Edward B. Clark

Hemodynamics of the stage 12 to stage 29 chick embryo.

The heart is the first functioning organ in the embryo and provides blood flow during cardiac morphogenesis from a muscle-wrapped tube a few cells thick to the four-chambered pump. We described the hemodynamics of the chick embryo from stage 12 (50 hours of a 21-day incubation) to stage 29 (6 days), during which the embryo weight increased 120-fold. We measured ventricular, embryo and extraembryonic vascular bed wet weights, dorsal aortic blood flow with a directional pulsed-Doppler velocity meter, and ventricular and vitelline arterial blood pressures witfa a servo-null micropressure system. The data are reported as mean ± SEM. With rapid development and morphogenesis, dorsal aortic blood flow increased from 0.015 ± 0.004 to 2.40 ± 0.20 mm3/sec parallel to the geometric increase of wet embryo weight from 2.22 ± 0.10 to 267.5 ± 9.7 mg. Dorsal aortic blood flow normalized for embryo and extraembryonic weight remained relatively constant (Y=2.13+0.02X, r=0.23, SEE=0.03). Stroke volume increased from 0.01 ± 0.003 to 0.69 ± 0.03 mm3, and heart rate doubled from 103 ± 2 to 208 ± 5 beats/min. Systolic, diastolic, and mean vitelline arterial pressure increased linearly from 0∼32 ± 0.01, 0.23 ± 0.01, and 0.28 ± 0.01 mm Hg at stage 12 to 2.00 ± 0.06, 1.22 ± 0.03, and 1.51 ± 0.04 mm Hg, respectively, at stage 29. Ventricular peak systolic and end-diastolic pressure increased from 0.95 ± 0.04 and 0.24 ± 0.02 at stage 12 to 3.45 ± 0.10 and 0.82 ± 0.03 at stage 29, respectively. The hemodynamic waveforms were similar to those found in the four-chamber heart of the mature animal. These data are integral to understanding the interrelation of function and form during cardiac development.

Residual strain in the ventricle of the stage 16-24 chick embryo.

Residual stress and strain, i.e., the stress and strain remaining in a solid when all external loads are removed, may be produced in biological tissues by differential growth. During cardiac development, residual stress and strain may play a role in cardiac morphogenesis by affecting ventricular wall stress. After a transmural radial cut, a passive ventricular cross section opens into a sector, and the size of the opening angle provides a measure of the circumferential residual strain. Residual strains were characterized in this manner for the apical region of the diastolic embryonic chick heart for Hamburger-Hamilton stages 16, 18, 21, and 24 (approximately 2.5, 3.5, 4.0, and 4.5 days, respectively, of a 21-day incubation period). The average opening angle at these stages was 107 +/- 10 degrees, 79 +/- 10 degrees, 73 +/- 11 degrees, and 74 +/- 7 degrees, respectively (n > or = 5 for each stage). These measured angles were correlated with changes in ventricular morphology. Scanning electron micrographs of the apex revealed that the wall of the ventricle is smooth at stage 16. Then at stage 18, myocardial trabeculae develop, forming ridges with primarily a circumferential orientation. By stage 21, the trabeculae develop into a mesh, giving the ventricular wall a spongelike appearance, and the preferred orientation is lost by stage 24. The large decrease in opening angle between stages 16 and 18 corresponded to the onset of trabeculation, which is the greatest change in form during the studied stages. We speculate that residual strain is an important biomechanical factor during cardiac morphogenesis.

Diastolic Filling Characteristics in the Stage 12 to 27 Chick Embryo Ventricle

ABSTRACT: Cardiac output is affected by the diastolic filling characteristics of the ventricle. We hypothesized that the relative contributions of passive and active filling change as the ventricle develops from a smooth-walled tube to a trabeculated four-chamber heart. In stage 12 to 27 white Leghorn chick embryos, we simultaneously measured ventricular pressure with a servo-null micropressure system and dorsal aortic and atrioventricular velocities with a 20-MHz pulsed-Doppler velocity meter. The analog waveforms were sampled at 500 Hz and converted to digital format via an analog/digital board. We partitioned diastole into passive and active components. The passive phase began with the return of the pressure curve to baseline and extended to the onset of the a-wave. The active phase began with the upstroke of the atrial velocity curve and extended to the upstroke of the ventricular pressure curve at end-diastole. Data are presented as mean ± SEM (n ≥ 6 at each stage) and analyzed by analysis of variance and regression analysis. At similar cycle lengths ranging from 480 to 600 ms (p > 0.05), end-diastolic pressure increased from 0.24 ± 0.02 mm Hg at stage 12 to 0.55 ± 0.01 mm Hg at stage 27. Passive and active filling volumes were 92 (0.0038 ± 0.0005 mm3) and 8% (0.0004 ± 0.0002 mm3), respectively, at stage 12 and changed to 24 (0.23 ± 0.08 mm3) and 76% (0.62 ± 0.08 mm3), respectively, at stage 27. The ratio of passive to active filling volume decreased from 7.89 to 0.35. Thus, active ventricular filling became dominant as the trabeculae formed in the embryonic ventricle. These observations define the diastolic filling characteristics of the embryonic heart during primary cardiac morphogenesis.

