed films were projected frame by frame and their overall photographic magnification was measured by photographing a millimeter scale in place of the embryo and measuring its projected size on the screen. Measurements were made at either x 71 or x 142 linear magnification. Heart rates were counted visually by means of a stop watch at the time of the experiments in order not to be dependent on the exact speed of the tine camera. Cardiac outlines were retraced on paper. At least 50 consecutive frames were traced
FIG. 1 a. Cine recording of embryonic chick heart at 50 frames/set. in Fig. 1 b. Third frame shows end diastolic volume.
Copy from color film. Part of sequence 2
DEVELOPMENTALBIOLOGY
i
VOLUME 41, 1974
A Sequence I (control) 0 Sequence 2 0 Sequence 3
I
I
I
I
I
I
I
I
I
I
I
I
50
25
Frame Number FIG. 1 b. Computed cardiac volumes of embryo whose sequences 2 and 3 after injection of 6% and 16% of body wt after injection of 28% and 38% have been deleted for clarity. mms, respectively, but the last sequence had to be rejected 209/min to 18l/min (- 13%).
for each measurement of stroke volume. In most recordings, the boundary between the blood in the ventricle and the inside of the ventricular wall was not as clear as the boundary between the outside of the ventricular wall and the thoracic cavity of the embryo. This was due to the well documented presence of peculiar multiple pouches on the endocardial surface of the heart at this stage 9f development, which make the interior surface of the heart almost spongelike in appearance (Romanoff, 1960). The epicardial boundary was traced. The calculated cardiac volumes were, therefore, the combined volumes of myocardium and its contained blood. However, since the difference between the end
heart is shown in Fig. 1 a. Sequence 1 is control, respectively of dextran solution. Sequences 4 and 5 Stroke volumes were 0.43, 0.54, 0.77, 0.93, and 0.97 because heart rate dropped from its control value of
diastolic volume and the end systolic volume was due to the difference in the amount of contained blood, it represented true stroke volume. Two methods were used to convert a projected image of the heart to volume. At first, long axis (2a) and short axis (2b) of the ventricle were measured, suitably corrected for optical magnification, and converted to volume (V) according to the formula for a prolate spheroid V = (4/3) x ab2. Later, when an automatic planimeter (Hewlett Packard) became available the surface area (A) of the projection was measured, because this measurement was less sensitive to subjective error. Area was converted to volume by the formula for the
FABER. GREEN AND THORNBURG
sphere: V = (4/3) r-0.5 A’.5. The two methods yielded statistically indistinguishable results (see Results), and a series of ten sequences of cardiac contours analyzed for stroke volume by both methods showed a coefficient of correlation of 0.991 (P << 0.01) and a regression coefficient that did not significantly differ from its ideal value of one. Control tine recordings were made in 52 embryos. In 19 of these, known amounts of fluid not exceeding 50% of embryo body weight were then injected into the embryonic circulation and further tine recordings were made. No recordings were analyzed if there were any signs of bleeding from the site of cannulation. Such bleeding was easily detected against the light yellow background of the yolk. Usually, heart rate after injection remained between + 10% of its control value but nine recordings (out of a total of 49) were rejected because heart rates changed by more than 10%. In no case was the time between the control recording and the last recording more than 16 min and during this period the embryos cooled by only a few degrees centigrade as confirmed by thermocouple measurements. Finally, the embryos were dissected out, gently blotted and weighed. RESULTS
AND
DISCUSSION
The embryos selected for these experiments have an atrium, a ventricle and a truncus arteriosus that are clearly separated from each other by narrow junctions. All three chambers contract. There are as yet no valves and no separation of left and right hearts. The chambers contract in sequence. There is no reflux from ventricle to atrium or from truncus arteriosus to ventricle since the junctions close during the appropriate moments of the cardiac cycle. This is easily verified by observation or by slow motion analysis of the tine recordings of the (transparent) embryonic hearts. These observations are in full agreement with those of Patten et al.
