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Functiogenesis of cardiac pacemaker activity
The Journal of Physiological Sciences volume 66, pages293–301(2016)
Throughout our investigations on the ontogenesis of the electrophysiological events in early embryonic chick hearts, using optical techniques to record membrane potential probed with voltage-sensitive dyes, we have introduced a novel concept of “functiogenesis” corresponding to “morphogenesis”. This article gives an account of the framework of “functiogenesis”, focusing on the cardiac pacemaker function and the functional organization of the pacemaking area.
Conventional physiology has dealt, primarily, with functions in completed living systems, as stated in standard textbooks of physiology. In the late 1970s, and the beginning of the 1980s, we introduced the idea of the ontogeny of electrophysiology and related functions associated with morphogenesis in the early embryo: a new combination of embryology and electrophysiology through our newly developed knowledge and technical skills with multiple-site optical recording of membrane potential using voltage-sensitive dyes (VSD) [1–3]: we later termed this interest “functiogenesis”, a coined word corresponding to “morphogenesis”: “functiogenesis” is the physiology of process (becoming) rather than state (being). Thus, “functiogenesis” is far more interesting than the stereotyped description of the growth of functions: it was intended to break through the conventional frameworks of physiology.
Functiogenetic approaches using VSD-optical recording has been a powerful method for complex functions such as heart and/or central nervous systems: it allows us to progressively analyze complex functional organization and architecture, in a manner reminiscent of the expansion of a complex function in a power series in mathematics, as a natural ontogenic progress.
We describe quasi-classically here, on the bases of our own original works, the functiogenesis of electrophysiological events, and of pacemaker activity in the cardiac primordia and the precontractile chick heart [see also reviews 2, 4].
Morphological characteristics and methodological problems
In the early phases of cardiogenesis of the chick embryo, paired cardiac primordia are brought together at the midline and begin to fuse with each other, progressively caudally at the 7-somite stage (corresponding to an incubation time of about 26–35 h) (Fig. 1). This process results in the formation of the primitive tubular heart in which first spontaneous beating appears at the middle period of the 9-somite stage: before this stage, contraction has not yet appeared in tubular hearts . A review article by Patten  on the embryonic origin of the heart beat in vertebrate embryos appeared in Physiological Reviews, and is a comprehensive summary of earlier studies on the ontogenesis of cardiac functions.
In these very early embryonic developmental stages, because the cardiac primordium and primitive embryonic cells are extremely small, fragile, and possessed of non-specific topology, it is technically difficult or impossible to make experiments on these subjects by using conventional electrophysiological methods with microelectrodes, including even modern patch-clamp methods. For these reasons progress in the study of the embryonic origin (onset) of physiological function in cardiac tissue has been hampered.
In this situation, with the introduction of VSD-optical recording of membrane-potential activity, we were able to answer many of the questions that had plagued the earlier developmental physiologist for decades. As a consequence of this achievement, we also showed that the VSD-optical technique [2, 4, 7] is the optimal method for studying embryonic primitive hearts: it is important, therefore, to emphasize that “functiogenesis” of electrophysiology has been made possible by a breakthrough in the VSD-optical recording technology. In this context, we introduced multiple-site optical recording with VSDs as the leading principle.
Embryonic onset of cardiac electrophysiology
The Greek philosopher Aristotle already understood that “the heart is the first organ of the embryo to reach a functional state” more than two millennia ago . However, on the bases of the results obtained using conventional electrophysiological measurements, Manasek, in Handbook of Physiology published in 1979 , tentatively concluded that the onset of electrical activity occurs at stage 10 of Hamburger–Hamilton (H–H)  (corresponding to the 10-somite stage, and to about 35 h of incubation). Afterwards, in 1987, introducing the VSD-optical monitoring technique, we first recorded spontaneous electrical activity in the pre-fused cardiac primordium in 6-somite chick embryos  (Fig. 2).