Ventricular pressure-area loop characteristics in the stage 16 to 24 chick embryo.

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 greater than or equal to 4, and were tested by regression analysis and analysis of variance (p less than 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 less than 0.05), while isometric relaxation time was 124 +/- 12 and 120 +/- 10 msec (p greater than 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.

A nonliner poroelastic model for the trabecular embryonic heart.

A theoretical model is presented for the primitive right ventricle of the stage 21 chick embryo. At this stage of development, the wall of the heart is trabecular with direct intramyocardial blood flow. The model is a pressurized fluid-filled cylinder composed of a porous inner layer of isotropic myocardium and a relatively thin compact outer layer of transversely isotropic myocardium. The analysis is based on nonlinear poroelasticity theory, modified to include residual strain and muscle activation. Correlating theoretical and experimental pressure-volume loops and epicardial strains gives first-approximation constitutive relations for stage 21 embryonic myocardium. The results from the model suggest three primary conclusions: (1) Some muscle fibers likely are aligned in the compact layer, with a fiber angle approximately + 10 deg from the circumferential direction. (2) Blood is drawn into the wall of the ventricle during diastolic filling and isovolumic contraction and is squeezed out of the wall during systolic ejection, giving a primitive intramyocardial circulation before the coronary arteries form. As the heart rate increases, the transmural blood-flow velocity increases, but the volume of blood exchanged with the lumen per beat decreases. (3) Residual strain affects transmural stress distributions, producing nearly uniform stresses in the porous layer, where the peak end-systolic stress occurs. These results improve our understanding of the relation between form and function in the developing heart and provide directions for biological experiments to study cardiac morphogenesis.

Prenatal detection of cardiovascular malformations by echocardiography: An indication for cytogenetic evaluation

Prenatal diagnosis of congenital cardiovascular malformations by echocardiography may signal associated chromosome abnormalities. The exact proportion of these associations is not known but is expected to be higher than that with live-birth. To estimate the risk that a fetus with an echocardiographically detected heart defect has an autosomal trisomy or Turner syndrome, we adjusted the known frequency of aneuploidy in live-born infants with congenital cardiovascular malformations by the reported rate of spontaneous abortion, with data from a population-based case-control study of congenital cardiovascular malformations in which 268 cases (12.7%) had both congenital cardiovascular malformations and a chromosome abnormality. Included in the present analysis were 188 aneuploid infants with congenital cardiovascular malformations that would have been detectable by fetal echo. When data are adjusted for the high spontaneous abortion rate of aneuploid fetuses, we estimate that there would have been more than a threefold increase in aneuploidy over the 13% seen at live-birth. Thus cytogenetic analysis is appropriate in a fetus with echo-diagnosed congenital cardiovascular malformations.

Cardiac mechanics in the stage-16 chick embryo

A theoretical model is presented for the tubular heart of the stage-16 chick embryo (2.3 days of a 21-day incubation period). The model is a thick-walled, pseudoelastic cylindrical shell composed of three isotropic layers: the endocardium, the cardiac jelly, and the myocardium. The analysis is based on a shell theory that accounts for large deformation, material nonlinearity, residual strain, and muscle activation, with material properties inferred from available experimental data. We also measured epicardial strains from recorded motions of microspheres on the primitive right ventricles of stage-16 white Leghorn chick embryos. Relative to end diastole, peak axial and circumferential Lagrange strains occurred near end systole and had similar values. The magnitudes of these strains varied along the longitudinal axis of the heart (-0.16 +/- 0.08), being larger near the ends of the primitive right ventricle and smaller near midventricle. The in-plane shear strain was less than 0.05. Comparison of theoretical and experimental strains during the cardiac cycle shows generally good agreement. In addition, the model gives strong stress concentrations in the myocardial layer at end systole.

