Keywords
Heart ablation studiesEmbryo heart reversalEmbryo heart explantsCardiac mutantsMexican salamanderDefective cardiac contraction coupling5.1 Heart Ablation Studies
The premise that lower vertebrate embryos not only survive but continue to develop after the removal of their heart goes against the grain of the widely accepted physiological concept that the heart is the essential “motor” of circulating blood. Buried within the literature are reports which, despite the fact that they were primarily designed to demonstrate a host of related cardiovascular phenomena, clearly point to the primary role of the peripheral circulation.
Well over a century ago, Loeb demonstrated that fish embryos continue to develop after their hearts have been arrested with KCl [1] and frog embryos continue to live up to 2 weeks after the removal of their heart primordia [2]. Stöhr carried out a series of experiments in bullfrog larvae to determine the relative morphogenic potential inherent in the early embryo’s heart, as compared to the effect of flow-induced forces of the circulating blood [3]. An attempt was made to separate out the hemodynamic variables in two ways, either by isolating the heart from the stream of blood or by altering inflow/outflow conditions of the existing heart. In the first experiment, the heart primordium of another embryo at the stage of the tube heart was implanted into the abdomen of the host embryo and turned 180° in respect of longitudinal embryonic axis. Stöhr noted that the transplanted heart cells not only developed into a normally oriented heart, integrated into the existing circulatory stream, but grew in size beyond normal and even assumed the dominant role. The original heart, on the other hand, continued to beat, but deprived of blood, soon became pale and failed to develop further.
To determine the effect of blood flow on the heart’s morphology, Stöhr explanted the tube heart of an early bullfrog larva and re-implanted it in the same embryo in its normal place, but turning it 180°, against the stream of the inflowing blood. To Stöhr’s surprise, the blood continued to flow in the normal direction, despite the fact that the heart now contracted in the opposite direction, i.e., from the arterial toward the venous pole. A significant hemodynamic disturbance was created, resulting in gradual yolk sac edema and the embryo’s death over the next couple of days. Stöhr commented that while it is possible to rotate the heart, it is evidently not possible to reverse the direction of the circulating blood. We will see that yolk sac or whole embryo edema is a characteristic feature, not only of the failing heart, but also in conditions where the heart fails to beat, or is absent altogether. It points to the fact that the blood possesses its own movement and that it continues to move for a considerable amount of time, even in the absence of the heart.
In the 1950s, Kemp showed that frog embryos continue to develop for 4–5 days after the heart had been surgically removed. In spite of significant blood loss sustained during surgical intervention, Kemp was still able to observe that, “some ebb and flow of blood occurred in the vessels near the heart.” During the first day after excision of the heart, the larvae were as active as controls, but became less vigorous thereafter [4]. Gradually, generalized swelling of the head and of the peritoneal cavity of the larvae occurred, leading to their demise. In a different paper, Kemp reported the survival of salamander larvae for up to 15 days, after removal of their pre-cardiac mesoderm [5].
5.2 Cardiac Mutants
In 1965 a mutant gene c (“cardiac lethal”) was discovered as a natural variant in Mexican salamander (Amblyostoma mexicanum), in which the heart seemingly develops in normal fashion, but fails to beat. The affected larvae show normal swimming and righting movement and survive up to 2 weeks, until the exhaustion of the yolk nutrients. The larvae homozygous for gene c are easily recognizable by their grossly edematous, pear-shaped forms, resulting from the accumulation of hemolymph in the heart, the pericardium, and the cephalic portion of the trunk. Microscopically, the thin-walled, distended heart exhibits sparse, disorganized myofibrils [6]. The extended survival of heartless larvae and embryos has been a source of conjecture and surprise among physiologists, since lack of circulation, the primary means of oxygen and nutrient transport, is by all accounts, not compatible with survival. In order to determine just how important the delivery of oxygen is for survival and development, Mellish et al. measured oxygen consumption and circulatory transport in the (1) normal salamander larvae (Amblyostoma mexicanum) and compared them to embryos at the same developmental stage, (2) whose hearts’ primordia were explanted, and (3) to naturally heartless embryos that were homozygous for gene c. The operated larvae were given 8–9 days to recover from the procedure. To their surprise the results showed that oxygen uptake and transport were the same for all three groups. They concluded that the circulatory system is not required for oxygen transport during early embryonic period and that the embryo’s needs are met solely by diffusion. They moreover suggested that the heartless embryo succumbed due to extensive edema, which might have hindered diffusion of oxygen [7].
Because of the similarity of the early vertebrate heart development in lower and higher vertebrates, the zebrafish mutants have been used extensively as a model for abnormal human heart development. In the course of normal development, the paired zebrafish heart primordia fuse into a single tube by 19 h post fertilization (hpf), and the heart starts beating at 22 hpf and undergoes the process of looping by 33 hpf. At about 36 hpf, a vigorous circulation is established [8]. A number of zebrafish heart mutants have been identified, collectively named the silent heart, in which the morphology of the early heart seems normal, but fails to contract. In spite of this gross aberration, some of the embryos show normal motility and response to touch, but in the course of 3 days, the heart gradually disintegrates and the embryos die [9].
In addition to morphological aberrations, several zebrafish mutants have been described, which mimic a group of phenotypic conditions, resembling cardiomyopathies of the higher vertebrates. The embryos with hypertrophic or obstructive type have hearts with increased thickness of cardiac jelly, which severely restricts endocardial lumen. The resulting defect hinders the blood’s return to the heart, causing regurgitant flow in the region of sinus venosus and atrium. In the course of several days, the cardiac function in these embryos invariably deteriorates, resulting in whole body edema. Gross edema is likewise a common terminal feature of various hypokinetic and dilated zebrafish heart mutants [9, 10].
A variety of avian and mammalian heart mutants exist which show, depending on severity, a similar overall pattern of circulatory phenomena. For example, a genetic strain of mice has been developed in which a targeted inactivation of Na/Ca exchange gene (Ncxl−1−) results in a defective cardiac contraction coupling mechanism and an abnormal myofibrillar organization [11]. The homozygous mice fail to initiate the heart beat on embryonic day E8.25, as is the case in normal embryos, but nevertheless continue to develop through E10 when heart looping takes place. Beyond E11, the embryos become severely retarded in growth and die by E11.5. In addition to proving that survival is possible without a beating heart, this model has been used to demonstrate the migration of hematopoetic progenitor cells from the yolk sac to the embryo, indicating that at least a rudimentary circulation exists between the two.
