Paper - Initiation and early changes in the character of the heart beat in vertebrate embryos
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Patten BM. Initiation and early changes in the character of the heart beat in vertebrate embryos. (1949) Physiol Rev. 29(1): 31-47. PMID 18125910
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Initiation and Early Changes in the Character of the Heart Beat in Vertebrate Embryos
Bradley M. Patten
From the Department of Anatomy, University of Michigan Medical School ANN ARBOR, MICHIGAN
From the Department of Anatomy, University of Michigan Medical School ANN ARBOR, MICHIGAN
In dealing with advances in science there is a natural tendency to emphasize the newer findings appearing in the current literature, and pass too lightly over the long series of foundational steps that preceded them and made them possible. In assessing our present knowledge as to when and how the embrypnic vertebrate heart begins to beat, we should, therefore, not fail to cast at least a brief glance backward.
The unaided eyes of early observers could make of the beating heart of a transparent living embryo little more than a pulsating red fleck. With the discovery of the circulation in the adult and the development of the microscope, the embryonic heart began to be studied with new understanding. Haller (33) saw the beating heart and the moving blood in chicks as young as 45 hours of incubation, and was impressed by the inherent rhythmicity of the pulsations of the embryonic heart. Schenk (67) excised the heart of a 3-day chick embryo, kept it beating outside the body and noted its increased rate when he raised the temperature of the warm stage. Moreover, he cut the heart into fragments and noted that after a quiescent period following the operation these resumed rhythmic contractions. One of Schenk’s statements (p. 111) should be quoted verbatim: “Die Bewegungen des embryonalen Herzens in einer bestimmten Periode der Entwickelung, ist eine jener Bewegungserscheinungen die vom Einflusse des Centralnervenssystems unabhfingig ist.”
Wemicke (1876) observed that the heart rate increased during the early stages of development, and began to try out the eflects of different chemical solutions on the rate of its pulsation. Fano and Badano (23) in an exceptionally penetrating study of the embryonic chick heart crushed it at the atrio-ventricular sulcus and noticed that die atrium continued to beat without interruption.
The ventricle stopped altogether for a time and when it did resume beating its rate was slower than the rate of the atrium. They also cut the heart in a zig-zag ribbon and noted that the beat was transmitted along this ribbon from the atrium toward the ventricle. They commented on the peristaltoid character of the contraction of the young tubular heart and estimated the spread of transmission of the wave of contraction at 6 to 8 cm. per second. In their studies, heart movements were recorded photographically by throwing the shadow of the heart against a narrow aperture in a camera containing moving film. They also tried the eflect of various gases on the rate of cardiac pulsation noting particularly the striking effects of oxygen and carbon dioxide.
Another unusually interesting paper of about the same period is that of Pickering (62). He built a special water—jacketed chamber for studying the heart action of chicks under better controlled temperature conditions than previous workers had employed. In addition to studying the effects of temperature changes he investigated the effects of drugs such as cafiein, xanthine, theobromine, digitalis, nicotine, and hydrocyanic acid. Of special interest is his report of the reversing of the direction of the heart beat by treatment with dilute hydrocyanic acid and, sometimes, under the influence of amyl nitrite or morphine. These experiments I have tried to repeat without success, but incidental to other studies, I did see one case of spontaneous reversal of the direction of heat in a. chick embryo of about 55 hours. Unfortunately, this embryo reverted to normal beat propagation before a microcinematographic record could be made. Bremer (8) records having seen two cases of similar spontaneous reversal of the heart beat.’ These observations lend support to the reversals reported by Pickering, despite the lack of success up to the present in inducing such reversals under controlled conditions. Perhaps the most striking of Pickering’s experiments, and one which has since been repeatedly confirmed, was his production of a ventricular block by exerting pressure from a small piece of silk fiber across the atrio-ventricular junction.
Thus, before the end of the nineteenth century, we find well established some of the most significant facts as to the general character of the embryonic heart beat. Its inherent rhythmicity had been remarked upon by Haller (3 3). The definitely myogenic character of the early embryonic heart beat had been clearly enunciated by Schenk (67). Wernicke (76) had noted the change in heart rate due to temperature changes; Fano and Badano (23) had established the fact that there was a difference in the inherent rate of contraction of different parts of the young tubular heart and that its beat was peristaltoid in character. Pickering (62) had attempted the first comprehensive study of the effect of drugs on the embryonic heart, and had made the significant observation that heart block could be caused by pressure applied at the region of narrowing between atrium and ventricle.
