Download The early development of the atrioventricular node and bundle of

Development 102, 623-637 (1988)
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The early development of the atrioventricular node and bundle of His in
the embryonic chick heart. An electrophysiological and morphological
study
CARLOS ARGUELLO 1 , JESUS ALANIS2 and BLANCA VALENZUELA1
1
Secci6n de Patologia Experimental, Centro de Investigation y de Estudios Avanzados del Institute) Politecnico National, Apartado
Postal 14-740. Cod. Postal 07000, Mexico, D.F.
2
Unidad Experimental de Electrofisiologia Arturo Rosenblueth, Apartado Postal No. 15. Jiutepec, Morelos, 62550, Mexico
Summary
The development of the atrioventricular node and
bundle of His of embryonic chick hearts was studied
by electrophysiological and morphological techniques.
The dorsal wall of the AV canal and the interatrial
septum were explored to determine if they contribute
to the formation of the AV node and bundle of His.
The resting membrane and action potentials of the
interatrial septum cells were systematically analysed
and found to undergo progressive differentiation with
development. The earliest identification of the AV
node and upper bundle of His group of cells was
achieved at 5^-6 days of development by the electrical
recording of their corresponding characteristic action
potentials, from a circumscribed area located in the
lowest and dorsal segment of the interatrial septum.
The morphological and anatomical characterization of
the cells was made following electrical recording and
labelling with charcoal particles. The earlier AV node
and bundle of His responses had similar characteristics to those of the adult heart. It is concluded that
the AV node and upper bundle of His cells derive from
the low interatrial septum. The possibility that AV
canal cells contribute to this event was discarded. The
functional relationship of the AV node and bundle of
His with other cardiac tissues during the early development of the heart is discussed.
Introduction
mammals (Davis, 1930; Yousuf, 1965) but its origin
has not been clarified. Patten (1956) was of the
opinion that the AV node in the chicken embryo
starts its development as a counterpart of the left
sinus horn and Kim & Yasuda (1979) concluded that
it derived from the atrioventricular ring. With regard
to the origin of the bundle of His in mammalian
embryos, even more controversy exists in the literature, (a) Some investigators (Viragh & Challice,
1911 a,b, 1982) proposed that the bundle of His
develops from the interventricular septum, (b) On
the other hand, Retzer (1908), Tandler (1912),
Shaner (1929) and Walls (1947), considered that the
bundle of His is formed by cells from the AV node,
(c) Truex et al. (1978) and Marino et al. (1979),
suggested that cells from the dorsal wall of the AV
Different explanations have been advanced for the
embryological origin of the atrioventricular node (AV
node) and bundle of His in mammals. The AV node
was suggested to develop from the dorsal wall of the
atrioventricular canal (AV canal) in embryonic hearts
of mouse, rabbit, calf and man (Sanabria, 1936;
Walls, 1947; Viragh & Challice, I911a,b, 1982). Other
investigators considered the AV node to rise from the
atrioventricular ring (AV ring) in man (Benninghoff,
1923; Wenink, 1976; Anderson et al. 1976). More
recently, it was postulated that the AV node initiates
from two primordia located in the dorsal wall of the
atrium of the ferret heart (Marino et al. 1979). The
AV node of birds is in many aspects similar to that of
Key words: embryonic chick heart, action potential,
interatrial septum, cellular differentiation,
atrioventricular node, bundle of His, conductive system
origin.