Although we suggested earlier that, in the embryonic chick heart, the spontaneous action potential would be generated initially during the middle period of the 7-somite stage of development, corresponding to the initiation of fusion of paired right and left primordia [12, 13], additional experiments indicated that this speculation was not reliable. As described above, the multiple-site optical recording method with a 100-element photodiode array detected spontaneous electrical activity in the pre-fused cardiac primordia at the 6- and early 7-somite stages, before the morphological formation of the heart. This finding was an epoch-making discovery for the study of cardiac functiogenesis .
The size of the VSD-optical action signals (optical action potentials) detected from the pre-fused cardiac primordium was very small, and the signal-to-noise ratio was smaller than 1.5:1–2.0:1. Nevertheless, the signals appeared rhythmically and were nearly synchronized among the different positions where the signals were detected. The optical signals related to spontaneous action potentials (spontaneous optical action potentials) in the early 7-somite prefused cardiac primordia seem to be somewhat larger than those observed at the 6-somite stage. In addition, in the 6-somite embryos, the signals were always detected from only one side of the primordia .
Furthermore, a remarkable finding was that spontaneous action potential-related signals (optical action potentials) occur rhythmically in the pre-fused cardiac primordium. This strongly suggests that the genesis of excitability, spontaneity, and rhythmicity appear to be inherently coupled in the cardiac primordium: it was thus deduced that pacemaking ability is incorporated by nature in the cardiac primordium. Further, the size of the optical action potentials obtained from the pre-fused cardiac primordium was often distinct, though very small, and the electrical activity appeared in a limited area. This result suggests that a few cells with very immature (not yet fully developed) electrical activity, differentiated in a limited area in the right or left cardiac primordium, before fusion begins .
Excitation spread in primitive tubular hearts
In precontractile primitive hearts of chick embryos, during the 6- to 9-somite stages, the size of optical action potentials and their area of spread increased dramatically .
Figure 3a shows an example of a simultaneous optical recording of a spontaneous action potential from 91 different contiguous regions of a 9-somite embryonic precontractile chick heart stained with a VSD (NK2761) using a 12 × 12-element photodiode array. The spontaneous optical action potentials are nearly synchronized among the different regions. Figure 3b demonstrates an example of a simultaneous multiple-site optical recording of spontaneous action potentials from 128 adjacent areas of the same preparation as that shown in Fig. 3a. The grid superimposed on the drawing of the heart in Fig. 3b indicates the relative size and position of the photodiode array elements, which correspond to positions of the optical signals measured simultaneously. In this recording, short delays between the signals are clearly recognized, and the action potential first appeared in position J8 (indicated by an arrowhead). Thus, it is likely that, in this heart, the pacemaking area was located at this position, and the excitatory wave spread over the entire region of the heart from this pacemaking area .
Similar measurements were carried out on 7-, 8-, 9-, 10- and 11-somite embryonic hearts . At the 7- and 8-somite stages, the initial optical action potentials were detected from various positions, such as the right and left ventricular and pre-fused atrial portions. They were usually first detected from the left atrial portion at the 9- to 10-somite stages and the left sinoatrial primordium at the 11-somite stage. It is thus concluded that at the 7- to 8-somite stages, the location of the pacemaker was not uniquely determined, and was situated randomly in various positions, such as the left and right ventricular and atrial portions, and, as development proceeds to the 9-somite stage, the pacemaker area becomes localized in the left preatrial portion. Subsequently, the pacemaker area shifts caudally along the atrium to the sinus primordium at the 11-somite stage.
Pacemaking area and excitation spreading
Using multiple-site optical recording, we assessed the size of the pacemaking area with the following procedure . We measured the delays in the feet of the signals between the position of the first firing and other positions in which each signal was detected, and the delays were plotted against the distance from the center of the firing position of the grid. An example of the results is shown in Fig. 3c. This relationship between the delay and the distance indicates that the excitation spreads over the heart radially and at a uniform rate, from the pacemaking site. In this graph, from the slope of the straight line, the conduction velocity was calculated to be about 1.3 mm/s in this heart; we extrapolated the straight line at the distance axis, and as a result, the extrapolated value of approximately 21 μm was obtained (indicated by an arrowhead). This graphical characteristic indicates that, in this heart, the pacemaking area is circular in shape, and that this extrapolated value on the distance axis corresponds to the radius of the circular pacemaking area. Therefore, the pacemaking area in this heart was calculated to be about 1500 μm2. We have named such a procedure the “graphical micro-survey method” of the pacemaking area .