Sequential Formation of the Primary Regions of the Heart
Almost concurrently with the studies of Fano and Badano and of Pickering dealing with cardiac activity, Born (6, 7) was developing the wax-plate method of reconstruction which was destined to add many new pieces to the growing mosaic of our knowledge of the embryonic heart. Earlier workers in their observations of heart action had taken too little account of the radical changes that were occurring in the structure of the heart itself as its development progressed. The plate-reconstruction method brought an infinitely greater accuracy to our analysis of the morphogenesis of the heart. Although this review is concerned primarily with the initiation of cardiac pulsation and the early changes in the character of the heart beat, the fact that the embryonic heart does not begin at all as a miniature of the adult organ makes it imperative that we should review its early structural changes before attempting to interpret its activities. For the plan of the embryonic heart, even the chambers present, changes with age in a manner which makes physiological observations meaningless unless they are properly correlated with the changing structure of the heart itself.
Utilizing the Born wax-plate method, the structure of the embryonic mammalian heart has been carefully studied in a large number of forms. To mention but a few of the more detailed papers that deal with the critical early stages in the fusion of the paired cardiac primordia there is the beautiful work of Davis (18) on the human heart; that of Schulte (68) on the cat; Wang (75) on the ferret; Yoshinaga (79) on the guinea pig; Patten (57) on the pig; Girgis (24, 25) on the rabbit; Goss (27) and Burlingame and Long (9) on the rat. In forms below the mammalian level the fusion of the cardiac primordia and the establishment of the early regional divisions of the heart have been studied in the chick by Patten (56), in the duck by Yoshida (78), in Amblystoma by Copenhaver (14) and in fishes by Senior (69) and Armstrong (2).
- Gowanloch (’ 2 2) reported having induced reversals in the heart of teleosts “by the action of various chemical and physical agents during early development.” Unfortunately, these observations seem to have been presented only in the form of an abstract without any details as to the age of the material or the kinds of “agents” employed.
There are, naturally, detailed differences in the development of even closely related forms, but they do not particularly concern us in the present connection. One developmental trend that is of basic physiological importance is shared by the above group as a whole. Because this particular phase in the morphogenesis of the heart has been too often slighted in studies dealing with cardiac activity it should be reemphasized here. It concerns the sequential formation of the primary regional divisions of the embryonic heart. The embryo of primitive vertebrates is endowed with a considerable amount of stored food material and the body must develop prone on this inert mass, as if it had been split open midventrally and closely applied to the relatively enormous spheroid constituted by the food material stored as yolk. This means that certain structures such as the heart which are destined to be midventral in location are formed from paired primordia which arise on either side of the mid line while the embryonic body still lies spread out, open ventrally, on the surface of the yolk sphere. These paired cardiac primordia can not meet each other in the mid ventral line until the embryonic body has grown sufficiently to pull away from the yolk mass, establish a floor under the foregut, and complete the ventral body-wall.
This process proceeds cephalo-caudally toward the mid-body region. This manner of completion of the ventral part of the body in the thoracic region means that the paired cardiac primordia must of necessity fuse in the same cephalo-caudal sequence. The tubular embryonic heart, therefore, has its cono-ventricular region established first, its atrial region at a somewhat later time, and its sinus venosus last of all. This sequential formation of the heart is summarized for the chick in figure 1. The forma tion of the heart in fishes and in amphibia is similar in its sequential character. Even in the higher mammals, which have ceased to store yolk in any quantity, the yolk-sac remains as a phylogenetic imprint on their ontogeny, so their embryos develop spread out on an empty yolk-sac, and their hearts exhibit the same paired primordia and the same cephalo-caudal sequence in their formation. This origin of the heart from paired primordia may be dramatically emphasized by procedures interfering with mid-line fusion. Cuts between the as yet unfused primordia, or extirpation of a wedge of mid-line tissue, or even pressure exerted between the growing primordia, cause the development of double hearts. Such experiments have been carried out on chicks (32), on amphibian embryos (20, I 3, 22) and on rat embryos (26).
If one realizes that cardiac pulsations begin in an embryo before all the parts of the heart have been formed, the importance of the sequential nature of the fusion of the cardiac primordia, and of the resultant sequential formation of the several regions of the heart as a basis for any studies of early embryonic heart action is at once apparent. In the earliest stages of its activity the heart is essentially nothing but a primitive ventricle, with a conical discharging end. Later the atrial region is added caudal to the ventricle, and last of all the sinus venosus is formed caudal to the atrium.
As each new region is added it brings tissue of different physiological properties into the complex, and the character of the heart beat shows correlated changes.