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C. Arguello, J. Alanis and B. Valenzuela
canal contribute to the formation of the bundle, while
(d) Anderson et al. (1976, 1977) and Wenink (1976)
have proposed that the bundle of His is derived from
the bulboventricular ring. The above studies were
based solely on morphological features and, at the
early stages of heart formation, cells do not show
enough differences between the various regions to
permit their accurate distinction. It would then be
useful to apply a more precise and complementary
identification procedure, such as membrane action
potential recording, to characterize the different
groups of cardiac cells. In previous work (Arguello et
al. 1986), we used this procedure and found that even
at early stages of heart formation (li-5 days) there
exist regional differences in some electrophysiological properties. In particular, the cells from the AV
canal, besides being responsible for the atrioventricular delay, generated slow and long-lasting action
potentials. These characteristics make the AV canal a
probable precursor of the AV node. However, as we
did not follow the further development of the AV
canal region until the AV node appeared, we were
not able to ascertain whether or not these cells
contributed to the formation of AV node and bundle
of His. On the other hand, since in the adult heart
both structures are located in the low and dorsal
segment of the interatrial septum (IAS), we could
consider this region as another possible source of cells
for the AV node and bundle of His. The aim of the
present investigation was to determine whether the
AV node and bundle of His develop from either the
AV canal, the IAS or from another tissue. To clarify
this problem, we have studied the morphological and
electrophysiological changes taking place in the IAS
from its initial development until the characteristic
action potentials of the AV node and bundle of His
appeared. A similar survey was made on the dorsal
wall of the AV canal and neighbouring tissues at
different stages of heart development.
Materials and methods
Isolated and perfused chick embryonic hearts were obtained from fertilized Rhode Island eggs incubated at 37 °C
and appropriate humidity. To follow the development of
AV canal and IAS, chick embryos of 60-168 h were used.
The endocardial surface of the IAS was exposed by an
incision made on the left atrial wall in the younger embryos
(2i-4i days) and on the right side in the older ones (5-7
days). The small size of the right atrial wall of the younger
limited the surgical procedure. The dorsal wall of the AV
canal was explored either from the external surface or from
the interior of the heart. The hearts were placed in a
constant temperature chamber (37°C) containing a semisolid culture medium plate (1-5 % Agar-Agar and 10 % egg
albumin). To impale cells from different segments of the
exposed IAS, spontaneously beating hearts were affixed
with fine glass needles placed in the apical region of the
ventricle and the truncus. The hearts were perfused with a
modified Tyrode's solution, pH7-2 (in ITIM: NaCl 140; KC1
5-6; CaCl2 1-37; MgCl2 0-5; NaHCO3 10; NaH2PO 1-8;
dextrose 5-5) gassed with a mixture of 95% O2 and 5 %
CO2. Conventional electrophysiological and electron microscopy techniques were applied as previously described
(Arguello et al. 1986). For intracellular recording, glass
micropipettes filled with 3M-KC1 and a resistance of
40-100 Mil were used and connected to DC capacitancecompensated preamplifiers (Grass P18; AM-system 1600).
The records were displayed on a two-beam oscilloscope
(Tektronix 5440) and photographed with a Grass C4
camera.
In order to detect on serial histological sections the
precise location of cells from which the AV node and
bundle of His action potentials were recorded, small
charcoal particles were placed on the corresponding sites.
Subsequently, the specimens were fixed with glutaraldehyde and prepared for light and electron microscopy study.
In other experiments, hearts of the same age as those
explored electrophysiologically were used for light, transmission and scanning electron microscopy. For histochemical studies of the cell surface and extracellular matrix
components of the AV node and neighbouring tissues, the
hearts were fixed with 2-5 % glutaraldehyde in cacodylate
buffer 0-1 M, pH7-2, containing either 1 % of tannic acid
(Singley & Solursh, 1980), lmgml" 1 of ruthenium red
(Luft, 1966), or 1 % of alcian blue 8G-X (Spicer & Henson,
1967), to retain the glycosaminoglycans of the extracellular
matrix.
Results
IAS development
At the 3rd day of incubation, the IAS is seen as an
incipient invagination of myocardial atrial cells.
These cells are surrounded by a small local cushion
formed by mesenchymal cells embedded in an extracellular matrix (Fig. 1A). At 3 | days, the IAS has
grown further down and fused with the dorsal endocardial cushion of the AV canal (Fig. IB). This
growing process persisted during days 4-5 until the
distal end of the septum almost reached the AV canal
level (Fig. 1C,D). On day 4, in the dorsal and
superior portion of the IAS, small foramina form
constituting the ostium secundum (Fig. 1C,D).
