INTRODUCTION

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VENTRICULAR-VASCULAR coupling allows the low-pressure, energetically limited embryonic cardiovascular system to adapt to acute and chronic alterations in its biochemical (nutrient) and biomechanical (loading) microenvironment while undergoing simultaneous structural and functional maturation (17, 18). The embryonic ventricle can acutely alter end-diastolic volume (EDV) and stroke volume (SV) in response to altered preload, similar to the mature circulation (19). Peripheral vascular resistance, as measured by arterial impedance, drops significantly in response to increased blood flow (32). In contrast to the relatively linear mature end-systolic pressure-volume (PV) relation (15, 19), the highly curvilinear embryonic end-systolic PV relationship at increased EDVs may be due to almost instantaneous changes in arterial impedance after altered SV (32). Of note, curvilinear end-systolic PV relationships have been noted for the puppy left ventricle and for small animals (26). Thus the purpose of this study was to generate embryonic ventricular PV relations during ejection into the arterial circulation and during acute uncoupling from the arterial circulation to define maximal ventricular contractile reserve.

This study shows that the embryonic ventricle generated a significantly higher systolic pressure when ejection into the arterial tree was prevented by acute conotruncal (CT) occlusion, resulting in a more linear end-systolic PV relation. However, the increased end-systolic pressure generated after acute CT occlusion was associated with a prolonged rate of ventricular relaxation (), suggesting prolonged calcium reuptake by immature myocytes. As expected, the increase in ventricular afterload associated with CT occlusion was associated with decreased SV for matched EDVs. Thus embryonic myocardial contractile reserve is masked in the ejecting state by rapid changes in arterial impedance that occur in response to altered systemic blood flow.

	METHODS

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Embryo preparation and developmental staging. Fertilized white Leghorn chicken eggs were incubated blunt end up in a forced draft incubator to Hamburger-Hamilton stage 21 (3.5 days) of a 46-stage (21-day) incubation period (12). This embryonic stage corresponds to 11.5 embryonic days in mice (23) and to Streeter Horizon XIV in humans (25). The egg was positioned on a photomacroscope stage, and then access to the embryo was gained by opening the shell and removing a small region of extraembryonic membranes. Ambient temperature between 37 and 38°C was maintained using ambient heat lamps and a double-walled glass chamber (custom design;, Radnotti Glass Technologies, Monrovia, CA) perfused at 15 l/min with 40°C water using a circulating water bath (model EX221; Neslab Instruments, Portsmouth, NH). All studies were performed in ovo.

Hemodynamic preparation. We measured ventricular pressure and dimensions using an integrated physiology and morphometry workstation that has been previously described in detail (19). Briefly, video images were acquired using a photomacroscope (model M400; Wild Leitz, Rockleigh, NJ), video camera (model 70-newvicon tube; Dage-MTI, Michigan City, IN), a video recorder (model VR9670; Magnavox), and a time-date generator (model VTG-33; FOR.A, West Newton, MA). A 50-µm division scribed glass standard was recorded in the plane of each embryo after imaging for planimetry software calibration.

We simultaneously measured intraventricular pressure with a servo-null system (model 900A; World Precision Instruments, Sarasota, FL). A fluid-filled glass capillary pipette was positioned with the use of a micromanipulator (Leitz, Wetzlar, Germany) to puncture the ventricle. The servo-null system is linear (y = 0.995x  0.23, r = 0.99, SE = 0.11 mmHg) to a standing water column over the range of 0-10 mmHg (6). The frequency response of the servo-null pressure system was determined by pop test, and the actual pressure decay of the system can be approximated as a second-order system (33). Intraventricular pressure was calculated as the difference between measured pressure and the pressure recorded when the tip was placed in extraembryonic fluid adjacent to the ventricle.

Preload alteration. Acute volume alterations were produced using a 10-µl graduated syringe (Hamilton, Reno, NV) connected by plastic tubing and a three-way stopcock to a reservoir of warmed, oxygenated Krebs-Henseleit buffer (KHB) and a polished, 5-µm-tip diameter glass pipette. We inserted the glass pipette into the sinus venosus to alter ventricular preload. After the recording of baseline pressure and video data, an event marker was triggered, and ventricular preload was increased by a single 3-µl injection of buffer. Ventricular preload was acutely decreased by severing a fourth-order vitelline vein, which results in a gradual reduction in ventricular preload over 180 s or by the acute withdrawal of 3 µl of venous blood (19, 32).