Using this method, we also compared the sizes of the pacemaking areas among 100 preparations during the 8- to 9-somite stages. The mean radius was 25.2 ± 5.3 μm for the 8-somite stage (35 preparations), and 24.9 ± 5.1 μm for the 9-somite stage (65 preparations). At the 8- to 9-somite stages, the size of the pacemaking area was estimated to be 1200–3000 μm2. These results suggest that, although the size of the heart increases gradually throughout the 8- to 9-somite stages, the size of the pacemaking area does not increase .
Here, in the 7- to 9-somite embryonic chick hearts, the myocardial cells nearly form a monolayer, and, since the diameter of the cells is about 5 μm [16–18], the pacemaker in early embryonic chick hearts is probably composed of 60–150 cells. It is thus strongly implied that, not a single cell, but a population of cells having pacemaker ability serves as a rhythm generator in the heart, and that these cells form a strong functional syncytium . As a consequence, it is implied that the functionally organized group consists of pacemaker cells, which are the rhythm generator, and is located dramatically in the right sinus primordium from the atrium, as development proceeds from the 7-somite to the 11-somite stage . This is surely the ontogenetic process of the functional organization of the cardiac pacemaking area.
On the other hand, the spontaneous optical action potentials spread widely at a uniform velocity over the entire area of the primitive tubular heart in the shape of concentric circles from the pacemaking area . Accordingly, it is important to emphasize that no specific conduction systems are yet differentiated in early developmental chick hearts, and that there is electrical coupling among heart cells even in the early stage of cardiogenesis. Also, the slow conduction velocity of the excitation-spread reflects the fact that the electrical coupling among the early embryonic heart cells is very weak, though they are uniformly distributed.
Early embryonic rhythm
As described above, rhythmicity in recurrence of spontaneous action potentials is evident in the pre-fused cardiac primordium at the 6-somite stage, and during the 7-somite to 8-somite stages, the rhythm is labile, but is stably organized by the early period of the 9-somite stage [11, 19], just before the first rhythmical contraction . Corresponding to such a process, the rhythm of the spontaneous action potentials is increased in frequency as development proceeds from the 6-somite to the 9-somite stages [5, 13, 15, 20].
Regional rhythm gradient
When an early embryonic primitive tubular heart was microsurgically separated into right and left or anterior and posterior parts, the spontaneous action potential synchrony between the two separated halves was blocked, although the synchrony and the intrinsic rhythmicity of the spontaneous action potential recurrence remains in each part .
Similar to these experimental results, in the congenital double heart, which was formed by a block of fusion of the right and left cardiac primordia, and developed independently in each half-heart, rhythmical spontaneous action potentials were detected from both the right and left hearts, and differences were detected in their rhythm. Usually, the frequency in the right half-heart was slower than that in left half-heart [22, 23].
Using cultured double- and multiple-heart embryos, we examined regional differences of rhythmicity in more detail. The multiple-heart embryos were produced by whole embryo culture of 1- to 6-somite embryonic hearts in which the tissue of anterior intestinal portal was microsurgically cut into two, or several shreds. In this case, embryos having two or four to six hearts were obtained [24, 25].
In these double- and multiple-heart embryos, regional differences in the rhythmicity were observed. In the fragmented hearts produced in the anterior portion, irregular spontaneous action potentials appeared, and in the fragmented hearts produced in the left posterior position, the most rhythmical spontaneous action potentials were detected in the great majority of the multiple-heart embryos [24, 25].
