On account of relative ease of accessibility, the early embryo circulation is an eminently suitable model, which can help unravel the old question of the relative importance of the peripheral circulation, versus that of the heart. Its “simplified” morphological plan, i.e., the absence of valves and lack of innervation, serve as additional advantages over its mature counterpart. Finally, the embryonic and extra-embryonic circulations occur on a single plane, rendering the force of gravity almost negligible, in comparison to a horizontally placed animal, or vertically oriented human circulatory system. We now examine several studies where the fundamental question of heart vs. circulation has been addressed by the investigators.

To assess the influence of blood flow on early (37 hpf, hours post fertilization) and fully formed (4.5 dpf, days post fertilization) zebrafish heart development, Hove et al. [1] inserted a single 50 μm glass bead into the sinus venosus of the transgenic zebra fish embryo hearts, in order to either (a) partially or (b) completely obstruct the inflow of blood into the heart. In a separate experiment (c) a bead was implanted into the ventricle to obstruct the outflow from the heart (Fig. 7.1). A group of sham-operated embryos, in which the bead had been placed and removed within 1 h, was run as a control. The embryos were checked after 20 h of bead implantation. The sham-operated embryos showed normal blood flows and patterns of heart development. In the group of partially obstructed embryos, there was a reduced blood flow through the heart, but no morphological changes. A very different set of events occurred in the case of completely obstructed embryo hearts. At both levels of occlusion, i.e., at the inflow into the heart and at the outflow, severe regurgitation and accumulation of blood corpuscles were noted at the inflow to the heart. In addition, the ventricular outflow obstruction resulted in atrial and ventricular distension. Significantly, in all cases of complete obstruction, a marked yolk sac edema was noted, indicating that the blood continued to move toward the heart, in spite of its alleged propulsive action being blocked. The primary importance of flow was further demonstrated by significant morphologic abnormalities, such as failure to develop the outflow tract, the defect of looping, and finally, by the collapse and fusion of the heart walls. The authors concluded that the lack of shear forces of the flowing blood, rather than the lack of transmural pressure, was responsible for the severe cardiac morphologic defects.

Another important observation made by the authors was that both inflow- and outflow-blocked 4.5 dpf hearts failed to develop valves. Does this suggest that the primary role of the heart is indeed to set itself against the flow of blood, working as an organ of impedance? Furthermore, does the presence of glass beads remove the mechanical stimulus needed for normal formation of the valves supplied by the sheer stress of the flowing blood? This view is further corroborated by the fact that the absence of blood flow through the heart results in complete obstruction of the lumen by the proliferation of cardiac jelly [2]. A similar, possibly related phenomenon was also observed in the above-mentioned obstructive zebrafish heart mutants [3, 4] (Chap. 5).

It should be noted, parenthetically, that during the preliminary flow observations in the 37 hpf embryo heart, the authors found it “intriguing” that despite the lack of valves, “the early embryonic heart can act as both a valve and a pump” with ejection fraction of up to 60%, which is comparable to adult mammalian hearts [1].

The primacy of the peripheral circulation in the overall embryonic hemodynamics is further demonstrated by experiments where selective clipping of major venous conduits causes perturbance in flow. It has been shown that chronic unilateral vitelline vein ligation results in the diversion of vitelline blood flow and alters intracardiac flow patterns, adversely affecting normal cardiac morphogenesis [5, 6]. Stekelenburg-de Vos et al. found an acute decrease in heart rate, stroke volume, peak systolic velocity, and mean blood flow, by clipping the right lateral vitelline vein of HH stage 17 chick embryo. As expected, there was a damming-up of blood in the vitelline vascular bed with concomitant decrease in venous return. During the course of several hours, the venous, normally drained by the right vitelline vein, was redirected to the posterior vitelline vein via a small capillary vessel, which had, in due course, expanded into a conduit large enough to accommodate the excess flow. Within the study period (5 h), the heart rate and mean blood flow had returned to control values [7]. A similar study performed on stage 17 chick embryos showed a decreased rate of passive ventricular filling as a function of decreased venous return. When observed a day later, at HH stage 24, the stroke volumes and dorsal aortic flows were within the range of unclipped controls [8].

