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Experimental heart-block in the chick embryo.

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Experimental Heart-block in the Chick Embryo '
GEORGE H. PAFF, ROBERT J. BOUCEK AND HAROLD S. KLOPFENSTEIN
Departments of Anatomy and Medicine, University of Miami
School of Medicine, Coral Gables, Florida
ABSTRACT
While studying the electrocardiograms of developing hearts it was
noted that an adult type ECG could not be obtained in a n embryonic heart which
could not be blocked with digitalis. The need to know more about heart-block was
obvious. Intact embryos of 36 to 120 hours were floated on to glass plates and treated
with digoxin-Tyrode solution. Before 36 & hours no dissociation of beat could be
produced. The heart just stopped. At 40 -C hours a conal block followed by a midventricular block appeared. At 42 % hours conal and midventricular blocks were followed by the first appearance of AV block. It was intermittent. At 47 -C hours conal
block jumped to an incisive AV block. Midventricular block was rare. At 72 hours
conal and then AV block occurred. Combinations such as four atrial to two ventricular
to one conal beat could be readily produced while blood circulated.
Electrocardiograms and cinephotomicrographs were taken of 72 and 96 hour
hearts. Most striking was the fact that the typical Wenckebach phenomenon of adult
heart-block could be duplicated, i.e., an increase of PR interval preceding AV block
followed by a shorter PR interval which again gradually increased preceding AV
block. This sequence was repeated over and over. A n explanation of these events
is offered.
Heart-block, defined as defective transmission of impulses occurring in certain
important regions of the conduction system (Wiggers, '51), is a well recognized
entity accompanying abnormalities of the
post-natal heart. Not well known is the fact
that it is possible to block the embryonic
heart previous to the development of an
histologically recognized conduction system. The classical experiments of Erlanger,
'06 in which heart-block was produced in
the adult dog by compressing the atrioventricular (AV) bundle find their counterpart
in the work of Johnstone, '24 in the embryonic chick heart of two, three and four
days. By placing a ligature around the
heart at any level he demonstrated that the
part of the heart cephalic to the ligature
beat at a slower rate than that caudal to it.
Later Paff and Johnson, '38 described a
technique by which blockage of the isolated
48 hour embryonic chick heart could be
produced by ouabain. Also Paff, '40 and
Lehman and Paff, '42 used the time of appearance of block in digitalis treated 48
hour embryonic hearts as the basis for
assaying cardiac glycosides.
Our present interest in heart-block arose
during studies of the electrocardiogram
(ECG) in the embryonic heart. We had
been using digitalis to enhance the action
ANAT.
REC., 149: 217-224.
of the heart and noted that a distinct P
wave and QRS complex could not be obtained from an embryonic heart unless it
had developed the ability to undergo AV
block, i.e., dissociation of beat between
atrium and ventricle under the influence
of digitalis. This ability of digitalis t o
affect atrioventricular conduction was used
therefore to study block and the development of a functional conduction system
in the embryonic heart.
Technique
The procedures followed in producing
heart-block were simple. Between 37 and
120 hours of incubation chick embryos
with membranes intact were floated off the
yolk in a saline bath on to a glass square.
They were held free of wrinkles by small
pieces of filter paper placed outside the
perimeter of the marginal sinus. Drops of
digoxin-Tyrode solution, 1:1,000, were delivered over the cardiac area.
All observations were made with a m i m e
scope supplemented at critical ages, e.g.,
36, 40, 42, 47, 72 and 96 hours, with
moving pictures. Finally, at 72 hours
simultaneous pictures of the hearts and
their ECGs were taken before, during and
1
Study wa9 made possible by NIH grant HEQ
3565-06.
217
218
G. H. P A F F , R. J. BOUCEK AND H. S . K L O P F E N S T E I N
after heart-block had been established.
Although well over 200 hearts were studied, most of our observations were based
on a study of 19 hearts at 36 hours 5 one
hour; and 10 each at 40, 42, 47, 72 and
96 hours 2 one hour.
VISUAL OBSERVATIONS
At 36 t hours (fig. 1 A ) the heart responded to digoxin by beating progressively
slower and finally stopping. During this
period the depth of contraction was increased as indicated by decrease of the
internal diameter during systole. At no
time was dissociation of beat between the
primitive truncus arteriosus and the ventricle observed.