Stroke Volume
17
(1948). The ventricular stroke volumes are, therefore, “forward” stroke volumes. Stroke volume increased almost in proportion to the increase in embryonic weight. Figure 2 shows stroke volumes of the 52 embryos as a function of embryonic weight from 12.7 to 115.1 mg. The ventricular volumes of the first 29 embryos were measured with the ventricular shape approximated as a prolate spheroid. The ventricular volumes of the last 23 embryos were measured by conversion of planimetered ventricular areas. The regression coefficients relating stroke volumes to embryonic weight were not significantly different for the two groups (P r 0.3), and the mean stroke volumes normalized per mg body wt were 0.0055 (SE 0.00045) and 0.0062 (SE 0.00058) mm3/mg for the first and second group, respectively; these also were not significantly different. The results obtained with the two methods were therefore combined in Fig. 2 and Table 1. The increase of stroke volume with increasing embryo weight is substantial and statistically highly significant (Table 1). Within the restricted range of embryonic weights encountered in these studies, heart rate did not vary significantly with embryonic weight (r = 0.13, P z 0.35) although it is known to do so when large ranges are studied (Romanoff, 1960). The stroke volumes, even when normalized per mg embryo weight did not significantly correlate with heart rate (r = -0.08, P s 0.55). Cardiac output was calculated as the product of stroke volume and heart rate. Cardiac output increased with embryonic body weight as is shown in Fig. 3 and Table 1. In contrast to the normalized stroke volumes, which did not depend on heart rate, normalized cardiac output increased reliably with increasing embryonic heart rate (Table 1). We concluded from these relationships that in the ranges of heart rates that naturally occur in chick embryos, the diastolic period is sufficiently long to allow close to complete filling of the empty
18
DEVELOPMENTALBIOLOGY
0
25
FIG. 2. other lines (inner set) equivalent
75
50 Embryo
VOLUME 41, 1974
Wwght
150
100
(mg)
Stroke volume as a function of embryq body weight. Center line is least squares regression line. The are the 95% confidence limits for individual stroke volumes (outer set) and mean stroke volumes as functions of embryo weight (Snedecor and Cochran, 1967). One cubic millimeter is assumed to be to 1 mg.
ventricle. This is not to say that the filling of the ventricle cannot be increased, as will be shown below, but that it must be increased by increasing venous pressure rather than by increasing the duration of diastole. The variability in normalized cardiac output and in stroke volume exceeds the variability one finds in adults as is evident on inspection of Figs. 2 and 3. This variability must in part be due to unavoidable experimental inaccuracies in the present study. In the absence of another independent experimental method for determining cardiac output in embryos, one cannot determine the magnitude of this error. Figure lb shows that cardiac volumes determined by the present method are reproducible from cycle to cycle. Some of the observed variability is likely to be related to differences in the relative size of the extraembryonic circulations which are not
TABLE
1
STROKE VOLUME, HEART RATE, AND CARDIAC OUTPUT IN 52 EMBRYOSO Regressions as a function of embryo weight: S.V. = 0.120 + 0.00326 (SE 0.00067) wt. C.O. = 13.9 + 0.65 (SE 0.13) Wt. H.R. does not correlate significantly with weight S.V. and C.O. normalized per mg body wt; regressions as a function of heart rate. Mean (S.V./Wt.) ,100 = 0.58 (+ 0.036 SE) Mean (C.O./Wt.). 100 = 95.6 (+ 6.1 SE) (S.V./Wt.) does not correlate significantly with heart rate (C.O./Wt.). 100 = 28.1 + 0.41 (SE 0.206) H.R.
P < 0.01 P < 0.01 PxO.35
P= 0.55 P5 0.05
“S.V., stroke volume (mm”); C.O., cardiac output (mmJ.min-I); H.R., heart rate (min-I); Wt., embryo body weight (mg).
Stroke Volume
FABER. GREEN AND THORNBURG
accurately reflected in body weight. Upon inspection, the extraembryonic circulation appears to us to accommodate more than half of the cardiac output of the early chick embryo. Figure 4 shows the changes in stroke volumes that were observed in 19 embryos after the injection of Ringer’s or dextran solution into a vitelline blood vessel. (All of these stroke volumes were analyzed by planimetry.) The stroke volumes were expressed as percent of the control (preinjection) values and injected volumes were expressed as percent of embryo weight. (Rychter et al. (1955) estimated that blood volume of 3-5 day old embryos is about 40% of body wt.) Thirteen measurements of stroke volume in embryos injected with Ringer’s solution showed that the stroke volume obeyed the least-squares regression: Stroke volume (%) = 106 + 2.21 (SE 0.41) injected volume (%) (r = 0.85, P < 0.01). Forty-six measurements of stroke
0
25
FIG. 3. Cardiac output as a function those in Fig. 2.
