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Artificial cardiac stimulation has been performed for almost 40 y; however, only a few studies dealt with changes in myocardial structure induced by chronic pacing. Prinzen et al.(1,2) showed that ventricular pacing in dogs induced thinning of the ventricular myocardium in areas close to the pacing site and thickening in remote areas. Because similar thinning of the early activated myocardium was also observed in patients with left bundle branch block, it was hypothesized that such thinning was caused by asynchronous electrical activation of the ventricular myocardium(3). The early activated fibers shorten markedly during the isovolumic phase, causing a prestretch in serially connected, as yet unactivated fibers. When finally activated, these late-activated fibers shorten vigorously during the ejection phase, counteracting further contraction of the early activated fibers. This results in regional differences in workload, a prerequisite for remodeling.

Much less is known about the effects of pacing in the immature heart. Apart from studies of Karpawich et al.(4,5) in puppies, which have shown perturbation of myocardial architecture associated with apical ventricular pacing but not with septal stimulation, no artificial electrical stimulation preparation has been reported to provoke changes in cardiac structure. To our knowledge, no study has dealt with pacing-induced remodeling in the prenatal heart. A model enabling analysis of responses of the embryonic or fetal heart to pacing might be relevant, as pacing is feasible in the fetus(6), and may be indicated for treatment of some refractory arrhythmias even in humans.

The chick embryo has often been used in studies of cardiovascular physiology(7), and the development of the myocardial architecture of its heart is well described(8,9). Dunnigan et al.(10) showed that electrical pacing is feasible in stage 24 chick embryo. However, the experiments were performed only for a short time (<1 h) to study its hemodynamic effects. To induce structural remodeling of cardiovascular system longer periods are needed. We demonstrated in a previous report that the continuous apical ventricular pacing induces ventricular dilatation in the chick embryonic heart(11). Recently, Kappenberger et al.(12) have shown that intermittent ventricular pacing can be supported by the chick embryonic heart for a period of 48 h, which is sufficient to produce a decrease in apical ventricular wall thickness comparable to that seen in the adult canine heart(2).

The adult heart reacts to increased pressure load by hypertrophy or dilatation; on the other hand, the immature heart adapts to the same stimulus by hyperplasia rather than hypertrophy(13,14). The switching between hyperplastic and hypertrophic response seems to happen within the early postnatal period, and correlates with the loss of ability of cardiomyocytes to enter the cell cycle(15). It is therefore likely that any remodeling during the embryonic period will be based on regulation of the cell number. The proliferation patterns in the embryo are controlled by genetic and epigenetic mechanisms. PDGF might be one of the candidates, inasmuch as it is strongly expressed in the embryonic myocardium(16) and was found to be strongly up-regulated by increased pressure loading in the chick embryonic heart(17). FGF-2 is also strongly expressed from the earliest stages of cardiogenesis(18), and inhibition of its expression by antisense oligonucleotides resulted in decreased cell proliferation(19).

All these facts led us to formulate the hypothesis that the myocardial remodeling of the chick embryonic heart caused by asynchronous activation should be based on hypoplasia. Therefore, we studied the patterns of cell proliferation and cell death in the myocardium, together with expression of growth factors likely to participate in control of proliferation rates. To exclude involvement of cellular hypertrophy, we also measured cell size in the normal and remodeled ventricular myocardium. We demonstrated that the localized remodeling of the ventricular wall near the pacing site was preceded by local decrease of cell proliferation, accompanied by down-regulation of FGF-2.

METHODS

Embryo preparation. Fertilized eggs from Warren hens were incubated at 37.5°C and 94% humidity for 3.5 d up to Hamburger-Hamilton stage 21(20) with two rotations per day. Access to the heart was gained by windowing of the shell, removing the inner and outer shell membranes, and opening microsurgically the amniotic cavity and developing thoracic wall. The incubation of the windowed eggs was continued in an acrylic glass incubator with integrated dissecting microscope, stimulator, and charge-coupled device camera at the same temperature and 80% humidity. Platinum wires (diameter 0.2 mm) isolated with a plastic sheath were used as electrodes. With use of a micromanipulator (Narishige, Tokyo, Japan), the anode was placed on the vitelline membrane, and the cathode, used for unipolar stimulation, was positioned at the ventricular apex (Figs. 1 and 2).