Although the IAS cells develop from the atrial wall,
they differ from the latter in possessing a higher
content of glycogen and an irregular arrangement of
myofibrils, as previously described by Morse (1981).
When the electrical activity of the exposed IAS cells
was recorded, it was found that both the resting
membrane potential (r.m.p.) and action potential
amplitude (a.p.a.) progressively increased during
development, the membrane potential values of the
IAS cells being lower than those of the atrial wall cells
at a given stage of development (Fig. IE). For
Development of the atrioventricular node and bundle of His
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Fig. 1. Histological and electrophysiological development of the IAS of embryonic hearts. (A-D) Perpendicular
sections of IAS at 3, 3i, 4 and 5 days, respectively, showing the continuous downward growth of the septum and its
fusion with the dorsal endocardial cushion (dec). At, atrial wall; AVC, atrioventricular canal, os, ostium secundum.
Bar, 100^m. (E) The upper traces are the transmembrane action potentials of the atrial wall (At) at different stages of
development (3-5 days) and the lower correspond to the intracellular responses of the interatrial septum (MS). Notice
the different shape of the responses of the atrial wall as compared to those of the septum at each stage of development.
Calibration 50 mV, 50 msec.
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C. Argiiello, J. Alanis and B. Valenzuela
Fig. 2. Scanning electron micrograph of the
interior of the heart at 5 days showing the
interatrial septum, ostium secumdum (os) and
dorsal and ventral cushions (dec, vec).
Electrical activity of the atrial wall (At), the
upper (WAS), lower (LIAS) segments of the
interatrial septum and the atrioventricular
canal (AVC) at 5 days. No action potentials of
the slow-rising type were recorded either from
the upper or from the lower and dorsal
interatrial septum. Bar 100jum. Calibration
50 mV, 50 msec.
instance, on the 3rd day the r.m.p. and a.p.a. of IAS
cells were — 43 mV and 51 mV respectively, while
those of the atrial wall were of — 60 mV and 75 mV.
On day 5, the IAS cell membrane potential values
were —53 mV and 75 mV as compared to those of the
atrium that reached — 62 mV and 83 mV. In addition,
the rising phase of the action potentials from atrial
wall cells was faster and the repolarization was slower
than those of the IAS (Fig. IE). The only slow type of
a.p.a. recorded from 5-day embryonic hearts was
obtained from the AV canal myocardial wall (Fig. 2).
This action potential appeared earlier (45 h), before
the septum had even initiated its development. It was
previously demonstrated (Arguello et al. 1986) that
the AV delay at early stages is brought about by the
AV canal. Septation of the atria is complete by the
end of day 5 due to interatrial septum development
and fusion with the dorsal and ventral endocardial
cushions (Fig. 2A). At this period, the a.p.a.s from
different segments of the upper interatrial septum
(UIAS) and lower interatrial septum (LIAS) were
recorded. These responses have a rapid upstroke and
a repolarization phase without plateau (Fig. 2).
Although various segments of IAS had somewhat
differently shaped a.p.a.s slow-rising and long-lasting
responses were never observed.
Development of the AV node and bundle of His
At more advanced stages of heart development (5i-6
days), the upper and medium IAS cells generated
fast-rising APAs (Fig. 3, UIAS, MIAS). When a
micropipette was gradually displaced from the endocardial side of MIAS downwards by 35 jum steps,
smaller and slower action potentials were recorded
from the low and dorsal portion of the IAS (Fig. 3,
LIAS; Table 1). Impalements lower than the precedent elicited a.p.a.s with even slower rising phase.