These results clearly show that there is a regional gradient in the ability to generate electrical rhythmicity in the early phases of cardiogenesis, and suggest that the rhythmicity is highest in the left posterior portion of the early precontractile heart.
As described before, we note that the regional pacemaking priority is very labile in early embryonic hearts at the 7- to 8-somite stage of development and, later, as the development proceeds to the 9-somite stage, it becomes stable and the pacemaking area confined to left pre-atrial tissue as a result of the developmental increase in a regional gradient of pacemaker activity, i.e., special gradient of inherent rhythmicity. Such a process would be summarized as following four steps: (1) maturation of spontaneous active pacemaker cells which have their own inherent rhythmicity in the cardiac primordia; (2) formation of the regional gradient of rhythmicity and emergence of the regional priority of the pacemaking function: at this stage the regional gradient of rhythmicity is very small and the regional priority of pacemaker function is unstable; (3) developmental increase in the regional gradient of rhythmicity results in the functional organization of a single pacemaking area; and (4) localization of the pacemaking area [14, 15, 20, 26].
This developmental sequence appears to provide an example of the scheme of formation of order from instability, which often appears in biological systems, and of functional self-organization both in time and in space. In this case, time corresponds to the development of rhythmicity, and space to the localization of the pacemaking area. This event is shown as a phase diagram, which is named a Kamino diagram (Fig. 4) .
The correlation between development of rhythm generation and development of pacemaker potential is of physiological interest. In the 8- to 9-somite embryonic precontractile primitive tubular hearts, optical signals resembling the pacemaker type action potential with a slow (diastolic) depolarization phase (i.e., optical pacemaker potential) are detected from the pacemaking area and its near neighbors [19, 26]. The slope of the slow depolarization phase is largest in the pacemaking area, and it decreases gradually towards the periphery. In mature cardiac physiology, it is known that the heart rate is determined by the slope of the diastolic depolarization phase (e.g., Weidmann ). In the same way, it is shown that early development of heart rate is coupled with development of the slope of the slow depolarization phase [19, 26, 28, 29].
The maximum slope of the slow depolarization also increases as development proceeds, and is related to the early developmental increase in the heart rate . On the other hand, the regional gradient of the slope of the slow depolarization phase is increased as development proceeds . Those results precisely suggest that formation of a gradient in pacemaker activity results in the morphological and functional organization of the pacemaking area in the early phases of cardiogenesis. Furthermore, in the 7- to 8-somite embryonic primitive heart, the regional lability of the pacemaking area is produced by fluctuations in the tiny slope of the slow depolarization phase.
The events in generation of spontaneous rhythmic electrical activity (i.e., pacemaking function) in early phases of cardiogenesis of chick embryo are summarized in Fig. 5.
In the early developing chick hearts, the temporal origin of generation of morphology and function is not absolutely determined with physical time-scaling of the incubation, because of individual differences among preparations. On the other hand, there is a direct correspondence between the number of pairs of somites and morphological and physiological generations. Thus, the number of pairs of the somite stage is indicated as the time-scale within the biological dimension in the early embryonic process: in this biological time-scaling, there are generally not individual differences in ontogenetic emergence times in the morphological and physiological events .
Accordingly, morphogenesis and functiogenesis are closely coupled. In addition, it is shown that in the 6- to early 8-somite stages, the early development of morphogenesis proceeds symmetrically about the median line of the embryo. In the same fashion, the development of function, e.g., electrical activity, also proceeds symmetrically on the right-to-left axis. In contrast, in the later 8- and 9-somite stages, the pacemaker function is asymmetrical, and becomes dominant on the left side [11, 14, 15, 26], while morphological development of the right side of the tubular heart proceeds at a different rate [30, 31]. In other words, around the 8-somite stage of the chick embryo, the symmetry of the primitive tubular heart is broken. Consequently, the asymmetrical developmental progression of the tubular heart, with “morphogenesis-functiogenesis coupling”, results in the localization of the pacemaking area in the primitive atrial tissue  and further proceeds to reside in the sinoatrial primordium tissue [30, 31]. Eventually, this process organizes the sinoatrial node, forming its complex topology.