Figure 1
figure 1

Scheme of the measurements performed on the chick embryonic heart at d 5.5. Left, Contours of the heart cavities, site of stimulation, and levels of horizontal sections as shown on the right. Right, Transversal sections of the ventricles (not to scale) showing where the thickness of the compact layer was measured (circles) in SEM and histologic sections. RA, right atrium; LA, left atrium; CT, conotruncus; RV, right ventricle; LV, left ventricle.

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Figure 2
figure 2

Effect of stimulation on the heart beat. A, Heart of stage 22 chick embryo in late diastole. The white line indicates the pixels used for construction of the spatiotemporal maps. Plus and minus signs indicate the platinum stimulation electrodes. RA, right atrium; V, ventricle; Ct, conotruncus. B-D, Spatiotemporal maps (20 s) from control period (B), and periods of pacing at 150/min (C) and 200/min (D). Each light band corresponds to one ventricular contraction, and its slope indicates the propagation velocity of the contraction. The spontaneous rate was 164/min, and stimulation at 150/min (shown only as an example) provoked rhythm perturbation (interference of spontaneous and paced rhythm). Pacing at 200/min resulted in complete and very fast ventricular capture. Scale bar 1 mm (A); the spatiotemporal maps are reduced 1:2.

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Pacing protocol. As we have demonstrated previously(12), continuous apical pacing led to ventricular dilatation, heart failure, and death of embryos in approximately 8 h. We have therefore adopted an intermittent pacing protocol. The best compromise between the pacing duration and survival was switching between a 5 min on-5 min off regimen during the day (12 h) and 5 min on-15 min off during the night (12 h), which allowed for 9 and 18 h of effective pacing in 24 and 48 h, respectively, with survival rate 60% at 48 h(12). The loss of embryos was gradual, and survival at 24 h was 80%. To follow the normal developmental rise of heart rate (from about 140 to 190 beats/min during the study period), the pacing frequency was maintained at 110% of the intrinsic rate, i.e. between 155 and 210 cycles/min, according to case. The stimulus of 2-ms duration and amplitude 2 times the diastolic threshold (2-5 mA) was delivered by the Medtronic 5320 atrial pulse generator. Ventricular capture was assessed visually, and verified by video image analysis. Stimulation threshold did not change significantly during the pacing period(11,12).

Video image analysis. Images were recorded on a standard VHS video tape during normal and paced rhythm. End-diastolic and end-systolic endocardial surface areas (A) and ventricular length (L) were traced on representative fields from three cardiac cycles, and measured by planimetry(21,22). End-diastolic and end-systolic volumes (V), together with ejected fraction, were calculated from the above measurements considering the ventricle as a prolate ellipsoid with major axis L and equal minor axis using the area-length formula V = 8A2/3πL To study the ventricular entrainment, wall motion was assessed using edge movement detection technique on stacks of 500 images (20 s of recording, 25 images/s) in National Institutes of Health Image software run on a Macintosh Power PC 8500/180X. In the first image of the stack, a reference line was traced along the ventricular and conotruncal axes (Fig. 2A). The gray level values from the pixels corresponding to the line were extracted and arranged as a linear array.

Such an operation was repeated for each image, and the value array from the subsequent image was added, pixel-by-pixel to that from the previous image. In this manner, a pseudo three-dimensional display was obtained with the distance along the heart on the abscissa, the time on the ordinate, and the gray level value at each coordinate. In such spatiotemporal maps (Fig. 2, B-D), contraction and relaxation waves are respectively represented as light and dark bands. These patterns greatly facilitate the analysis of rhythmicity and propagation of heart contractions.