This a.p.a. will be referred to as the upper atrioventricular node (UAVN) depicted in Fig. 3. In close
proximity (20-40 fim) to the last point and going
downwards, distinctive a.p.a.s were recorded. These
responses have slow S-shaped rising and decay
phases, their duration being the longest of all the
cardiac a.p.a.s recorded from the 6-day-old embryos
(Fig. 3, AVN). This latter type of action potential
closely resembles that of the AV node of adult
mammalian heart and it was only recorded from a
circumscribed area with an approximate diameter of
50-70nm, located in the same anatomical region as
the AV node of the adult heart. It can be considered,
therefore, the same as the earlier electrical activity of
the AV node group of cells (Fig. 3, AVN). When the
exploring microelectrode was displaced ventrally
from this area by steps of a few microns, one observes
a response with a larger amplitude, a slow foot with a
fast-rising depolarization, followed by a slight plateau. This a.p.a. was recorded within a region of
about 100 fxm length and 50 ;tim wide as illustrated in
Fig. 3 (BH). These action potentials would represent
the earlier electrical activity of the bundle of His cells,
since they resemble those reported for the adult heart
(Hoffman & Cranefield, 1960; Alanis & Benitez,
Development of the atrioventricular node and bundle of His
1964, 1975; Benitez et al. 1973) and were generated
by cells in close continuity with those of the AV
node. When the micropipette was displaced further
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down from the circumscribed regions of the AV
node and bundle of His, no electrical activity was
recorded. This was to be expected since around
Fig. 3. The early electrical activity of the AV node and bundle of His. Scanning electron micrograph of the interior of
the 6 days embryonic heart showing the sites from which intracellular records were taken. At, atrial wall; UIAS, upper
interatrial septum; MIAS, medium interatrial septum; LIAS, low interatrial septum; UAVN, upper atrioventricular
node; AVN, atrioventricular node; BH, bundle of His; fVS, interventricular septum; V, ventricle and AVC,
atrioventricular canal. Note that in the lower and dorsal segment of the septum the APs start to have a slow-rising phase
(LIAS and UAVN). Below this segment there is a circumscribed area (broken lines) from where the early AV node and
bundle of His action potentials were recorded. Bar 200^m. Calibration 50mV, 50msec.
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C. Arguello, J. Alanis and B. Valenzuela
Fig. 4. Histological section
parallel to the interatrial
septum of the heart of 6
days, showing that the AV
node (AVN) and bundle of
His (BH) group of cells are
separated from the dorsal
AV canal wall (AVC) by a
tissue formed exclusively by
mesenchymal cells (enclosed
area, me) and extracellular
matrix components in which
propagated responses are
not recorded. Their
corresponding action
potentials are depicted in
the upper panel. Note that
the AV canal and AVN
action potentials being of
the slow type are easily
distinguished from each
other. Bar, 100urn.
Calibration 50 mV, 50 msec.
Table 1.
Tissue
(6-7 days)
r.m.p.
(mv)
Auricle
UIAS
LIAS
Upper AV node
AV node
Bundle of His
AV canal
-59-0
-55-0
-57-0
-44-0
-38-0
-50-0
-67-0
-60-0
-67-0
IVS
Ventricle
a.p.a.
(mv)
(14)
(20)
(20)
(8)
(6)
(13)
(18)
80-0
71-0
70-0
53-0
55-0
70-0
(14)
(20)
(20)
650
(8)
(6)
(13)
(7)
75-0
(8)
(?)
(15)
930
(15)
56-0
55-0
5-0
1-6
1-0
5-1
3-0
42-0
42-0
(4)
(6)
(6)
(5)
(4)
(4)
(6)
(4)
(8)
Comparative average values of resting membrane (r.m.p.),
action potential (a.p.a.) and rate of rise (Vsec"1) of different
cardiac cells. Note that the lowest values correspond to the AV
node cells, (n) number of measurements.
these structures only mesenchymal cells and the
extracellular matrix components of the cushions were
present.
The analysis of the electrical membrane characteristics of the different cardiac tissues revealed that the
cells from the LIAS have a lower r.m.p. and slower
rising phase compared to those of the atrium, the
ventricle, the UIAS and interventricular septa (IVS)
(Fig. 3); the AV node response having the slowest
rising phase (see Table 1 and Figs 3, 4).