volume in embryos injected with dextran solution obeyed the least-squares regression: Stroke volume (%) = 104 + 3.86 (SE 0.32) injected volume (%) (r = 0.88, P < 0.01). Since nine measurements were discarded, because heart rates changed by more than 10% from control value, changes in cardiac output closely paralleled the changes in stroke volume. End diastolic volumes increased significantly after the injection of Ringer’s solution (r = 0.75, P < 0.01) or dextran solution (r = 0.92, P < 0.01). The increase in stroke volume is, therefore, a form of heterometric autoregulation. End systolic volumes did not significantly correlate with injected volume in the series injected with Ringer’s solution, but correlated reliably in the larger series injected with dextran solution (r = 0.57, P < 0.01). We conclude, therefore, that the heart emptied less completely after the injection of dextran solu-
50 Embryo
19
75 Weight
100
125
(mg)
of embryo body weight. The indicated
lines have the same functions
as
DEVELOPMENTAL
BIOLOGY
Infused Solution
VOLUME 41. 1974
(mg/mg)
FIG. 4. Increases in stroke volumes as a function of infused volumes. Stroke volumes are expressed as % of control stroke volumes and infused volumes are expressed as % of embryo body weight. The lines are the 95% confidence limits of the mean stroke volumes as functions of injected volume (1 mm3 = 1 mg).
tion. Ejection fractions could not be calculated as the measured cardiac volume included myocardial volume. The regression coefficients 2.21 and 3.86 relating stroke volume to injected volume in the Ringer’s and dextran injected series respectively were statistically highly significantly different from each other (P < 0.01). The experiments showed, therefore, that the increase in stroke volume produced by injection of dextran exceeded the increase produced by the injection of an equal volume of Ringer’s solution. This was most likely due to rapid loss of Ringer’s solution from the vascular compartment to another, unidentified, embryonic or extraembryonic compartment. These compartments are too numerous to permit identification (interstitial fluid of the embryonic soma, seroamniotic fluid, amniotic fluid, allantoic fluid, subgerminal yolk or albumin). To our knowledge, no measurements of the colloid osmotic pressure of embryonic chick plasma are available. It cannot be ruled out, therefore, that the
colloid osmotic pressure of the injected dextran solution was relatively hypertonic. If so, it may have attracted extra vascular water to the vascular compartment of the embryo causing an increase in blood volume in excess of the volume of water already injected. The injected volumes shown in Fig. 4 do not, therefore, either in the case of Ringer’s solution or in the case of dextran solution, necessarily reflect the actual change in circulating embryonic blood volume. We conclude that embryonic stroke volume depends on embryonic blood volume and increases with embryonic growth. This increase must be in part dependent on the known increase in blood volume. The increase in embryonic cardiac output with growth is due to the known increase in heart rate and the increase in stroke volume. A continuous and rapid equilibrium of the colloid osmotic and hydrostatic pressures in the blood plasma and an unidentified extravascular compartment would account for the observed differences between
FABER. GREEN AND THORNBURG
the effects of injected Ringer’s and dextran solutions. The authors thank Mrs. Alice Fitzgerald, Robt. P. Roth and Dr. E. Louise Kremkau.
Mr.
REFERENCES FABER, J. J. (1968). Mechanical function of the septating embryonic heart. Amer. J. Physiol. 214, 475481. HIBBS, R. G. (1956). Electron microscopy of developing cardiac muscle in chick embryos. Amer. J.
Anat. 99, 17-52. HUGHES, A. F. W. (1949). The heart output of the chick embryo. J. Roy. Microsc. Sot. 69, 145-152. MANASEK, F. J. (1968). Embryonic development of the heart. J. Morph. 125, 329-366. MANASEK, F. J. (1970). Histogenesis of the embryonic myocardium. Amer. J. Card. 25, 149-168. MICHAL, F., EMMETT, F., and THORP, R. H. (1967). A study of drug action on the developing avian cardiac muscle. Comp. Biochem. Physiol. 22, 563-570.
Stroke Volume
21
NEW, D. A. T. (1966). “The Culture of Vertebrate Embryos.” Academic Press, London. OLIVO, M. O., LASCHI, R., and LUCCHI, M. L. (1964). Genesi delle miofibrille de1 cuore embrionale di’ poll0 osservate al microscopio elettronico e inizio dell’ attivita contratille. Lo Sperimentale 114, 69-78. PAITEN, B. M., KRAMER, T. C. and BARRY, A. (1948). Valvular action in the embryonic chick heart by localized apposition of endocardial masses. Anat. Rec. 102, 299-311. PAITEN, B. M. (1949). Initiation and early changes in the character of the heart beat in vertebrate embryos. Physiol. Reu. 29, 31-47. PAT~EN, B. M. (1951). “Early Embryology of the Chick.” McGraw-Hill, New York. ROMANOFF, A. L. (1960). “The Avian Embryo.” Macmillan, New York. RYCHTER, Z., KOPECK?, M., and LEMEZ, L. (1955). A micromethod for determination of the circulating blood volume in chick embryos. Nature 175, 1126-1127. SNEDECOR, G. W., and COCHRAN, W. G. (1967). “Statistical Methods,” 6th ed. Iowa State University Press, Ames, IA.