Morphologic analysis. The numbers of embryos submitted to different protocols are summarized in Table 1. Six control and six intermittently paced hearts at the 48-h interval were submitted for SEM examination. The processing was performed essentially as described in detail previously(9). The hearts were fixed in diastole by perfusion with 2% glutaraldehyde-1% formaldehyde in isotonic (280 mOsm/L) 0.1 M cacodylate buffer and postfixed in 1% osmium tetroxide(23). The dissection was performed in frontal and transverse planes. The observations were made on a JEOL JSM 6300 F (Tokyo, Japan) scanning electron microscope.

Table 1 Numbers of embryos submitted for different protocols
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Three hearts per group were prepared for TEM at both intervals. The fixation protocol was the same as for SEM. Dehydration was performed with ethanol with 5% uranyl acetate, and the hearts were embedded into Epon in a manner allowing slicing in the transverse plane, starting from the apex. One-micrometer sections were observed with an Olympus BH-2 light microscope. Ultrathin sections from selected areas were stained with standard uranyl acetate-lead citrate method, and observed with a Philips CM 12 transmission electron microscope (Eindhover, Netherlands).

Using a digital caliper (Mitutoyo, Tokyo, Japan), the thickness of the compact layer was measured on SEM photographs of transversely dissected hearts (three per group) in apical slices, formed by the left ventricle, and at the midportion slices, containing both right and left ventricles. The measurements were done at standard points as shown in Figure 1. Comparable, carefully matched slices and the same measuring points were used for measurements of thickness (ocular micrometer, precision ± 1 µm) in histologic cryostat sections (additional three hearts per group at 48-h interval, see below). Atrial thickness was measured on transverse histologic sections across the atrioventricular junction, which showed both atria separated by the interatrial septum. Cell width was measured on high-power (40× objective) photographs of semithin sections. Only cardiomyocytes in the compact layer in which the nucleus was encountered were considered for this measurement to avoid profiles of incompletely cut cells.

Evaluation of proliferative activity. At the 24-h sampling interval, five intermittently paced and five control embryos were injected with 30 µg of 5-bromodeoxyuridine (Sigma, Buchs, Switzerland) in 10 µL of distilled water. After 30 min of reincubation (during which pacing was continued), the embryos were rapidly removed from eggs, and the hearts were perfused with 4% neutralized formaldehyde in PBS and immersed in the same fixative for 24 h at room temperature. After washing with PBS, thoracic blocks containing hearts were processed for routine serial paraffin sectioning at 5 µm in the transverse plane. The incorporation of 5-bromodeoxyuridine into DNA of cells was detected using in situ cell proliferation kit AP (Boehringer). Fluorescent Hoechst 33342 dye (Sigma Chemical Co) was used as a nuclear counterstain. The cells were counted on sister photographs taken under bright-field illumination (labeled nuclei stained with Fast Red) and fluorescent image of the same field, in which all nuclei were visible. The percentage of proliferating cardiomyocytes was then calculated, taking care not to include epicardium, endocardium, or erythrocytes.

Immunohistochemistry and detection of apoptosis. Three intermittently paced and three control hearts at the 48-h interval were used for the detection of PDGF immunoreactivity. The hearts were perfusion-fixed with ice-cold 4% paraformaldehyde and postfixed by immersion in the same solution. Fixed embryos were incubated with sucrose-RNase 30%-protease inhibitor cocktail for 4 h, rinsed with PBS, embedded in Jung medium, and frozen. Serial transversal 10-µm cryosections were cut from the apex to the base, fixed with 4% paraformaldehyde, rinsed again with PBS, dried, and stored at -20°C. Thereafter, the sections were rehydrated with PBS, blocked with normal horse serum, and incubated overnight with goat monoclonal anti-human PDGF BB (Becton Dickinson, San Jose, CA) at 4°C. The biotinylated horse anti-mouse IgG was used as secondary antibody. Final detection was done by avidin-alkaline phosphatase complex using nitroblue tetrazolium and 4-bromo-5-chloro-3-indolylphosphate as a substrate.