At 40 -C hours (fig. 1B) the heart first
showed evidence of ability to become
blocked. The conus dropped occasional
beats and progressed through such relationships with the ventricle as five ventricular to four conal beats then three to one,
two to one, etc. Following the appearance
of conal block, the block progressed sometimes gradually but usually suddenly to
the midventricular region of the heart. At
this time the caudal end of the ventricle
beat faster than the cephalic end. Activity
was observed to progress through two to
one block to complete dissociation between
the two parts of the heart, i.e., the sinuatrium and caudal half of the ventricle
beat as one unit and the cephalic half of
the ventricle beat as another.
At 42 -t hours (fig. 1C) the block appeared first in the conus region, jumped to
the midventricular region ( a s it did in the
40 hours heart) and then jumped to the
AV junction. This was the earliest age at
which the AV block was consistently observed. However, neither the midventricular nor the AV block was permanent.
Instead, block oscillated between the AV
junction and the midventricular region as
indicated by the two headed arrow in
figure 1C.
At 47 2 hours (fig. 1D) conus block
could be elicited but midventricular block
was seldom seen and then only in hearts
which were below average in development.
The usual sequence was conal block followed by the sudden appearance of AV
block. When AV block appeared the conus
block often disappeared. Sometimes four
atrial to two ventricular to one conal beat
could be observed.
At 72 and 96 hours (fig. 1E) the conus
blocked first and was followed by a n AV
block which left no doubt that the entire
ventricle was unresponsive to some of the
impulses originating in the sinu-atrial region. Midventricular block was never observed. I n these relatively advanced stages
the interference in the normal flow of blood
as AV block occurred presented a striking
picture which became more so when a
relationship such as four sinu-atrial to two
ventricular to one conal beat developed.
ELECTROCARDIOGRAPHIC OBSERVATIONS
Electrocardiograms of 72 hour embryonic hearts induced to block by digitalis
are shown in plate 1. The film strip (fig.
2 ) indicates two things: first, that the exploring electrode was placed at the junction of the right duct of Cuvier with the
sinus venoms; second, that the ECGs were
synchronously recorded with the activity
of the heart.
Figures 3, 4 and 5 are ECGs from a
single heart. That shown in figure 3 was
taken at the start of the experiment and
well before digitalis had time to act. The
negative P wave was followed by a positive
QRS complex after a time interval of 0.11
second (the PR interval). The heart rate
was 136 per minute. Figure 4 shows the
ECG of the same heart at a time when
the sinu-atrium was discharging at the rate
of 150 times per minute and the ventricle
was responding to only one of each two
impulses reaching it. This was obviously a
two to one AV block as evidenced by
the fact that only alternate P waves were
followed by a QRS complex. When the
ventricle responded to excitation in the
sinu-atrium the PR interval remained
unchanged at 0.11 second (compare figs.
3 and 4 ) . Note that the QRS complex was
reduced in amplitude. A few minutes later
the heart had undergone complete AV
block and the ventricle was inactive. As
seen in figure 5 the orderly procession of
P waves was not followed by any evidence
of electrical activity in the ventricle, i.e.,
the QRS complex was absent. At this time
the sinu-atrial beat was 150 per minute.
Electrocardiograms from a second heart
are shown in figures 6, 7 and 8. Figure 6
219
BLOCK I N THE EMBRYONIC HEART
Fig. 1 Outline tracings from cinephotomicrograms of hearts age 36 & hours ( A ) , 40 &
hours ( B ) , 42 2 hours ( C ) , 47 f hours ( D ) and 96 ? one hour ( E ) showing by broken
lines and arrows the position and sequence of block. Please see text for explanation.
shows the ECG before digitalis began to
influence the heart. The rate of beat was
148 per minute and the PR interval was
0.07 seconds. Within a few minutes after
application of digitalis evidence of AV
block appeared. The PR interval gradually
increased in length; AV block occurred, as
evidenced by the absence of the QRS complex and a dropped ventricular beat; the
PR interval then became shorter but gradually increased, until AV block occurred
again. This sequence was repeated over
and over. Beginning with the first cardiac
cycle shown in figure 6 the PR intervals
interrupted by dropped ventricular beats
are as follows: 0.09 second; 0.10 second;
0.12 second; AV block; 0 0 9 second; 0.10
second; 0.12 second; AV block; 0.9 second; 0.11 second, etc., etc. The rate of
beat of the sinu-atrium was 165 per minute.