If the dorsal AV canal cells were the precursor of
the AV node one would expect that they would be
histologically and electrophysiologically connected at
some stage of their development. To determine if the
dorsal AV canal electrical activity propagates to and
excites the AV node group of cells, we performed the
following experiment. After exposing the inner side
of the right auricle of spontaneously beating hearts
(6th day of development) successive impalements
were made in the corresponding regions. When a
micropipette was placed in the dorsal AV canal wall,
its typical slow action potential was recorded (Fig. 4,
AVC). Displacements of the microelectrode by 30,um
steps from the AV canal, towards the AV node
region, showed that no propagated electrical activity
was recorded within an area of about 200 jum. Only
when the microelectrode reached the group of AV
node and bundle of His was it possible to observe the
APA characteristic of these cells (Fig. 4, AVN, BH).
Histological study of the region where no propagated
electrical activity was recorded, revealed that it was
mainly formed by mesenchymal cells from the dorsal
cushion, and lacked myocardial cells (Fig. 4).
Morphological features of the early developing A V
node and bundle of His
Once the AV node and bundle of His were identified
and localized by electrophysiological means, small
particles of charcoal (25-40 ^m) were placed on the
endocardial surface as near as possible to the region
of impalement to facilitate their anatomical and
histological characterization. In order to know the
spatial relationship of the AV node and bundle of His
with other cardiac tissues, 6-day hearts were sectioned in three perpendicular planes (Fig. 5A-D).
Development of the atrioventricular node and bundle of His
Plane B, parallel to the IAS, showed that in its dorsal
and lowest segment there was a distinct group of
myocardial cells with larger intercellular spaces
(AVN, BH) than those of the septum (Figs 4, 5,
LIAS). The AV node myocardial group has an
approximate length of 150/im and is formed by five to
seven irregular-arranged rows of cells distributed
near the endocardial surface. The group is in continuity in its upper end with the IAS cells and in the
lower part with those of the bundle of His (Figs 4,5).
The AV node and bundle of His cells, are immersed
in the upper part of the dorsal cushion. Plane C,
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perpendicular to IAS, shows that the AV node region
is in continuity with the IAS, and localized subendocardially and distant from the IVS (Fig. 5C). Plane D
perpendicular to the preceding planes shows that the
AV node cells are included in the dorsal endocardial
cushion in proximity to the dorsal atrial wall cells but
not connected to them (Fig. 5D).
The ultrastructural study of the AV node and
bundle of His cells showed that they have large
intercellular spaces (Fig. 6B), contain scanty and
poorly organized myofibrils and a great amount of
glycogen granules in the cytoplasm (Fig. 6C). In
Fig. 5. Spatial relationships of the AV node (AVN) and bundle of His (BH) group of cells with other cardiac tissues of
6-day-old embryonic heart. (A) Three different perpendicular planes of serial sectioning (B-D). (B) The AV node is
formed by a group of cells that are in continuity with those of the low and dorsal interatrial septum (LIAS) and are
surrounded by mesenchymal cells of the dorsal endocardial cushion (dec). The bundle of His cells forms part of the
lower and ventral part of the AV node group of cells. In planes C and D, the AV node is located subendocardially to
the right side of the interatrial septum, ec, endocardial cushion; vec, ventral endocardial cushion; ivs, interventricular
septum; At, atrial wall. Bars (A) 100/im; (B-D) 100 fim.
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C, Arguello, J. Alanis and B. Valenzuela
Fig. 6. Some ultrastructural characteristics of the AV node cells from a preparation in which the site of their
impalement (x) was labelled with charcoal particles (Arrows in the histological section of A). (B,C) AV node group of
cells with few myofibrils (mf) and a great amount of glycogen granules (g) separated by large intercellular spaces (ics)
and extracellular matrix components (emc) which are closely associated to the cell membrane (arrows). Bars (A) 45,um;
(B) 5f«n; (C)
preparations in which ruthenium red was used to
retain and visualize the extracellular matrix components, the complex network of collagen fibrils was
observed to surround the AV node and the surfaces of
the cells in the bundle of His (Figs 6C, 7A,C, 9C).