Wagman et al. measured the effect of acute increase of the circulating blood volume in stage 18, 24, and 29 chick embryos, by injecting isotonic Ringer’s solution in aliquots ranging from 0.9% to 14.9% of circulating blood volume. (The circulating blood volume ranges around 80 μL in stage 18 embryo.) The acute decrease of intravascular volume was affected by the withdrawal of 10 μL of blood from the circulation at the rate of 0.5 μL/s and re-injected 15 s later. Heart rate, dorsal aortic blood flow, and arterial and ventricular pressures were measured and stroke volume index (SVI, SV per cardiac cycle) and peripheral vascular resistance (PVR) were derived. While there was no increase in heart rate, compared to controls, the dorsal aortic flow, stroke volume, SVI, and arterial blood pressure increased linearly in response to volume of infusion. There was a concomitant decrease in calculated PVR. The opposite changes were observed during blood withdrawal. After a short time, all changes equilibrated toward baseline values, pointing to an efficient hemodynamic control of hemodynamic parameters [9].

Given the fact that the heart undergoes such significant morphologic changes during this developmental period, i.e., from being a simple tube to a four-chambered heart, the authors were at a loss to explain such a large and linear response in SVI across stages when injected volumes were normalized for average weight of the embryo at each stage. Moreover, the lack of innervation, together with immaturity of the myocytes and their myofibrillar disarray, points to the fact that the heart does not in fact exert the primary control over hemodynamics at this stage.

Atrial natriuretic peptide (ANP) is widely distributed in embryonic myocardium [10, 11]. It exerts a profound effect on embryonic hemodynamics by causing venous dilation, possibly affecting vascular permeability across the capillary beds. Nakazawa et al. demonstrated a dose-dependent fall in arterial pressure and dorsal aortic blood flow, when stage 21 chick embryos were infused with an increasing dose of ANP. Despite some 50% drop in aortic flow and arterial pressure and a decrease in vitelline venous pressure to one third of control, the heart rate remained the same as in control animals [12]. Hu et al. quantified the effect of ANP on the heart in stage 21 chick embryo and showed that the drop in above parameters occurs due to decrease in passive diastolic filling. In addition, the atrial component of ventricular filling, known as active ventricular filling, was likewise shortened [13]. Hu et al. concluded that the lack of heart rate and adaptive contractility response point to the importance of ventricular preload (venous return) in embryonic hemodynamics. Unchanged heart rate and shortened phase of passive ventricular filling, in response to vitelline vein clipping, was also reported by Ursem et al. [8].

The importance of the peripheral circulation was further demonstrated by Bowers et al., who tested the effect of NO on embryonic vascular tone and ventricular function, by using nitroprusside as a source of exogenous nitric oxide in stage 21 chick embryo and compared it to hemodynamic response in embryos of the same developmental stage, in which venous hemorrhage was induced by severing a fourth-order vitelline vein. They found that, like the venous hemorrhage, nitroprusside linearly reduces end diastolic volume (preload), stroke volume, and cardiac output. There was no change in heart rate except for a 7% drop at the highest rate of nitroprusside infusion and no effect on the arterial circulation as defined by arterial elastance. They concluded that the embryonic cardiovascular system is dynamically regulated at the tissue level, possibly through vascular tone, as is the case in fetal and adult circulations [14].

The above studies demonstrate that should the blood be impelled by the heart, the heart rate and the peak systolic velocity would increase to compensate for such a large acute drop in venous return. In spite of relatively rapid compensation, this however does not occur. It has been proposed [5] that cardiovascular equilibrium in the embryo, at least until stage 24, is maintained by vascular and endocardial endothelium, which respond directly to local pressure. Thus, altered shear stress caused by change in blood flow can release a variety of endothelins, which modify contractile characteristics of the adjacent vessels and the myocardium [15–17].