Fig. 1. Formation of the fundamental regions of the chick heart by progressive fusion of its paired primordia. A, at the 9—somite stage, when the first contractions appear. The canoventricular part of the heart is the only region where the fusion of the paired primordia has occurred and their myocardial investment has been formed. B, at the I6-somite stage, when the blood first begins to circulate. The atrium and ventricle have been established, but the sinus venosus exists only as undifferentiated primordial channels, still paired and still lacking myocardial investment. C, at the I9-somite stage. Fusion of the paired primordia is just beginning to involve the sinus region. D, at the 26-somite stage. The sinus venosus is-' definitely established and its investment with myocardium well advanced. While schematic as to manner of drawing, these figures are_ based on projection outlines of actual preparations, and such structures as the somites and the cranial ganglia V, VII—VIII, and -IX-X are shown in their exact relationships to serve as landmarks in following -the progress of fusion of the cardiac primordia. As an additional aid in following this fusion, arabic numerals have been placed against approximately corresponding locations. The 6 is located at the point of entrance of the duct of Cuvier as determined from injected specimens, and serves as a precise indication of the level of future sinus territory. The heavy black outlines and the crosshatched contours indicate the extent to which the heart is invested by differentiated myocardium. Note especially the absence of anything like an effective myocardial layer encasing the sinus region until a considerable time after the heart has begun to beat and the blood has been set in motion.
First Contractions in the Myocardium
The recent advances in our knowledge of heart action in young embryos have stemmed largely from new techniques which have made observation possible for longer periods under better controlled conditions, and from the development of recording methods of greater accuracy. Chief among these are the applications of tissue-culture methods (10, 48) to the handling of Sauropsidan embryos. Such techniques have made it possible to keep the same embryo alive under reasonably good physiological conditions for periods sufficiently long to permit observation of the changes in activity which occur as the primitive tubular heart progresses toward its adult condition. One of the most valuable of the new recording techniques has developed from the utilization of micromoving pictures of cardiac activity. Such records offer unequalled means of direct comparison of data obtained from experiments carried out on different occasions. Moreover, the possibility of controlling the time factor by speeding up the rate at which growth processes appear on the screen , and conversely, the use of the ‘slow-motion’ technique for the analysis of processes too rapid to follow with the unaided eye, make micromoving pictures peculiarly valuable in the study of the embryonic heart.
Because of the widespread use of chick embryos as a basis for both morphological and experimental studies it is not surprising that they were the subjects of the first detailed observations on the beginning of cardiac activity. Even in this muchstudied form it was not until work with tissue-culture methods had paved the way that continuous observation of the same embryo for considerable periods of time made it possible to see the very first signs of myocardial activity. The first record of the actual beginning of cardiac contraction as it can be seen in living embryos kept under continuous observation in vitro appears to have been made by Sabin (66). Incidentally to her beautifully detailed observations on the origin of the blood vessels of the chick, she noted the time at which the first beating of the heart commenced and that this first pulsation was not in the venous part of the heart, but in the ventricle. She says (p. 255):
- “The very first beats of the heart can be made out in these hanging drop specimens. They occur at the stage of ro somites and always in the same position. The first twitching is along the right margin. . . . It is interesting to note that there is no movement whatever in the vein, the entire twitching being confined to the ventricle proper. . . . The beat is at first slow but rhythmical, and gradually involves the entire wall of the ventricle, spreading from the posterior to the anterior end.”
These observations Sabin herself did not pursue beyond putting them thus on record. They were, however, repeated and confirmed by Johnstone (40, 41) in two striking papers which we shall have occasion to consider further in connection with the early changes in the location of the pace-making center.
Olivo (52, 53) studied the chick heart intensively at the time it first exhibited contractile activity. Like Sabin, he reported the first contractions as involving only very small areas of the myocardium. In agreement with Sabin, also, he regarded the beating as rhythmic from the outset although its rate was at first no more than 8 per minute. Olivo reported the first contractions as occurring in 9-somite embryos which agrees, well within the range of individual variability, with Sabin’s findings.
More difficult to reconcile is Olivo’s statement that the first contractions appeared on the left more frequently than on the right.