Thus, we have described, phenomenologically, “morphogenesis-functiogenesis coupling” in the early embryonic tubular heart. Genesis of the structural factor would stand between “morphogenesis” and “functiogenesis”. This is a future problem in the triple conjugation among “morphogenesis”-“structural-genesis”-“functiogenesis”. For this study, a novel experimental methodology is needed. It is not developed in the present step.
The concept of “functiogenesis” described above in this article consequently proceeded to the ontogenesis of excitation–contraction coupling [5, see also review 32], initial heart beat , and hemodynamics in the tubular heart . Furthermore, this concept has been generalized further in respect to “functiogenesis of the central nervous system”, as methodology and as strategy [35, see also reviews 36, 37]. The concept of “functiogenesis” should give useful tips for research on systems physiology.
Hamburger and Hamilton
Cohen LB, Salzberg BM (1978) Optical measurement of membrane potential. Rev Physiol Biochem Pharmacol 83:35–88
Kamino K (1991) Optical approaches to ontogeny of electrical activity and related functional organization during early heart development. Physiol Rev 71:53–91
Kamino K (2015) Personal recollections: regarding the pioneer days of optical recording of membrane potential using voltage-sensitive dyes. Neurophotonics 2:021002-1-9
Kamino K (1990) Optical studies of early developing cardiac and neural activities using voltage-sensitive dyes. Jpn J Physiol 40:443–461
Hirota A, Kamino K, Komuro H, Sakai T, Yada T (1985) Optical studies of excitation-contraction coupling in the early embryonic chick heart. J Physiol Lond 366:89–106
Patten BM (1949) Initiation and early changes in the character of the heart beat in vertebrate embryos. Physiol Rev 29:31–47
Sakai T, Kamino K (2001) Optical mapping approaches to cardiac electrophysiological functions. Jpn J Physiol 51:1–18
Thomson W (1910) The work of Aristotle. Oxford, Clarendon
Manasek FJ (1980) Organization, interactions, and environment of heart cell during myocardial ontogeny. In: Berne RM, Sperelakis N, Geiner SR (eds) Handbook of physiology sect 2, The cardiovascular system vol 1. The heart. Williams and Wikins, Baltimore, pp 29–42
Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92
Hirota A, Kamino K, Komuro H, Sakai T (1987) Mapping of early development of electrical activity in the embryonic chick heart using multiple-site optical recording. J Physiol Lond 383:711–728
Fujii S, Hirota A, Kamino K (1980) Optical signals from early embryonic chick heart stained with potential sensitive dyes: evidence for electrical activity. J Physiol Lond 304:503–518
Fujii S, Hirota A, Kamino K (1981) Optical recording of development of electrical activity in embryonic chick heart during early phases of cardiogenesis. J Physiol Lond 311:147–160
Kamino K, Komuro H, Sakai T, Hirota A (1988) Functional pacemaking area in the early embryonic chick heart assessed by simultaneous multiple-site optical recording of spontaneous action potentials. J Gen Physiol 91:573–591
Kamino K, Hirota A, Fujii S (1981) Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye. Nat Lond 290:595–597
Manasek FJ (1968) Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo. J Morphol 125:329–366
Ojeda JL, Hurle JM (1981) Establishment of the tubular heart role of cell death. In: Pexieder T (ed) Perspectives in cardiovascular research. Mechanism of cardiac morphogenesis and teratogenesis, vol 5. Raven, New York, pp 101–113
Hiruma T, Hirakow R (1985) An ultrastructual topographical study on myofibrillogenesis in the heart of chick embryo during pulsation onset period. Anat Embryol 172:325–329
Fujii S, Hirota A, Kamino K (1981) Optical indications of pace-maker potential and rhythm generation in early embryonic chick heart. J Physiol Lond 312:253–263