Rate of coronary vascularization during embryonic chicken development is influenced by the rate of myocardial growth.

OBJECTIVE We tested the hypothesis that the degree of coronary microvessel formation in the embryonic heart is regulated by the magnitude of myocardial growth. METHODS The outflow tract of Hamburger-Hamilton stage 21 chicken hearts (prior to the onset of coronary vasculogenesis) was constricted in ovo with a loop of 10-0-nylon suture, and the hearts were studied at stages 29 and 36. RESULTS At stage 29 ventricular mass was 64% greater in the pressure-overloaded than in the hearts of sham-operated controls, but vascular volume density and numerical density, determined by electron microscopic morphometry, were identical. As demonstrated by histological morphometric evaluation, the compact region of the left ventricle at stage 29 was 43% thicker than the shams. However, by stage 36 heart mass, thickness of the compact region, and overall wall thickness (demonstrated by scanning electron microscopy) were significantly less than in the sham group of this stage, but vascular volume density was virtually identical in the two groups. Formation of the two main coronary arteries was clearly impeded in the banded hearts, i.e., the coronaries were stunted in their development or failed to completely form coronary ostia. CONCLUSIONS Vascular growth is proportional to myocardial growth in the embryonic, overloaded heart, but the persistence of the pressure overload results in a failure of or severe limitations in coronary artery development. These data support the hypothesis that vascular growth during this period of development is regulated, at least in part, by the rate and magnitude of myocardial growth.

Epicardial strains in embryonic chick ventricle at stages 16 through 24.

Embryonic cardiac development depends, in part, on the local biomechanical environment. Tracking the motions of microspheres attached to the embryonic chick ventricle, we computed two-dimensional epicardial strains at Hamburger-Hamilton stages 16, 18, 21, and 24 (2.5, 3.5, 4.0, and 4.5 days, respectively, of a 21-day incubation period). First, in a cross-sectional study, strains were measured in separate embryos at each stage (n > or = 19 per stage). Then, in a longitudinal study, strains were measured serially on the same heart, with the eggs resealed and reincubated between successive stages (n > or = 4 per stage). Although the heart undergoes major changes in mass, morphology, and loading during the studied stages, both studies showed that peak circumferential and longitudinal strains relative to end diastole were similar in magnitude (0.13 to 0.16) and did not change significantly across the stage range. The peak principal strains also showed no significant changes, with magnitudes of approximately 0.11 and 0.18. The shear strains were small, and their signs varied from one heart to another. These results suggest that wall strain is maintained within a relatively narrow range during primary cardiac morphogenesis.

Effect of Heart Rate Increase on Dorsal Aortic Flow before and after Volume Loading in the Stage 24 Chick Embryo

ABSTRACT: In the stage 24 chick embryo, a paced increase in heart rate reduces stroke volume, presumably by rate-dependent decrease in passive filling. We hypothesized that rate-dependent stroke volume reduction could be abolished by volume loading. Dorsal aortic blood velocity was measured with a 20 mHz puIsed-Doppler meter from a 0.75-mm piezoelectric crystal (eight embryos), and atrioventricular velocity was simultaneously measured from the ventricular apex (six embryos). Sinus venosus pacing (stimuli of 1 ms duration and <4 mA) was performed at intrinsic rate (P:I) and at 150% of intrinsic rate (P:150%I). Volume loading was performed during P:150%I by intravenous injection of 7.5 ixL of chick Ringers solution. Using atrioventricular velocity profile, stroke volume was divided into the proportion due to passive (E-phase) and active (Aphase) filling. Stroke volume was compared during P:I, P:150%I, immediately (P:150%I′) and 30 s after (P:150%I) volume loading. Data (mean ± SEM) were compared by ANOVA. During pacing, stroke volume (mm2/cycle) decreased but increased after volume loading (I, 0.43 ± 0.03; P:I, 0.37 ± 0.03; P:150%I, 0.19 ± 0.03; P:150%I′, 0.24 ± 0.05; P:150%I, 0.28 ± 0.04 (p < 0.005). During P:150%I, E-phase filling disappeared and was not restored by volume loading, whereas, A-phase filling diminished but was restored by volume loading. In stage 24 chick embryos, rate-dependent stroke volume decrease is reversed by volume loading that restores stroke volume due to an increase in active filling but not passive filling. Thus, even at rapid heart rate, the embryonic ventricle responds to volume loading, indicating that the Frank- Starling relationship functions during tachycardia in the embryonic heart.