Their membranes are covered by granular matrix
Development of the atrioventricular node and bundle of His
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*
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Fig. 7. Extracellular matrix components associated to the AV node cells (A,C-E) and to the mesenchymal cells of the
dorsal cushion (em in B). (A) Scanning electron micrograph of the AV node region (head arrows) showing a complex
network of collagen fibrils surrounding the cells. (C) Enlarged area of A, showing collagen fibrils (co) attached to two
AV node cells (AVN). (D,E) Transmission electron micrograph that illustrates collagen fibrils (co) and granular matrix
components close to the cells surface (arrows). Note in B that the mesenchymal cells have more abundant matrix
components (em) than the AV node cells depicted in A. Bars (A) lOjum; (B) 5/itn; (C) 2;tm; (D,E) 500nm.
materials (Figs 6C, 7E, 8A, 9C) that are almost
absent in the IAS cells. The extracellular matrix of
endocardial cushion cells differ from that of the AV
node and bundle of His in the higher content of thin
fibrillar and granular components and fewer collagen
fibres (Fig. 7B). The AV node cells mainly associate
with each other by small desmosomes and the rest of
the opposing membrane is separated by a regular
space of about 20 nm. The existence of gap junctions
among the AV node cells was intensely surveyed but
only undefined focal junctions were seen (Fig. 8C).
By contrast, cells from the low portion of the IAS
have abundant and large gap junctions (Fig. 8B,D).
The cells from the bundle of His have similar ultrastructural features to those of the AV node
(Fig. 9B,C). The only noticeable difference is that the
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C. Argiiello, J. Alanis and B. Valenzuela
mti
Fig. 8. Transmission electron
micrographs of (A,C)
atrioventricular node cells
(AVN) and (B,D) the lower
segment of interatrial septum
(IAS) of 6-day embryonic
hearts. Note that the AV
node cells are loosely
associated and have focal
undefined junctions (arrow,
C). The interatrial cells are
closely associated (B) and
have large gap junctions
(arrow D). Bars (A,B) 1 fim;
(C,D) 100 nm.
bundle of His cells are more slender and seem to be
aligned and associated with each other by their ends
(Fig. 9A.C).
time at which they get their peculiar electrophysiological characteristics during the process of heart
development, were unknown.
Discussion
The embryological origin of the AV node
For the experimental analysis of the present data, the
AV canal and the IAS regions were considered as the
precursors of the AV node. Our experimental results
favour the hypothesis that the IAS cells give rise to
the AV node as shown by the following facts. (1) The
slow intracellular action potentials with the characteristics of the AV node responses were generated by
cells from a circumscribed area located in the lower
In the adult mammalian heart, the propagation of
impulses travelling from the auricle to the ventricle is
known to be delayed by the AV node cells that
generate small and slow action potentials (Hoffman et
al. 1958; Alanis & Benitez, 1964a) and have scanty
cell-cell junctions (Marino et al. 1979). However, the
embryonic origin of the AV node cells, as well as the
Development of the atrioventricular node and bundle of His
X
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and dorsal segment of the IAS (Fig. 3, AVN). This
occurred between the 54 and 6 days of heart development at the time the cells of the septum penetrated
deeply into the dorsal cushion (Fig. 5B-D). No
action potentials of the slow type were recorded from
the IAS before the 5\ days of incubation. (2) The AV
node group of cells are activated exclusively by
Fig. 9. Some morphological
features of the bundle of His
cells previously identified by
their action potentials. (A)
Histological section parallel
to the IAS illustrating that
the cells are aligned and
associated with each other
mainly by their ends. (B,C)
Transmission electron
micrographs of bundle of His
cells characterized by the
small content of myofibrils
and a few desmosomes
(arrows). The cell membrane
has abundant granular matrix
components (arrows, C). Bars
(A)22^m; (B) 500nm; (C)
1/im.