After four minutes the block had progressed to a three to two relationship between the sinu-atrium and ventricle. The
PR intervals were 0.09 second; 0.13 second; AV block; 0.9 second; 0.13 second;
AV block, etc. (fig. 8 ) . Obviously AV block
was progressing toward complete asystole
of the ventricle. About five minutes later
the QRS complex had disappeared. Except
for an initial increase in heart rate from
148 to 165 per minute the rate of beat of
the sinu-atrium remained constant at 165
per minute.
DISCUSSION
There is an element of frustration in
attempting to analyze block in the early
embryonic heart because the structural
foundation has not been laid. For example, what change begins in hearts of
40 to 42 hour embryos which accounts for
220
G . H. PAFF, R. J. BOUCEK AND H. S . KLOPFENSTEIN
the appearance of hesitant blocking of
impulses between atrium and ventricle, a
change which progresses so rapidly that
within five or six hours the block is incisive
and its location can be pinpointed?
The limitations imposed by instrumentation make it difficult to record electrical
activity in hearts younger than 47 hours
but a conduction pattern in older hearts
can be demonstrated with ease by ECGs.
Pertinent to our present problem, for example, is the fact that the time between
electrical and mechanical events is relatively equal for the isolated sino-atrium,
ventricle and conus of 72 hour hearts when
each chamber is permitted to function at
its own intrinsic rate (Paff and Boucek,
’62). In the intact 72 hour heart, however,
the time between electrical and mechanical
events is longer in the ventricle than it is
in the sinu-atrium and markedly longer
in the conus than in the ventricle. Since,
in the intact heart, the intrinsic rate of
beat of the ventricle, and especially the
conus, is greatly exceeded under the stimulus of the pace-making sinu-atrium the
conclusion was drawn that the electromechanical delay of ventricle and conus,
relative to that of the sinu-atrium becomes
progressively longer when a rapid heart
rate exists (Boucek, Murphy and Paff, ’61).
The action of digitalis in producing block
suggests that either an increase in electromechanical delay or a prolongation of the
recovery period occurs in step-like fashion
between ventricle and conus and also between the caudal and cephalic halves of
the ventricle in 40 hour hearts; between
ventricle and conus and between sinuatrium and ventricle in 47, 72 and 96 hour
hearts. The usual disappearance of conus
block following the appearance of digitalis
induced AV block is explainable on the
basis that the reduction in ventricular rate
permitted the “recovery” of the conus region before the next impulse arrived.
The ECGs (figs. 3-8) are records from
two hearts which illustrate common types
of activity observed in block produced by
digitalis. In both hearts the P wave was
negative because the exploring electrode
was placed over the sinus venosus, the site
of impulse origin, and the summated action current moved away from the electrode. However, the PR interval, the pause
between excitation of the sinu-atrium and
that of the ventricle, was markedly different in the two hearts. In the first heart
no change in PR interval preceded the
sudden appearance of AV block (figs. 3
and 4). This despite the fact that digitalis
is known to prolong the pause between
atrial and ventricular depolarization, i.e.,
the PR interval (at least in the adult heart).
However, in the second heart (figs. 6-8)
the PR interval gradually increased in
length with each successive cardiac cycle
until the impulse which originated in the
sinu-atrial region was blocked and a QRS
complex failed to appear. The period of
ventricular quiescence favored resumption
of ventricular activity and the next cardiac
cycle showed a shorter PR interval which
again increased in length with successive
cycles until another block occurred. This
pattern was repeated over and over and is
analogous to the Wenckebach phenomenon
seen with excessive digitalis in adult hearts
(Wenckebach and Winterberg, ’27). This
variation in the sensitivity of AV junctional
tissue to digitalis is unexplained but the
phenomenon is frequently seen in the adult
human heart. Most commonly a progressing AV block is observed following digitalis
therapy, however, an idio-atrial focus can
develop causing a shortening of the AV
pause, or digitalis can induce a sudden
refractoriness of the AV junctional tissue
causing a sudden blocking similar to that
seen in the ECGs from the first 72-hour
chick heart (figs. 3-5).
