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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 Downloaded from http://circres.ahajournals.org/ by guest on April 30, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on April 30, 2017 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. Downloaded from http://circres.ahajournals.org/ by guest on April 30, 2017 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 Downloaded from http://circres.ahajournals.org/ by guest on April 30, 2017 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) Downloaded from http://circres.ahajournals.org/ by guest on April 30, 2017 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 References 1. Pappano AJ, Loeffelholz K: Ontogenesis of adrenergic and cholinergic neuro-effector transmission in chick embryo hearts. J Pharmacol Exp Ther 1974;191:468-478 2. Higgins D, Pappano AJ: Development of transmitter secretory mechanisms in adrenergic neurons in the embryonic chick heart ventricle. Dev Biol 1981;87:148-162 3. Clark EB, Hu N: Developmental hemodynamic changes in the chick embryo from stage 18 to 27. Circ Res 1982;51:810-815 4. Rychter Z, Kopecky M, Lemez L: The blood of chick embryos: IV. On the circulating blood volume from the 2nd day of incubation till hatching. Cesk Morfol 1955;3:11-25 5. Clark EB, Hu N, Dummett JL, Vendekieft GK, Olson C, Tomanek R: Ventricular function and morphology in the chick embryo stage 18 to 29. Am J Physiol 1986;250:H407-H413 6. Downing SE, Talner NS, Gardner TH: Ventricular function in the newborn lamb. Am J Physiol 1965;208:931-937 7. Friedman WF: The intrinsic physiologic properties of the developing heart. Prog Cardiovasc Dis 1972;15:87-111 8. Kirkpatrick SE, Pitlick PT, Naliboff J, Friedman WF: FrankStarling relationship as an important determinant of fetal cardiac output. Am J Physiol 1976;231:495-500 9. Romero TE, Friedman WF: Limited left ventricular response to volume overload in the neonatal period: A comparative study with the adult animal. Pediatr Res 1979;13:910-915 10. Rudolph AM, Heymann MA: Fetal and neonatal circulation and respiration. Annu Rev Physiol 1974;36:187-207 11. Faber JJ, Green TJ, Thornburg KL: Embryonic stroke volume and cardiac output in the chick. Dev Biol 1974;41:14-21 12. Clark EB: Hemodynamic control of the chick embryo cardiovascular system, in Nora J, Takao A (eds): Congenital Heart Disease: Causes and Processes. Mt Kisco, NY, Futura Publishing Co, Inc., 1984, pp 377-386 13. Clark EB, Hu N, Dooley JB: The effect of isoproterenol on cardiovascular function in the stage 24 chick embryo. Teratology 1985;31:41-47 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 Downloaded from http://circres.ahajournals.org/ by guest on April 30, 2017 Circ Res. 1990;67:187-192 doi: 10.1161/01.RES.67.1.187 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1990 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/67/1/187 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. 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