Patten and Kramer (59, 6o), having designed and constructed an apparatus especially adapted for work with living embryos, made microcinematographic records of the first heart beats as seen in chick embryos cultured in vitro. By making preparations of specimens somewhat younger than the 9 to Io-somite stage at which Sabin and Olivo had noted the first pulsation, it was possible to keep embryos under continuous observation during the period when the beating of the heart was due to commence. In such preparations the first contractions of the myocardium were seen to occur in the tubular heart when it had reached the stage of development (9 to ID somites) indicated in figure I A. The only region of the heart which has at this time been established by fusion of its paired endocardial primordia and their complete investment by epimyocardium is the cono-ventricular portion. The atrium is represented only by paired endothelial tubes lying either side of the anterior intestinal portal and these tubes have as yet acquired little more than a partial covering of the splanchnic mesoderm which will later become differentiated into their epimyocardial mantle. The sinus venosus at this stage, is represented only by the paired endothelial tubes, diverging toward the yolk-sac, which show neither a recognizable local specialization setting them apart from the omphalomesenteric veins with which they are continuous, nor any differentiated myocardial investment.
In agreement with Sabin (66) and Johnstone (40, 41) the moving picture records of Patten and Kramer show the first contractions occurring along the right margin of the ventricle. The contractions were exceedingly small in amplitude, the earliest of them appeared to involve only a few cells at a time, and the impulse did not seem to spread by conduction. Superimposed tracings made by pulling the film, a frame at a time, through an enlarging apparatus, showed that these first flickers of contraction were not limited to any single sharply localized area. They occurred, rather, in unpredictable sequence in slightly different places along the right side of the ventricular myocardium. As the preparations were watched there appeared to be a gradual spreading of the areas involved in the contractions, until all of the right side of the ventricle began to contract. Soon thereafter the entire ventricular myocardium began to contract synchronously.
Sabin, Johnstone, and Olivo all described the first beats as being rhythmic. Patten and Kramer found the earliest contractions appeared intermittently. If observed during a period of activity the local fibrillar contractions could be seen to occur in rhythmic series as described by Sabin, Johnstone, and Olivo, but in observations extending over long continuous periods there were intervals of quiescence followed again by another series of pulsations. This sort of intermittent rhythmicity is sometimes spoken of by physiologists as ‘Lucciani grouping.’ Nordmann and Riither (51) regarded pauses in the beating of their cultures of embryonic heart muscle as signifying exhaustion, and it is possible that the pauses in rhythm reported by Patten and Kramer were indicative of unfavorable culture conditions. Certainly pauses in rhythm, once cardiac pulsations are well established, should be so interpreted. These very young embryos appeared, however, to go on to regular rhythmic beating suggesting that the culture conditions were at least reasonably favorable. Whatever interpretation future work may place on it, the phase of intermittent activity lasts but a brief time during which the periods of beating become progressively longer and the intervals of quiescence shorter, so there is rapid progress toward the establishing of sustained regular rhythmic pulsation. Armstrong (2) has carried out exceedingly interesting studies on the embryonic heart of the small marine teleost, Fundulus. His work shows that some of the developmental processes in this form differ in their timing when compared with corresponding processes in the embryos of higher vertebrates. Most striking is the fact that in Fundulus the myocardial primordia fuse in the mid-line before endocardial tubes have been formed. Thus the primordial heart is at first a solid cone of myocardium without any endothelially lined lumen. When the first cardiac pulsations occur at the I3-somite stage (personal communication) the heart still lacks any lumen. By the 16-somite stage endothelial cells have been difierentiated within the myocardium and with their belated appearance the embryonic heart of Fzmdulus is essentially similar to the tubular heart of other young vertebrate embryos.
It seems probable that the solid myocardial cone where pulsation first appears in Fzmdulus is homologous with the conoventricular portion of the chick heart, al though Dr. Armstrong tells me that the sequential formation of the several regions of the cardiac tube is by no means as clear-cut a phenomenon as it is in the chick and other higher vertebrates. The initial slow beat in the primordial myocardial cone and the subsequent establishing of a more rapid peristaltoid beat sweeping from sinus venosus to aortic outlet is, however, quite in line with conditions seen in the embryos of higher vertebrates.
The meticulous studies of Copenhaver (14, 16) on Amblystoma embryos, and Goss (27, 28, 30) on rat embryos, showed that in these representative species of am phibians and mammals the first heart beats appeared in the ventricle, as was the case in the chick. As might be. expected there were minor specific differences as to certain details. In Amblystoma the first contractions in the majority of the embryos studied appeared in the medial portion of the ventricular myocardium instead of first on the right side as in the chick. In rat embryos the first contractions appeared on the left. The situation as to activity on the right and left of the mid-line is particularly interesting in rat embryos because in their hearts pulsation begins before the paired primordia have fused with each other in the mid-line. It was some two hours following the first discernible contractions on the left before the right half of the ventricular myocardium showed any activity. For a time then each half of the heart beat independently and, surprisingly enough, at a somewhat different rate, that on the left being slightly higher. Dwinnell (19) reports similar observations on the hearts of young rabbit embryos. Under normal circumstances this dual rhythm is a transitory phenomenon, persisting only for the few hours before the right and left primordia fuse with each other to establish a single tubular ventricle. When this occurs unified contraction of the entire ventricular part of the heart is established.