FGF-2 (previously known as bFGF) expression was visualized on formaldehyde-fixed serial paraffin sections prepared for detection of cell proliferation at the 24-h sampling interval (see above) and in three additional hearts per group at the 48-h interval using rabbit anti-bFGF no. 147 (Santa Cruz Biotechnology) as a primary antibody detected by peroxidase-anti-peroxidase universal anti-rabbit detection kit (Dako) with diaminobenzidine as a color substrate.

Apoptosis was detected on the same series as proliferation and FGF-2 using an in situ cell death detection kit PO (Boehringer, Mannheim, Germany), on the basis of TUNEL staining protocol with diaminobenzidine as a color substrate.

Western blotting. Three hearts per group and sampling interval were quickly dissected out from the embryos and frozen rapidly in liquid nitrogen. Pooled paced or normal hearts were thawed by adding approximately an equal volume (90 µL) of 2× Laemmli buffer (4% SDS, 120 mM Tris, pH 6.8, 20% glycerol, 0.01% bromphenol blue, 2% DTT), sonicated, and heat denatured at 100°C for 5 min. Addition of Laemmli buffer directly to thawing hearts prevented protein degradation, but made estimation of total protein content impossible. After centrifugation at 10 000 × g, 15 µL of solubilized proteins per lane were separated on an 8-16% SDS-PAGE gel. PDGF was detected using anti-PDGF BB (Becton Dickinson, San Jose, CA), and FGF-2 by rabbit anti-bFGF no. 147 (Santa Cruz Biotechnology). Reactive proteins were detected by chemiluminescence using protein A linked to alkaline phosphatase (Sigma, Buchs, Switzerland). The relative amounts of detected proteins were quantified by densitometry of the films. The signal was normalized according to the total amount of proteins visualized by the Poinceau reaction.

Statistical analysis. All data are shown as means ± SD. The differences in quantifiable parameters between paced and control hearts were evaluated using Mann-Whitney nonparametric test, and for comparison of regional differences we used bilateral paired t test. Values of p < 0.05 were considered significant.

RESULTS

General response to pacing. Figure 2 shows an image of the heart after 6 h of experiment with the stimulation electrode positioned at its apex. Ventricular pacing at a supranormal rate resulted in a rate-dependent decrease of telediastolic, telesystolic, and stroke volume with no significant modification of the ejection fraction. However, the cardiac output calculated from heart rate and the approximated stroke volume showed about 5% decrease. Video image analysis demonstrated complete ventricular capture during pacing at supranormal rate. The paced beats started at smaller telediastolic volume and had a faster contraction rate than the normal beats. The contraction pattern was concentric in both cases, but the ventricular shortening amplitude was relatively increased at the apex level in the case of ectopic stimulation. In agreement with the previous results(11,12), the survival at 48 h was 60%. The embryos died out throughout the duration of the experiment; the survival at 24 h was 80%. The nonsurviving embryos had a progressive deterioration of cardiac function and heart dilatation before death. Ultimately, the cardiac output stopped but the heart still reacted to electrical stimulation. These hearts were not considered for the analysis presented here.

Morphologic analysis. The hearts of survivors of pacing at 48 h did not show any gross alterations of external morphology but had a mild dilatation of all compartments. Their internal trabecular structure was normal. The most significant alteration produced by pacing was a decrease in thickness of the compact layer of the myocardial wall at the apical region (Fig. 3). This was observed in both frontal and transverse sections in SEM as well as on histologic series. On average, the compact layer was reduced by 28% (Fig. 4A). This reduction was most pronounced in the anterolateral area, i.e. close to the position of the stimulation electrode (Figs. 1 and 2). The compact layer of the ventricular wall consisted on average of two to four cell layers versus five to 10 in the controls, but the cells were of the same size in both groups (Fig. 4B). In areas distant from the stimulation site, such as in the left ventricular midportion, the wall thickness as well as the number of cell layers and cell size did not differ between control and paced hearts (Fig. 4A; also data not shown). Right ventricular compact myocardium was thinner than in the left ventricle (p < 0.05), but no difference between control and paced hearts was found (paced 11.1 ± 2.6, control 12.2 ± 1.0 µm, p > 0.05).