From these studies, it would appear that
a conduction system consisting of a pacemaking sinu-atrium, AV junctional tissue,
ventricle and conus regions is developing
in the 42 hour chick embryo heart and is
fully operational by the forty-seventh hour.
SUMMARY
Excised 36, 40, 42, 47, 72 and 96 hour
embryos with membranes intact were
floated on to glass plates and treated with
drops of 1 : 1,000 digoxin-Tyrode solution
over the cardiac area. Microscopic and
cinephotomicrographic observations reveal
that at 36
one hour no dissociation of
beat occurred within the heart. At 40
hours a conus block was followed by a
midventricular block. At 42
hours the
conal and midventricular blocks were fol-
*
*
BLOCK IN T H E EMBRYONIC HEART
lowed by the first appearance of AV block
which oscillated between the AV junction
and the midventricular region. At 47
hours conal block progressed directly to
AV block. At this age midventricular block
was seldom seen. At 72 and 96 hours only
conal followed by AV block occurred.
Electrocardiographic studies of block in
the 72 hour embryonic heart demonstrated
that the Wenckebach phenomenon of the
adult heart could be duplicated, e.g., the
PR interval increased in length with each
successive cardiac cycle until the impulse
which originated in the sinu-atrial region
was blocked and a QRS complex failed to
appear. This sequence was repeated over
and over.
*
LITERATURE CITED
Boucek, R. J., W. P. Murphy and G. H. Paff 1959
Electrical and Mechanical properties of chick
embryo heart chambers. Circ. Res., 7: 787-793.
22 1
Erlanger, J. 1906 On the physiology of heartblock in mammals with special reference to the
causation of Stokes-Adams disease. Jour. Exp.
Med., 8: 8-59.
Johnstone, P. N. 1924 Studies on the physiological anatomy of the embryonic heart. Johns
Hopkins Hospital Bulletin, 35: 87-90.
Lehman, R. A., and G. H. Paff 1942 A practical
technique and design for the assay of digitalis
on the embryonic chick heart. J. Pharm. and
Exp. Therap., 75: 207-218.
Paff, G. H. 1940 A micro-method for digitalis
assay. J. Pharm. and Exp. Therap., 69: 311315.
Paff, G. H., and R. J. Boucek 1962 Simultaneous electrocardiograms and myograms of
the isolated atrium, ventricle and conus of the
embryonic chick heart. Anat. Rec., 142: 73-80.
Paff, G. H., and J. R. Johnson 1938 The behavior of the embryonic heart in solutions of
ouabain. Am. J. Phys., 122: 753-758.
Wiggers, C. J. 1951 Physiology in Health and
Disease. Lea and Febiger, Philadelphia.
Wenckebach, K. F., and H. Winterberg 1927
Die Unregelmassige Hertztatigkeit. Engelmann,
Leipzig.
PLATE 1
E X P L A N A T I O N O F FIGURES
222
2
Cinephotomicrogranis showing simultaneous recording of heart activity and ECGs. Position of exploring electrode indicated by round dot
over junction of duct of Cuvier with sinus venosus.
3
ECGs before evidence of block appeared. Note negative P waves and
positive QRS complexes. One PR interval is indicated by arrow.
4
ECGs from same heart a s figure 3. The P waves appear in uninterrupted sequence but only alternate ones are followed by y QRS complex. Two to one AV block ( B ) is obvious. The PR interval is of the
same duration as seen i n figure 3.
5
ECGs of same heart as figures 3 and 4. The steady progression of
P waves remains but AV block is complete.
6
ECGs from a second heart taken before block occurred. Each cardiac
cycle is complete. One PR interval is indicated by arrow.
7
ECGs showing the presence of 4 to 3 AV block. Note the gradual
increase in PR interval preceding the appearance of AV block ( B ) .
Same heart as in figure 6.
8
ECGS from same heart as in figures 5 and 6. AV block is 3 to 2.
Note again the long PR interval preceding block ( B ) .
BLOCK IN THE EMBRYONIC HEART
George H. Paff, Robert J. Boucek and Harold S. Klopfenstein
PLATE 1
223
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