Thus in all the forms as yet critically studied, the ventricle is the first part of the heart to show contractile activity. The first contractions are nonpropagating local twitchings involving only a few cells. In different species there are differences in the precise part of the ventricle to show the first activity but the gradual extension of the activity from the first areas involved until the entire ventricular myocardium begins regular rhythmic contraction is essentially similar in all forms. The striking peculiarities of this early ventricular beat are its slow rate as compared with that of the heart in older embryos, and its lack of peristaltoid character. These first ventricular pulsations are not adequate to set the blood in circulation.
It is perhaps worthy of comment that the first glycogen in muscular tissue of chick embryos (1) becomes recognizable in cardiac muscle at about the time its contraction begins. Glycogen can be identified in cardiac muscle, in keeping with its earlier assumption of activity, at considerably earlier stages than it can be seen in the developing somatic musculature. It is interesting also that M. R. Lewis (46) found that with special fixation involving the addition of osmic acid to a Zenker-acetic mixture, striated myofibrils could be demonstrated in chick embryos of about I0 somites. One should not conclude from these observations that the initiation of contraction in cardiac muscle is dependent on the presence of glycogen or on the formation of myofibrils. As a matter of fact in these independent observations, both the appearance of glycogen and the differentiation of myofibrils are reported as occurring in embryos about one somite older than those in which Patten and Kramer (60) noted the first evidence of contraction. Goss (29) made a very carefully controlled series of observations using rat embryos which had been kept under continuous observation until the primordial heart started to show its first contractions. When fixed according to the technique of M. R. Lewis, and by other appropriate histological methods, the myocardial elements which had thus been seen to exhibit their first contractions showed no evidence of cross-banding or even of definite myofibrils. Szepsenwol (7 3) has made similar observations on the explanted hearts of chick embryos. Thus it appears that the earliest contractions occur in cells that are but little specialized morphologically and which still lack the differentiated intracellular structures we are accustomed to think of as characteristic of cardiac muscle. It is, nevertheless, significant that by the time their contractile activity has developed to any degree of efliciency, crossbanded myofibrils can be demonstrated and glycogen becomes recognizable by the standard histochemical tests.
Shifts in the Location of the Pace-Making Center and Early Changes in the Character of the Embryonic Heart Beat
One of the most startling and significant facts about the developing embryonic heart is that its myocardium at different cephalo-caudal levels exhibits difierent inherent rates of contraction. It has been repeatedly demonstrated by fragmentation experiments that the atrial part of the young tubular heart beats faster than the ventricle, and that, later when the sinus venosus is added caudal to the atrium, its rate of contraction is faster than that of the atrium (23, 35, 47, 5o, 60, 14). Cohn (12) and Barry (3) have carried out similar studies in greater detail, segregating small fragments taken from a series of locations within each of the main cardiac regions. Their work clearly indicates that the gradient in contraction rate is intracameral as well as intercameral. The gradient in contraction rate can be demonstrated, also, by the less drastic experimental procedure of exerting pressure at the atrio-ventricular region of the cardiac tube sufficient to cause physiological disjunction of the sinoatrial beat from the ventricular beat (62, 40). This cephalo-caudal gradient in inherent contraction rate is the key to the establishing of a peristaltoid beat of suflicient efficiency to propel the blood through the heart. As might be suspected, the part of the cardiac tube with the highest contraction rate at any given phase of development sets the rate for the entire heart. This is clearly implied by the fact that in transec tion experiments the sino-atrial part of the heart continues to beat at essentially the rate of the intact heart, whereas any fragment derived from more cephalic regions, when isolated, reverts to the original slower rate of contraction it exhibited before more rapidly pulsating tissue was added to it caudally. The same phenomenon can be more strikingly demonstrated, as was done by Pail’ (5 5, 56) by cultivating the parts of transected chick hearts in such close proximity that a bridge of myocardial tissue could grow across from one piece to another. As long as a ventricular fragment remained independent it retained the slow rate it had assumed at the time the heart was transected. When even a slender bridge of tissue grew across connecting the two fragments the more rapidly beating sino-atrial region assumed dominance and caused the ventricle to beat at its own more rapid rate. Even more dramatic were the experiments of Copenhaver (15, 16) in which he grafted the sinus venosus of one species of Amblystoma to the ventricular part of the heart of another species with a characteristically different cardiac