Figure 3
figure 3

Pacing induces ventricular myocardial thinning. SEM pictures of the apical compact layer from control (A) and paced (B, severe case) transversely dissected hearts at the 48-h interval. Diminution of the compact myocardium (Co) thickness, indicated by arrowheads, is apparent. Tr, trabecula. Scale bar 100 µm.

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Figure 4
figure 4

Localized compact myocardial thinning is not related to decreased cell size. A, Left ventricular compact layer thickness in different locations. The compact layer of the left ventricle is significantly thinner in the apical area submitted to pacing whereas at the midportion level it is similar to control. B, As mean cell width in the apical compact layer shows, this decrease is related to decreased cell number (hypoplasia), because the cell size is similar between paced and control hearts (n = 53 and 93 cells, respectively). The cell size was similar in the midportion, with no difference between paced and control hearts (data not shown).

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Examination of cryostat histologic series confirmed the above data and afforded further information on increased left atrial thickness (50%, p < 0.05). This likely resulted from the left atrium occasionally working against already contracted ventricle during the pacing periods. The number of cell layers was systematically increased (on average, four versus two in controls, p < 0.05). A thickening (19%) was noted in the right atrium, but it was below the limits of statistical significance.

TEM examination revealed no abnormalities of nuclear morphology, mitochondria, contractile apparatus, and intercellular junctions at the pacing site or elsewhere in the ventricular myocardium in either sampling interval (data not shown). No signs of myocyte degeneration, apoptotic or necrotic, were detected more often than in one per 400 cells. Similarly, TUNEL staining failed to show any significant increase in percentage of apoptotic cells, which was <0.25% in both paced and control hearts at both sampling intervals.

Proliferation pattern of the embryonic heart. Proliferating cells were concentrated in the ventricular compact layer, while in the trabeculae, the values were significantly lower (on average by 47%, Fig. 5). There were no statistically significant regional differences in the left ventricle, but the interindividual variability was quite high. Proliferative activity was similar in the right ventricle (compact layer 16.6 ± 3.4, trabeculae 3.9 ± 2.5%), and lower in the atria (7.4 ± 4.8%) and conotruncus (10.0 ± 3.2%).

Figure 5
figure 5

Effect of pacing on cell proliferation. There is a significantly lower proliferative activity in the trabeculae in all locations. The regional differences (apex vs midportion) are not statistically significant. Proliferative activity is decreased compared with controls in the apical compact layer of the paced hearts.

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The difference between paced and control hearts was detected only in the apical compact layer, i.e. near the pacing electrode, where the proliferation was significantly decreased by 32% (Fig. 5).

Growth factor expression. Western blots using anti-PDGF antibodies failed to reveal any overall difference between paced and control hearts (data not shown). Immunohistochemistry also showed a homogeneous pattern of expression of PDGF within the myocardium, with no gradient between the compact layer and trabeculae or between different compartments, further confirming that PDGF localization was not influenced by pacing.

FGF-2 was detected in whole paced and control hearts by Western blotting (Fig. 6). The antibody used reacted with two bands of about 45 and 55 kD, which could correspond to its precursors or alternative splicing products(24). The detected products were specific, because preincubation of the antibody with pure FGF-2 peptide prevented detection of the bands. Pacing induced a dramatic reduction in the amount of the larger precursor, whereas the 45-kD band was slightly increased. Intensity scanning showed an overall reduction of FGF-2 by 50% already at the 24-h interval, and this difference persisted at 48 h.