Sakai T, Hirota A, Fujii S, Kamino K (1983) Flexibility of regional pacemaking priority in early embryonic heart monitored by simultaneous optical recording of action potentials from multiple sites. Jpn J Physiol 33:337–350
Fujii S, Hirota A, Kamino K (1981) Action potential synchrony in embryonic precontractile chick heart: optical monitoring with potentiometric dyes. J Physiol Lond 319:529–541
Fujii S, Sakai T, Hirota A, Kamino K (1983) Optical recording of the pacemaking activity of a congenital double-heart in an early chick embryo. Dev Growth Differ 25:193–200
Hirota A, Fujii S, Sakai T, Kamino K (1984) Optical recording of spontaneous action potentials from the congenital chick embryonic pre-contractile double-heart. Proc Jpn Acad Ser B 60:281–284
Yada T, Sakai T, Komuro H, Hirota A, Kamino K (1985) Development of electrical rhythmic activity in early embryonic cultured chick double-heart monitored optically with a voltage-sensitive dye. Dev Biol 110:455–466
Sakai T, Yada T, Hirota A, Komuro H, Kamino K (1998) A regional gradient of cardiac intrinsic rhythmicity depicted in embryonic cultured multiple hearts. Pflüger Arch-Eur J Physiol 437:61–69
Kamino K, Komuro H, Sakai T (1988) Regional gradient of pacemaker activity in the early embryonic chick heart monitored by multisite optical recording. J Physiol Lond 402:301–314
Weidmann S (1970) Electrical constants of trabecular muscle from mammalian heart. J Physiol Lond 210:1041–1054
Hirota A, Fujii S, Sakai T, Kamino K (1983) Temperature dependence of spontaneous electrical activity in early embryonic heart monitored optically with a potential-sensitive dye. Jpn J Physiol 33:85–100
Sakai T, Fujii S, Hirota A, Kamino K (1983) Optical evidence for calcium-action potentials in early embryonic precontractile chick heart using a potential-sensitive dye. J Membr Biol 72:205–212
Stalsberg H, DeHaan RL (1969) Precardiac areas and formation of the tubular heart in the chick embryo. Dev Biol 19:128–159
Patten BM (1971) Early embryology of the chick. McGraw-Hill, New York, pp 113–138
Kamino K, Hirota A, Komuro H (1989) Optical indication of electrical activity and excitation-contraction coupling in the early embryonic heart. Adv Biophys 25:45–93
Sakai T, Hirota A, Kamino K (1996) Video-imaging assessment of initial beating patterns of the early embryonic chick heart. Jpn J Physiol 46:465–472
Sakai T (2003) Video-imaging visualization of blood flow dynamics in early chick embryo. Jpn J Physiol 53:385–388
Kamino K, Katoh Y, Komuro H, Sato K (1989) Multiple-site optical monitoring of neural activity evoked by vagus nerve stimulation in the embryonic chick brainstem. J Physiol Lond 409:263–283
Momose-Sato Y, Sato K, Kamino K (2001) Optical approaches to embryonic development of neural functions in the brainstem. Prog Neurobiol 63:151–197
Momose-Sato Y, Sato K, Kamino K (2015) Monitoring population membrane potential signals during development of the vertebrate nervous system. In: Carpenter M, Zecevic D, Bernus O (eds) Membrane potential imaging in the nervous system and heart. Springer, New York, pp 213–242
The experiments on the embryonic hearts were carried out in collaboration with many members in the laboratory in Tokyo Medical and Dental University. We are grateful to Brian Salzberg for his improvement of the English version.
Conflict of interest
The authors declare that there are no conflicts of interest.
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Sakai, T., Kamino, K. Functiogenesis of cardiac pacemaker activity. J Physiol Sci 66, 293–301 (2016). https://doi.org/10.1007/s12576-015-0431-2
- Voltage-sensitive dye
- Early embryonic chick heart
- Cardiac pacemaker potential
- Pacemaking area
- Optical technique