CT occlusion. Acute occlusion of the embryonic ventricular outflow tract, CT, was produced using a single strand of 10-0 nylon placed around the CT using microforceps and then loosely tied in an overhand knot (7). The loose CT suture was placed before instrumentation for ventricular pressure measurement. After the recording of 15 s of baseline ventricular pressure and dimension data with or without acute alterations in ventricular preload, the CT knot was then tied tightly to occlude the outflow tract, preventing ventricular ejection.

Experimental protocols were performed as follows: group I, pressure data recorded at baseline and then after isolated acute CT occlusion (n = 15); and group II, pressure and image data recorded at baseline and then after increased ventricular preload (KHB infusion), reduced preload (venous hemorrhage or blood withdrawal), and acute CT occlusion (n = 20). After data was recorded for each embryo, ventricular tetany was induced by the topical application of 50 µl of 2 M NaCl to calculate ventricular wall volume (19).

Data acquisition and signal processing. In the first experimental protocol, we continuously recorded analog pressure waveforms at a sampling rate of 500 Hz with an analog-to-digital board (AT-MIO16; National Instruments, Austin, TX). We used custom analysis software (Labview; National Instruments) to simultaneously view the recorded pressure waveforms in sequential 5-s windows. Individual waveforms were reviewed for signal stability, signal noise, and waveform reproducibility. Three consecutive cardiac cycles were then chosen for each window. These data files were used to calculate time derivative of pressure (dP/dt) and . In the second experimental protocol, a separate analog device sampled the waveform at 15.75 kHz and superimposed the analog pressure waveform onto the video image in real time (model PONV; Ogden Scientific, Spencerport, NY; see Ref. 19). The device also placed zero, full-scale, and pressure baseline markers onto video fields for pressure scale calibration. Composite video fields were recorded on VHS tape. These data files were used for all calculations of PV loop data.

Video image processing. Individual video fields were analyzed at workstations that included a minicomputer, frame-grabbing board, and image analysis software (19). Intra- and interobserver error of area measurement by planimetry is not significant (P > 0.29 and P > 0.96, respectively; see Ref. 19). The video measurement protocol for each embryo included 1) calibration of measurement software for length (mm) and area (mm²) from the recorded standard; 2) measurement of the absolute horizontal position of zero, full-scale, and instantaneous pressure markers on vertical lines 50 or 51; 3) conversion of positional values into millimeters mercury; 4) tracing of sequential video fields for epicardial ventricular cross-sectional area (mm²); 5) planimetry of epicardial ventricular area of the maximally contracted ventricle; 6) ventricular volume derived from area using a simplified ellipsoid geometric model V = 0.65 · A^3/2 where V is volume and A is area; and 7) cavity volume calculated as total volume minus wall volume calculated from the tetanized ventricle using the same model equation (19). The ellipsoid equation was derived from equations for the cross-sectional area of an ellipsoid (A = ab), where a is the semimajor axis and b is the semiminor axis, and the volume of an ellipsoid of revolution [V = (4ab²)/3] and assumed a fixed aspect ratio (a/b = 4/3). End diastole was defined as the onset of ventricular contraction, and end systole was defined at the time of maximum pressure-to-volume ratio.

Basic hemodynamic parameters. Individual pressure waveforms were analyzed for cycle length, maximal +dP/dt (+dP/dt_max), and maximal dP/dt (dP/dt_max). dP/dt was calculated using numerical differentiation. We calculated , the rate of ventricular pressure decline as suggested by Braunstein et al. (4, 31), to include a pressure asymptote as follows: P(t) = a · e^(t/) + b where P is pressure, and t is time.

Ventricular pressure at the onset of relaxation, P₀, was defined at dP/dt_max for each cardiac cycle. Ventricular pressure data were then partitioned starting at P₀ for the subsequent 20, 30, and 40 ms and to P_min (Fig. 1). We calculated  by fitting raw pressure data using nonlinear least-squares regression using Sigmaplot (Jandel Scientific, Corte Madera, CA). Curve-fitting initial conditions were a = 2.5,  = 10, and b = 0. 

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Representative ventricular pressure and differentiated pressure (dP/dt) waveforms of a stage 21 chick embryo plotted on a time scale (top) in seconds (s). The expanded pressure decay and duration scale (bottom) displays ventricular pressure from the onset of diastole at P0 (pressure at the onset of relaxation), at 20, 30, 40 ms, and at Pmin (minimum pressure).