rate. The transplanted pace-making center took over control of the host ventricle and drove it at the rate of the intact heart of the donor species. Since the fastest beating part of the myocardium dominates the rhythm of the heart as a whole, and since each new part of the heart that is added behind the first formed cono-ventricular part has a higher intrinsic contraction rate than the part of the heart tube already formed, it follows that there is, during development, a succes sion of pace-making zones in the tubular embryonic heart. The first beats, as we have seen, appear in the ventricle. This idioventricular pulsation is slow and nonperistaltic in character. As foregut closure progresses caudad and permits more of the paired cardiac primordia to come together and fuse in the mid-line, myocardial tissue exhibiting a higher rate of contraction is added at the caudal end of the slowly beating ventricle. This newly added, faster beating tissue steps up the rate of the heart as a whole and, even more important, it establishes a peristaltoid sweep in the young tubular heart. Since the fastest beating tissue is at the intake end of the heart the waves of contraction there initiated sweep toward the outlet end of the heart, setting up for the first time pulsations of a type which are efficient in propelling the blood. This beat can be characterized as atrio-ventricular. The pace-making zone is, at this time, the atrial myocardium which has just been added caudal to the previously formed ventricle. Moreover, the atrial part of the heart is not formed all at once, but by progressive fusion of the paired primordia. This means that at any given phase of development the most recently added part of the atrium is the pacemaker. Later, as fusion involves still more caudally located parts of the paired cardiac primordia the sinus venosus is added behind the atrium. The myocardium of the sinus has an inherent contraction rate higher than that of the atrium and so in its turn takes over the pace-making function for the heart as a whole. The beat can now be characterized as sino-atrio-ventricular. This is the final major shift in the location of the pace-making center of the developing heart. With the formation of the sinus venosus caudal to the atrium the basic regional divisions of the heart are all established and the addition of new parts of the cardiac primordia with their pro gressively higher inherent contraction rates is no longer taking place. There are still minor changes to occur in the concentration and arrangement of the pace-making areas within sinus territory, but they are by no means as radical as the early changes just outlined and they appear in stages of development later than those being covered by this review.
Recently, attention has been directed to the functional importance of the socalled ‘cardiac jelly’ in the early pumping action of the heart. Davis (17) had applied this designation to the gelatinous material which lies between the endothelial lining of the tubular heart and its outer epimyocardial coat, and had emphasized its significance in giving mechanical cohesion to the two layers of the heart. Patten, Kramer, and Barry (61) in microcinematographic studies of the pumping action of the embryonic heart have shown by superimposed tracings from their moving picture films the manner in which the cardiac jelly is heaped up in local mounds which by their apposition give valvular closure of a hitherto unsuspected type in the embryonic heart. Such mounds appear at two levels, at the constriction between the atrium and the ventricle and in the ven tricular conus, at the point where the ventricle discharges into the truncus arteriosus. The time at which these valves close is controlled by the time the peristaltoid sweep of contraction reaches their level in the tubular heart. This means that they act in reciprocal fashion, the valve at atrio-ventricular level closing just after the sweep of contraction through the atrium has fully charged the ventricle with blood, and remaining closed while blood is being forced out through the truncus. At the end of ventricular systole the contraction wave reaches the valvular pads in the conal region which are apposed, thereby checking regurgitation of blood from the arterial stems into the ventricle. While the conal valves are closed the valvular pads at the atrio-ventricular canal are open, thus permitting the ventricle to be charged for its next contraction cycle. It is interesting that this primitive type of valvular action in the tubular embryonic heart appears at regions where the leaf-like atrio-ventricular valves, and the cup-shaped aortic and pulmonary valves are destined to be moulded at much later stages of development. Of possible far-reaching significance, also, is the fact that the mounds when they first appear are shaped from noncellular cardiac jelly, with a subsequent invasion by cells converting the cardiac jelly into a richly cellular connective tissue of the type which has long been called ‘endocardial cushion tissue.’ It is this readily moulded endocardial cushion tissue which plays such an important role in the later development of the cardiac valves and septa, and there arises the intriguing question as to the possible preliminary moulding influence of blood currents on the highly plastic cardiac jelly with the subsequent fixation of these configurations by the more firmly organized cellular tissue which later replaces it.