impulses propagated downwards through the IAS, as
shown by the systematic cell impalements made every
20-30nm along the IAS (Fig. 3). (3) When the AV
node cells appeared for the first time (51-6 days),
they were in histological continuity with those of the
IAS, since there exists cell-cell contacts between
both groups of cells but not with the cells of the AV
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C. ArgiXello, J. Alanis and B. Valenzuela
Fig. 10. Morphological and electrophysiological features of the ostium secundum from a 7-day embryonic heart. (A)
Longitudinal histological section of a trabeculae from the ostium secundum (05) formed by two or three rows of
myocardial cells covered by the endothelium. (B) Scanning electron micrograph of the ostium secundum (as) and
neighbouring regions of the interatrial septum (IAS) from where intracellular records were taken (arrows). (C)
Superimposed transmembrane action potentials from two different cells of a trabeculae of the os. Note their faster
repolarization phase as compared to that of the IAS neighbouring surrounding cells. The corresponding atrial wall
response (At) with the long-lasting repolarization phase is shown in C. Bars (A) 100fim; (B) 100^m. Calibration 50mV,
20 msec.
canal. (4) The ultrastructural characteristics of the
AV node cells are similar to those of the IAS, the
main difference being the scarcity of gap junctions
and the existence of large intercellular spaces filled
with abundant extracellular matrix components. (5)
We have described, in the intercalated region between the AV canal and the AV node, mesenchymal
and abundant extracellular matrix components. Evidence has accumulated pointing to the possibility of
conduction between muscle cells brought about by
interposed fibroblasts (Opthof et al. 1986). If some
fibroblasts are present in the intercalated region we
have not recorded propagated electrical activity.
Therefore the AV node and the AV canal cannot
excite each other through this region.
All the studies previously made to determine the
precursors of the AV node were based solely on
histological features (see Introduction) and have
serious inherent limitations. In this respect, our
extensive electrophysiological survey of the different
regions of the early developing heart, together with
their morphological identification, has provided a
solid base for the conclusion that the AV node
develops from the cells of the LIAS. It is our belief
that, in order to define the embryological origin of a
given group of cardiac cells, it is indispensable to
identify them first through the shape and magnitude
of their action potentials. This identification should
be complemented by a morphological analysis. The
paper in which Lieberman & Paes de Carvalho (1965)
studied the atrioventricular conduction would partially fulfil the latter requirements. They have considered the AV node to be 'a remnant of the embryonic AV ring cells'. This interpretation was based on
an extrapolation that postulated that the AV ring cells
from the chicken heart generate 'a slowly rising,
rounded action potential of reduced magnitude' with
striking resemblance to that of the adult rabbit AV
node. They were comparing two different cardiac
regions, namely, the AV ring and the AV node, that,
in addition, were from different species, the rabbit
and the chicken. These authors were recording the
slow-rising a.p.a. from the incipient lateral atrioventricular valve (AV ring) of 7- to 20-day-old embryos,
while the AV node cells, located in the low dorsal
interatrial septum, were already grouped and functioning at 51-6 days, as shown by our present results.
The development of the IAS
The most outstanding electrophysiological characteristic of the developing IAS cells is that they have the
capability to differentiate in such a manner that a
great variety of action potential shapes are generated
according to the region or to the age of the heart, as
illustrated in Figs 1-3. This was particularly evident
in the distal end of the IAS at 54-6 days when the
Development of the atrioventricular node and bundle of His
635
lowest IAS. This process probably occurs within a
short period of time, about 10-12 h, since on the 5th
day it was not yet possible to record any action
potential of the slow type, while, by 5|-6 days, a
typical AV node action potential appeared. Another
example of regional membrane differentiation of IAS
cells was the action potential of the ostium secundum.