We calculated end-diastolic and end-systolic pressures and volumes and SV from each PV loop. We calculated the EDV versus SV relationship from PV loops generated during alterations in ventricular preload and from PV loops generated during acute CT occlusion. We attempted to analyze end-systolic PV data using linear and second-order regression equations but were unable to adequately fit the data due to the limited range of end-systolic volumes (15).

Statistical analysis. All data were summarized as means ± SE. We used one-way repeated-measures analysis of variance to determine statistical significance for repeat measures within groups, e.g., EDV at baseline and after buffer infusion, hemorrhage, and CT clamp. Linear regression analysis was used to determine the relationship between EDV and SV during ventricular ejection versus during acute CT occlusion. Statistical significance was defined by a probability value of P < 0.05.

	RESULTS

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Baseline embryonic heart rate was 133.9 ± 2.3 beats/min, and heart rate was unchanged by alterations in ventricular preload (P = 0.88) or by acute CT occlusion (P = 0.88). Representative ventricular pressure and volume tracings at baseline and after buffer infusion, venous hemorrhage, and acute CT occlusion are shown in Fig. 2, and corresponding representative PV loops are shown in Fig. 3. During volume infusion, EDV increased dramatically with little increase in developed pressure.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Representative simultaneous ventricular pressure and volume waveforms at baseline, during buffer infusion and venous hemorrhage, and after conotruncal (CT) clamp in a stage 21 chick embryo. Pressure and volume waveforms are plotted on the same time scale.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Representative pressure-volume loops at baseline, during buffer infusion and venous hemorrhage, and after CT clamp in a stage 21 chick embryo.

As expected, EDV increased from 0.27 ± 0.02 to 0.39 ± 0.05 mm³ after volume infusion (P < 0.05), decreased to 0.19 ± 0.02 mm³ after preload reduction (P < 0.05), and then increased to 0.35 ± 0.03 mm³ after acute CT occlusion (P < 0.05, Fig. 4). EDV after CT occlusion was greater than baseline (P < 0.05) but was similar to volume infusion (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   End-diastolic volume and end-systolic pressure at baseline, during buffer infusion and venous hemorrhage, and after CT clamp. Values are means + SE. * P < 0.05 vs. baseline. n, No. of experiments.

End-systolic pressure calculated from PV loops was 2.32 ± 0.07 mmHg at baseline, 2.47 ± 0.12 mmHg after volume infusion (P > 0.05), and 2.12 ± 0.08 mmHg (P > 0.05) after venous hemorrhage. However, after acute CT occlusion, end-systolic pressure increased (3.60 ± 0.08 mmHg, P < 0.05, Fig. 4). Consistent with the increase in end-systolic pressure, +dP/dt_max increased from 44.2 ± 2.2 to 72.4 ± 6.8 mmHg/s after CT clamp (P < 0.001). As previously noted, SV varied linearly with EDV both during alterations in ventricular preload and after acute CT occlusion (Fig. 5).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Relationship between ventricular stroke volume and end-diastolic volume for stage 21 chick embryos during alterations in preload and after acute CT clamp. Linear regression curves fit to raw data.

After acute CT occlusion, there was no change in dP/dt_max from baseline (47.1 ± 2.8 vs. 45.3 ± 1.4 mmHg/s, P = 0.37). The  calculated from each cardiac cycle was significantly influenced by the duration of pressure decline analyzed (Table 1). After acute CT occlusion,  was prolonged as calculated for each duration of pressure decay (Table 1).

	DISCUSSION

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Ventricular contractile reserve. At each developmental stage, ventricular pump performance and ventricular-vascular coupling in the embryo are similar to fetal (9), neonatal (26, 29), and adult hearts (15, 24). Scanning electron micrographs of developing embryonic myocytes reveal myofibrils in varying states of maturation with a significantly smaller volume fraction of aligned sarcomeres than the mature cardiomyocyte (7). Despite myocyte immaturity and geometric simplicity, the embryonic heart alters SV linearly in response to changes in EDV, resulting in a moderate range of functional adaption to altered circulating blood volume (19, 32). However, the current study was performed to evaluate the embryonic contractile reserve that may be masked by dynamic changes in arterial load during altered SV. As expected, acute CT clamping significantly increased systolic pressure and +dP/dt_max consistent with increased afterload (21).