It is apparent from the material already reviewed that two of the basic factors necessary for eflicient propulsion of blood by a tubular pump have been adequately accounted for: (I) The pace-making portion of the heart, shifting in position at different ages but always located at the intake end, starts contraction waves which sweep through the tubular heart toward its outlet end thus providing an adequate propelling mechanism; 2) the development of valvular endocardial pads at the intake and outlet ends of the ventricle, by minimizing backflow, adds adequate efiiciency to the propulsive work of the peristaltoid sweep of contraction. Barry (4) has pointed out that the presence of cardiac jelly between the myocardium and the endocardium is a requisite for a third essential factor, adequate stroke volume. According to his analysis there are certain definite criteria which must be met if a tubular heart is to expel an adequate volume of blood with each beat. The systolic diameter of the heart must be sufiiciently reduced to practically obliterate the lumen if the contraction wave is to be efiective in forcing blood ahead of itself as it sweeps toward the cardiac outlet. The filling of the heart as well as the amount of blood expelled with each contraction obviously depends upon the diastolic diameter of the myocardial sleeve. Embryonic myocardium, like adult myocardium, can not shorten in systole more than a definite limiting proportion of its diastolic length. In embryonic chick hearts Barry gives this shortening as approximately 20 per cent of the diastolic length. It follows that the myocardial layer must be of relatively large diameter if the stroke volume is to be adequate. If the endocardial lining lay in immediate contact with the myocardial sleeve a functional dilemma would exist. Assuming the lumen were small enough to permit systolic closure by 20 per cent shortening of the myocardium, the stroke volume would be totally inadequate. On the other hand, if a situation were assumed such that the myocardial circumference would be sufficient to allow for adequate stroke volume, there would not be suflicient reduction of the lumen to provide efficient propulsion. The conflict between these two requisites is resolved by the presence of the thick layer of resilient cardiac jelly. This layer transmits the force of contraction of the relatively large myocardial sleeve radially down against the small endothelially lined lumen. Thus the circumference of the contracted myocardium is relatively large, even though the lumen of the heart is squeezed shut, for its radius is increased by the thickness of the layer of cardiac jelly. The increase in the diameter of the myocardial sleeve on diastole under these circumstances will be proportionately large, making it possible for the heart to pump with an adequate stroke volume.
Electrical Recordings from Early Tubular Stages of the Embryonic Heart
In view of the shifts in the location of its pace-making center exhibited by the young embryonic heart at various stages in its development it is obvious that electrocardiographic records obtained during the periods in which these changes are occuring would be of unusual interest, and there have been many attempts to secure such records. The technical problems involved, however, have been varied and troublesome, and until relatively recently the results have been disappointing. As is natural the earliest work in this field dealt with relatively old fetuses which, as might be expected, furnished records quite comparable to those of adults. The first to study the heart of really young embryos by electrocardiographic methods was Wertheim-Salomonson (77). Using the chick as an experimental animal he succeeded in taking records from embryos as young as 60 hours. He, like other early workers in the field, was greatly handicapped by the lack of amplification methods such as have since become available, and his records of these early stages showed only slow rises and falls with no clear-cut phases such as would be anticipated from what we know of the character of the heart action at this stage. Only when he worked with chick embryos as old as five to six days did his records show anything like a regular electrocardiographic pattern. Cluzet and Sarvonat (II) and Spadolini and Giorgio (71) encountered the same difiiculties, their records from young embryos showing nothing suficien tly consistent to warrant any attempt at interpretation.
That electrocardiograms of adult pattern are obtainable much earlier than was indicated by the foregoing results was shown by Ktilbs (43), who greatly improved the recording technique and obtained tracings from chick embryos showing the emergence of practically all of the adult characteristics as early as the third day of incubation. In agreement with Kiilbs’ results, Robb (65), in the abstract of her report to the International Physiological Congress, stated that the beginnings of P-waves and of the QRS-T complex were becoming recognizable between 50 and 72 hours.
Further progress in the technique of recording was shown in the work of Lauche and Schmitz (45) and that of Lueg and Htifer (49). Both these papers, however, dealt with explanted hearts or with cultivated heart fragments. Some of such observations, especially those of Lueg and Htifer, Katsunuma and Inada (42), and Szepsenwol and Odoriz (74), on isolated atrial and ventricular portions of the heart are exceedingly interesting, but they need to be evaluated in relation to records of the intact heart acting under more nearly normal conditions.
The work of Hogg, Goss and Cole (39) contains some exceedingly interesting electrical records obtained from cultured ventricular fragments from the heart of 16 day rat embryos. Their observations that diphasic tracings could be obtained by placing the microelectrodes near pulsating centers that were slightly out of phase with each other would seem highly significant. It suggests that one possible factor behind the polyphasic tracings obtained from young embryonic hearts may be an arrangement of the cardiac muscle which sets up a characteristic pattern of areas in a definite sequence of contraction phases. This at least seems like a lead worth fur ther investigation in opposition to the contention of Eyster, Krasno and Hettwer (21) that a polyphasic tracing is characteristic of heart muscle as such.