Figure 6
figure 6

FGF-2 expression in the chick embryonic heart. Western blot from pool of three control (Left) and paced (Right) hearts showing down-regulation of FGF-2 protein. Both lanes were loaded with equal amounts of protein.

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A closer examination by immunohistochemistry showed that FGF-2 was strongly expressed in the myocardium, confirming the results of Parlow et al.(18). There was no significant gradient between different compartments (atrium, ventricle, conotruncus) apart from weaker expression in the atrioventricular canal by the 24-h interval. Additional expression was observed in the macrophages of the conotruncal cushions. By the 48-h interval, the expression increased in the trabeculae, becoming slightly stronger than in the compact myocardium. The intensity of staining continued to be weaker in the atrioventricular canal. Consistent with the Western blotting result, staining was generally weaker in the paced hearts both after 24 and 48 h, with maximal reduction in the anterolateral apical region, i.e. close to the stimulation electrode (data not shown).

DISCUSSION

Modifications of heart beat. The decrease of stroke volume associated with pacing at increased rate was reported previously by Dunnigan et al.(10) in stage 24 chick embryo, and it correlated with the rate-dependent decrease in passive ventricular filling. Thus, despite increased frequency, cardiac output was decreased as the decrease in stroke volume predominated. Further diminution of filling resulted from the obvious loss of the active atrial component in the case of apical ventricular pacing, which starts to gain in importance from stage 18 onward(25). Taken together, these data suggest that the paced ventricle worked under decreased preload conditions in addition to ectopic activation.

Ectopic heart beat has an altered propagation of excitation wave that results in abnormal distribution of epicardial strain(3). It is quite difficult to measure precisely the activation sequence in the chick embryonic ventricle(26) because of its small size (1.5-3 mm at the stages examined), and further difficulties would be experienced in vivo because of mechanical activity interfering with precise positioning of the electrodes. The activation of the embryonic ventricle during the stages studied is in the majority of cases basoapical or concurrent(26), and the excitation is probably preferentially spread by the radially arranged trabeculae, which show higher conduction velocity(27) and likely contract before the compact layer(28). The contraction sequence of the ventricle determined from the video images from the right projection was, however, concentric and simultaneous in both normal and ectopic beats within the limited temporal resolution (25 images/s) used; nevertheless, the shortening was less homogeneous than during the normal beat with relatively increased amplitude at the apex, confirming the supposed asynchrony.

Cellular changes. Adomian and Beazel(29) observed disarray of myofibrils with enlarged and variable-sized mitochondria predominantly in the areas close to the pacing site in dogs chronically paced for 3 mo. Dedifferentiation and hypertrophy but no cell death were reported from the atrial cardiomyocytes of goats subjected to sustained atrial fibrillation(30). Stimulation performed in the developing canine heart(4,5) provoked myofibrillar disarray only when the activation arose from the ectopic (apical) site but not from the electrode implanted in the interventricular septum in the area of the bundle of His. These results show that although pacing does not kill the adult cardiomyocytes, it causes substantial changes toward the immature phenotype or disorganization of the myocardial architecture in the ectopically paced area. Absence of similar effects in the embryonic myocardium can be explained by its already low level of differentiation, e.g. myofibrils showing low level of preferential alignment(31). The levels of cell death are very low in the developing myocardium, as was noted already by Manasek(32) and later quantified by Pexieder(33). The resistance of the immature cardiomyocytes to cell death might be related to high levels of expression of bcl-2 proto-oncogene, which was shown to protect cells against apoptosis(15). Our findings of absence of increased levels of apoptosis by TUNEL method in the hypoplastic paced area correspond well to these data.