After acute outflow tract occlusion, embryonic myocardial contractile reserve is expressed as increased developed pressure and increased +dP/dt_max. The mechanism(s) responsible for this increased developed force likely relates to the obvious increase in afterload produced by acute CT clamping. Although direct calculations of end-systolic wall stress are of limited accuracy for the trabecular embryonic myocardium, acutely increased systolic pressure and end-systolic diameter likely produce increased end-systolic wall stress in the embryo (9, 20). In addition, the increased proximal resistance to ejection produced by CT clamping likely prolongs the time each myocyte generates force in an isometric mode before ejection and muscle shortening. This delay in "shortening deactivation" results in an increase in developed force toward a maximal "isometric" contraction (16). Thus we speculate that an acute increase in wall stress and force generation after CT clamping may be associated with an increase in intracellular calcium concentration, and further studies are needed to measure intracellular calcium concentration during acute alterations in ventricular preload and afterload and to correlate embryonic contractile reserve with myocyte excitation-contraction coupling.

Ventricular relaxation. In contrast to the increase in contractile function after acute CT clamping, diastolic function was affected adversely by acutely increased afterload. Our values for  before CT clamp were similar to previously published values for the chick embryo (4). Acute CT occlusion prolonged  in the embryonic heart, as has been noted for the mature left ventricle (13). Numerous studies have detailed the functional immaturity of the sarcoplasmic reticulum in developing embryonic and fetal rabbit, rat, mouse, and chick cardiomyocytes, highlighting the dependence of the embryonic myocyte on transarcolemmal calcium via Na⁺-Ca²⁺ exchange and L-type calcium channels (1, 2, 10, 14, 30). After acute CT clamp, the prolonged time course of pressure decay is consistent with prolonged calcium reuptake, likely due to the combination of increased intracellular calcium contraction after CT clamping and rate-limited reuptake. Of interest, chronic CT banding increases systolic pressure versus control embryos without prolonging  (13). When embryonic myocardial load is chronically altered, the embryonic myocardium compensates by altering the rate of myocyte division, e.g., resulting in myocyte hyperplasia after chronic CT banding (7) or hypoplasia after chronic calcium channel blockade (8) or left atrial ligation (28). Further studies of in vivo and in vitro embryonic muscle function are required to determine how altered myocardial growth rate influences the rate of maturation of the embryonic contractile apparatus.

Cycle length response. It is also worth briefly mentioning that acutely increased afterload was not associated with a compensatory change in cycle length. Previous studies of acute alterations in loading conditions in the embryo have shown a lack of chronotropic response to compensate for altered ventricular performance (3, 19, 31). However, the embryonic heart rate is linearly related to environmental temperature, likely due to direct effects on intracellular metabolic rates (5). The probable explanation for the lack of chronotropic responsiveness to altered ventricular load in the early embryo is the absence of a functional autonomic nervous system during primary cardiovascular development (22).

End-systolic PV relations. The basic model of a time-varying elastance assumes that arterial load is constant (24, 27). In contrast to the mature circulation, the embryonic CV system rapidly alters systemic vascular resistance in response to altered blood flow (31). Consequently, ventricular PV loops generated during alterations in ventricular preload display smaller changes in developed pressure than in SV, resulting in an extremely curvilinear end-systolic PV relationship (19). This rapid vasoactive response to altered blood flow in the embryo confounded our initial attempts to calculate a "maximum ventricular elastance." End-systolic PV points from PV loops generated for the ejecting and outflow tract occluded ventricle during alterations in preload show that acute "uncoupling" of the ventricle from the vascular system reduced the curvilinearity of the end-systolic PV relationship.

Adaptive mechanisms. With recognition of the importance of the acute functional response to increased ventricular afterload, the broader question is the mechanism by which the developing CV system (heart and vasculature) adapts to chronic alterations in hemodynamic loading conditions. After chronic CT banding, the embryonic chick heart accelerates myocyte division, resulting in a larger heart with an increased number of normal-sized myocytes (7). Ventricular pressure is increased after CT banding, but, interestingly, the rate of ventricular pressure decay is unchanged after myocardial adaptation to chronically increased afterload (13). Improved relaxation in the setting of accelerated growth is likely crucial for the embryo, because ventricular filling is very sensitive to changes in ventricular diastolic function in this relatively low-pressured system (18). Insights into the unique adaptive mechanisms present in the developing cardiovascular system may aid our understanding of cardiovascular adaption in the neonatal and mature heart.