A description of a carefully worked out technique for recording electrical changes in the embryonic heart was published by Pollack (6 3). This was followed by a paper in collaboration with Dionne and Schafer (64) on amplification technique, and a later paper by the same group giving some of the results obtained by these methods. The hearts studied by these workers were, however, for the most part rather too old to Show the most interesting changes involved in the development of an electrocardiogram of adult configuration.
In a short paper on the influence of digitalis on the embryonic electrocardiogram, Lagen and Sampson (44) state that multiphasic curves are obtainable from chick embryos as early as the 36th hour of incubation. Regrettably, no illustrations of their records were included, so that satisfactory comparison of their interesting find ings with other available data is not possible. Bogue (5) secured clear-cut and convincing records showing the early appearance of polyphasic tracings. Comparing his records with those of earlier workers, it is apparent that as amplifying techniques have improved the beginnings of the characteristic waves of the adult electrocardiogram have become recognizable in progressively younger embryos.
Taking advantage of the advances in amplification and tissue culture technique Hoff, Kramer, DuBois and Patten (38) obtained electrocardiographic records from a series of embryos in which the degree of heart development was carefully correlated with the tracings secured. The youngest embryo from which they secured satisfac tory records was a chick of I 5 somites. At this stage, which is reached on the average with about 33 to 36 hours of incubation, the nearly straight tubular heart con sists almost entirely of ventricle. The electrical record obtained from it shows none of the defiections characteristic of the adult electrocardiogram, but takes the form of a curve which first drops below, and then rises above, the isoelectric line. This configuration is consistent with the caudocephalic direction of the progress of contraction shown by superimposed tracings of successive frames made by Patten and Kramer (60) from micromoving pictures of the heart action at this stage.
Slightly older embryos (16 to 17 somites, average incubation age 37 to 40 hours) yielded records in which there appears a sharp downward deflection, followed by a rapid return to, or above, the isoelectric line. Because of its characteristic configuration and because correlated morphological studies indicate that the embryonic heart at this stage is practically all ventricle, they interpreted this as representing the QRS complex.
In the next three or four hours of development, fusion of the cardiac prirnordia progresses caudally, so that the atrial region becomes definitely difierentiated and the sinus venosus begins to take shape posterior to the atrium. Records from embryos in this age range showed a downward deflection coming about two twentyfifths of a second ahead of the QRS complex. This they intrepreted as an inverted P-wave.
During the next day of development the ventricular loop is bent backward so that it comes to be in its adult position caudal to the sinoatrial part of the heart. With this shift in relative positions the P-wave appears above the isoelectric line. Thus by the fourth day of development the electrocardiogram has assumed practically its adult configuration.
In connection with the study of the changes in the location of the pace-making center, and the propagation of the contractile impulse as indicated by the electrocardiographic records, the question of nervous control of heart rate naturally arises. The excision and transection experiments reviewed make the primary myogenic character of the pulsation so clear that belaboring of this old issue is uncalled for. Much is still to be learned, however, about the way in which the nervous mechanism secondarily assumes the regulation of the rate of the pulsations initiated within the myocardium. Although the general story of the development of the nerves to the heart is fairly well known (34, 36, 37, 72, 70) ; much more critical work is needed as to the exact stage of development at which the vagus and cervical sympathetic fibers estab lish, respectively, their retarding and accelerating action on the heart rate. Such information would be particularly valuable in the case of laboratory animals such as the fowl, the rat, and the rabbit, the embryos of which lend themselves so readily to experimental procedures. Although work of this type is urgently needed in following through the story of the control of heart rate in later stages, the already available studies clearly indicate that nerve control has not as yet been established in any of the stages here considered. Furthermore, ‘conduction tissue’ is not at these ages histologically distinguishable from the remainder of the cardiac muscle. There is considerable food for thought in the fact that it is possible to trace the appearance of all the major features of the adult electrocardiographic pattern in embryonic hearts well before they have differentiated a sinoventricular conduction system such as is familiar in the adult heart.
In addition to the work here covered there have been a number of studies on the effect of age difierences and temperature changes on heart rate, on the influence of various chemical substances on the character of cardiac pulsation, and, more recently, observations on the effects of hormones or hormone-like substances. Interesting and important as many of these studies are, it did not seem wise to divert attention from the main story of how the heart is established and first starts to function efiectively by attempting to cover such work in this review.
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