As illustrated in Fig. 10, the a.p.a.s from the trabeculae have a faster repolarization phase, without
plateau, than the responses of the neighbouring IAS
cells from which the trabeculae undoubtedly originated.
B
7-10 days
Fig. 11. Diagrammatic representation of possible
conductive pathways at different developmental stages of
embryonic chick hearts. (A) In 6-day hearts, the ventricle
is activated through the AV canal (AVC), since the
recently formed AV node (AVN) and bundle of His (BH)
are not yet connected to the ventricular fibres. (B) In
older embryos (9-10 days), when the bundle branches
have completely developed and the interventricular
septum (FVS) have been formed, the impulses traversing
the AV node are able to excite the ventricle, while those
travelling through the AV canal are delayed or blocked,
probably due to the enlargement of intercellular spaces
and increment of extracellular matrix components. IAS,
interatrial septum; ec, endocardial cushions; V, ventricle.
action potentials recorded from this area were exclusively of the slow type (Fig. 3, LIAS, UAVN, AVN).
The AV node action potentials would be a result of
the membrane cell differentiation taking place in the
The development of the bundle of His
The earlier electrophysiological identification of the
bundle of His was made simultaneously with that of
the cells of the AV node between 5i and 6 days of
development. The bundle cells were localized ventral
to the AV node as a continuation of its cellular
arrangement. Since there are no clear morphological
differences between these two groups of cells, one has
to rely solely on the shape of their action potentials,
in order to distinguish them (Fig. 3). The bundle of
His has not fully developed at this stage, since the
right and left branches are still absent. The bundle of
His and the AV node probably originate from the
same source of cells, that is, the lowest segment of the
IAS. The ridge of the IVS has been suggested (Viragh
& Challice, \911a,b, 1982) as a possible source of cells
for the formation of the bundle of His. This seems not
to be the case, however, since the IVS at this age
(5^-6 days) has not yet reached either the AV canal
cushions or the LIAS where the AV node and upper
bundle of His are located. In addition, the a.p.a.s
recorded from the upper IVS have a fast upstroke and
a peculiar triangular shape (Fig. 3), different to those
generated by the bundle of His cells.
With regard to the origin of the branches of the
bundle of His, it remains questionable whether they
are formed by the continuous growing of the bundle
of His group of cells or by the cellular differentiation
of the IVS occurring at more advanced stages of
development.
Functional organization of conductive pathways during
the embryological development of the heart
The present information allows us to envisage the
functions of the early embryonic AV node and bundle
of His as follows. At 6 days of heart development, the
ventricle is activated by impulses travelling through
the AV canal region, since the AV node and bundle of
His are not connected to the ventricular fibres, due to
the absence of the bundle branches, as shown in the
diagram of Fig. 11A. At further stages of development (9-10 days), impulses that travelled along the
AV canal would propagate progressively slower due
636
C. Argiiello, J. Alanis and B. Valenzuela
to the enlargement of the intercellular spaces and the
increment of extracellular matrix components among
the cells (Arrechedera etal. 1984). At the same time,
complete septation of the ventricle is accomplished
and probably the branches of the bundle of His are
then formed and connected to the ventricle. When
the ventricle is excited through the AV node and the
bundle of His system, a competence would be established between this system and that of the AV canal.
In other words, a situation would arise in which the
predominant pathway that activates the ventricular
fibres would be the AV node-bundle of His system,
leaving the AV canal cells ineffective for conduction
due to their electrical isolation from the ventricle by
the developing annulus fibrosis (Fig. 11B). If the
normal development of the atrioventricular conduction is complete at the early stages, all kind of rhythm
disturbances might be explained like Wolff-Parkinson-White syndrome.
We would like to express our gratitude to The Grass
Instrument Co. Quincy, MA, USA, for donation of equipment, to the group Alanis-Gonzalez, Mexico, for their
economical support and to Dr Lynn T. Landmesser from
the University of Connecticut, USA for her critical reading
of the manuscript.
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electrical activity of bundle of His. The fast and slow
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{Accepted 13 October 1987)