Cell proliferation. Our findings of preferential accumulation of proliferating cells in the ventricular compact layer correlates well with a study in the chick by Jeter and Cameron(34), who found higher proliferative activity at the heart periphery with maximal values around d 4. Similar observations of lower activity in the trabeculae were made by Tokuyasu(35), but no absolute values were provided. Lower proliferative activity of the trabeculae could be explained by their early activation(26,28). Within the period of 3 to 6 d, chick embryonic heart shows a rapid hyperplastic growth to meet rapidly increasing requirements of the developing embryo. Earlier studies performed in the chick embryo(13,36) have demonstrated that the embryonic heart adapts to decreased or increased load by hypoplasia or hyperplasia. This is also the case for the mammalian fetus(14). Our data showing that the decreased thickness of the apical compact layer of the paced hearts is accomplished by a localized drop in proliferation with no changes in cell size correspond well with these observations. The absence of thickening of the late activated ventricular wall at the midportion, which could be expected by analogy with the dog model(1) and as a result of slightly increased proliferation in this area, can be explained by overall dilatation resulting from the failing heart.

Growth factors. We tested whether PDGF could be involved in modification of ventricular wall thickness, because its upregulation was associated with accelerated ventricular growth induced in chick embryos by conotruncal banding(17). This is not the case inasmuch as PDGF gene expression and protein distribution were similar in paced and control hearts, and the patterns were essentially the same as described earlier(16). The expression of the protein correlated with its mRNA detected by RT-PCR (unpublished data) except for additional expression in the endothelial cells of the atrioventricular cushions. Thus, even though severe heart dysmorphogenesis was observed in mice with the PDGF pathway blocked(37,38), this growth factor does not seem to be involved in subtle regulations of myocardial architecture development.

FGF-2 was supposed to be involved in control of cardiomyocyte proliferation, because it is expressed in the chick embryonic myocardium from the earliest stages(18), and its blockade reduces cell division in vitro substantially(19). The overall reduction of its expression detected by Western blot analysis could be attributed to decreased preload owing to smaller ventricular filling. The finding of maximal changes by immunohistochemistry close to the pacing site where the morphologic changes occurred correlated with localized proliferation drop. These data are in agreement with our initial hypothesis, that the remodeling is accomplished by cell hypoplasia in response to decreased loading. However, the role of FGF-2 must yet be precisely determined, because of the detection of its precursors only by Western blot, and the semiquantitative nature of the techniques used.

Study limitations. Chronic apical ventricular pacing in the chick embryos leads to fairly rapid development of heart failure and death(11,12). This forced us to use an intermittent pacing protocol. It is not possible to extend the stimulation with the present setup beyond stage 29 (incubation d 6) because the rotation of the embryo deep into the yolk results in loss of heart accessibility. On the other hand, using an in vitro setup(39), it is possible to stimulate as early as the heart tube forms, i.e. from stage 11 until approximately stage 17. Differences between relatively autonomous chick embryo and mother-dependent mammalian fetus must be kept in mind when trying to extrapolate these data. For example, the relative importance of growth factors seems to be different in mammals; although FGF-2 is also expressed in the developing heart in appreciable amounts(40), its inactivation has no apparent effects on cardiac organogenesis(41). Nevertheless, basic mechanisms of the embryonic heart function are likely to remain the same(21,22). The chick embryo offers a possibility of manipulations on its circulatory system not (yet) possible in mammals at comparable stages, e.g. enabling the testing of the consequences of pacing in the conditions of increased afterload induced by conotruncal banding(13) or in a combination with pharmacologic interventions.

In summary, we have shown that the chick embryonic heart responded to intermittent ventricular pacing by remodeling of its myocardial architecture, namely by thinning of the ventricular wall in the vicinity of the pacing electrode. This hypoplastic change was accomplished by locally decreased proliferative activity probably caused by asynchronous activation and accompanied by down-regulation of FGF-2. The fact that structural modifications obtained in this work are similar to those induced by pacing of mammalian hearts suggests that the chick embryonic heart paced in situ represents a useful tool for investigating the effects of pacing on the immature heart.