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Nervous Mechanisms in Ventricular
Arrhythmias Induced by Calcium
Chloride in Rats
By MANUEL R E N E MALINOW, M.D., FERNANDO F. BATLLE, M.D., AND
BERNARDO MALAMUD, M.D.
The ventricular arrhythmias induced in rats by standardized doses of calcium chloride are largely
prevented by sympatholytic and autonomic blocking drugs, by encephalic and/or upper spinal
destruction and by medullospinal section. They are not prevented by bilateral vagotomy, bilateral
adrenalectomy, or by lower spinal destruction.
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O
a 10 per cent CaCUt solution was used in all animals.
During the injection and the following two minutes,
the cardiac mechanism was continuously studied
through a cardioscope connected to the animal with
two percutaneous clamp electrodes. An electrocardiograph recorded occasional tracings when so
desired.
Results were graded as follows: (o) isolated ventricular premature systoles; (6) frequent ventricular
premature systoles, but not runs or bigeminal
rhythm; (c) short runs of ventricular flutter or fibrillation, and (d) long runs of ventricular flutter or
fibrillation. In the final interpretation, results a and
b were considered as negative, c and d as positive.
Other methods used in this study are described in
the text. The control group of animals was taken
from a previous experimental series.3
PERATION of a nervous mechanism
in the genesis of cardiac arrhythmias
secondary to the injection of calcium
chloride was suggested by the observation that
electrical sympathetic stimulation consistently
induced ventricular arrhythmias only when
calcium chloride was given simultaneously.1
Insight into the problem was obtained when
sympatholytic and other autonomic blocking
drugs were reported to prevent ventricular
flutter and fibrillation induced by standardized
doses of calcium chloride.2 Further studies on
the involved mechanisms seemed to be in
order, since the suspected importance of the
central nervous system has not been fully
determined.
RESULTS
Effects of CaCI-i Control A nimals
MATERIAL AND METHODS
When 0.2 cc. per 100 Gm. of a 10 per cent
CaCl2 solution was injected into each of 20
rats, a positive response, that is, short or prolonged runs of ventricular flutter or fibrillation,
was found in 95 per cent of the cases (fig. 1).
Immediately after the injection, sinus tachycardia and increased voltage of the QRS appeared. Fifteen to 30 seconds afterwards, ventricular rate increased suddenly (up to ]200
per minute or above). The nature of this
arrhythmia is best shown in auricular electrograms,4 in which the independence of auricular
and ventricular waves is demonstrated. The
heart action may be regular or irregular, and
it is difficult to decide at these high rates
Seventy to 130 Gm. white albino rats of the
Williams strain were anesthetized with Nembutal*
intrnperitoneally, (6 mg. per 100 Gm. of a 0.6 per
cent solution). Only the main features of the method
for producing ventricular arrhythmias in the rat3
will be mentioned here. Using a clean but not sterile
technic, the femoral vein was isolated at the inguinal region in order to check the accuracy of the
injection. A standard close of 0.2 cc. per 100 Gm. of
From the Pabellon de Cardiologia "Luis H. Inchiiuspe," Chief: Prof. Bias Moia, Policlinico Ramos
Mejia, and the Servicio de Cardiologia, Chief: Dr.
F. F. Batlle, Policlinico T. Alvarez, Buenos Aires,
Argentina.
Partly supported by the Associaci6n Cardiol6gica
Inchauspe.
Received for publication July 14, 1953.
* Abbott Laboratories generously provided us
with the Nembutal used in these experiments.
t Ten per cent CaCU was obtained through the
kindness of Merck, Argentina.
554
Circulation Research, Volume I, November 195S
MALINOW, BATLLE AND MALAMUD
whether flutter or fibrillation is present. In
this paper, therefore, both of these mechanisms will indiscriminately be called ventricular arrhythmias. Other effects of CaCl2 on the
electrocardiogram, such as A-V or I-V block,
and S-T segment changes,4 will not be discussed in the present paper.
Influence, of the A ulonomic Nervous System
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In order to demonstrate the influence of the
autonomic nervous system in these ventricular
arrhythmias, several procedures were employed:
(a) Ganglion Blocking Drugs. Several drugs
reported to block sympathetic and parasympathetic pathways at the autonomic ganglia6• 6
all animals as evidenced by pallor of the skin,
collapsed femoral veins, and difficult breathing.
In nine of these animals, subsequent injection
of CaCl2 induced positive results.
In order to quantitate the results, the same
procedure was repeated in six additional animals, and the mean blood pressure was measured in the abdominal aorta with a mercury
manometer connected to the artery through a
0.8 mm. needle and heparinized physiologic
saline filled tubes. To avoid bleeding of the
animal into the system, the manometer was
previously set at 90 mm. Hg. The average control mean blood pressure was 73 mm. Hg
(range, 60 to 90 mm. Hg). Bleeding reduced
the average mean blood pressure to 22 mm. Hg
V'IV'V-
M
Fio. 1. (A) Fifteen seconds after the injection of 0.2 cc. per 100 Gra. of a 10 per cent CuCU solution,
short and prolonged bursts of ventricular fibrillation appear. Precordial electrodes; time in 0.05
second. (6) Ten seconds after the injection of a standard dose of CaCli solution, a short run of ventricular fibrillation. The auricular waves are clearly seen. Auricular electrogram, indifferent electrode
in the right paw; time in 0.05 second.
were used.2 Five mg. per 100 Gm. of tetraethylammonium chloride were injected into the
femoral vein of 10 rats, three minutes before
the injection of CaCl-j. In none of these animals
were positive results produced by the standard
injection of CaCU. The same was true in 10
rats that received 2.5 mg. per 100 Gm. of
hexamethonium bromide three minutes before
the injection of CaCl>.
Since this protection has been attributed to
the nonspecific effect of hypotension,7 experiments were performed to determine whether
the results could be explained through blood
pressure changes, per se.8
In 10 rats, 2 to 4 cc. of blood were withdrawn from the previously visualized abdominal aorta through a midline incision. This
massive bleeding induced a shock-like state in
(range, 20 to 30 mm. Hg). When the standard
close of CaCl2 was given to these six hypotensive animals, a positive result was found in
five. If both groups are added, 87.5 per cent
of the rats showed a positive reaction, a nonsignificant difference with the control group.
(b) Vagotomy. As the ganglion blocking drugs
influence sympathetic and parasympathetic
pathways, a further delimitation of the
involved mechanism was attempted by
vagotomy. In 10 rats both vagi were sectioned
in the neck. When CaCU was injected two
minutes after sectioning, ventricular arrhythmias appeared in all animals.
(c) Adrenalectomy. To rule out the influence
of adrenal hormones, 10 rats were subjected to
bilateral retroperitoneal adrenalectomy. In
nine animals ventricular arryhthmias were in-
NERVOUS MECHANISMS IN CALCIUM CHLORIDE ARRHYTHMIAS
556
duced when CaCU was injected two minutes
later.
(d) Sympatholylic Drugs. Sympatholytic
drugs might further delineate the pathways
involved. Three cc. of Hydergine,* a sympatholytic agent, were given intravenously to
10 rats. The injection of CaCU, three minutes
TABLE 1.—Influence of Nervous System on Ventricular Fibrillation and Flutter Induced by CaCh in Rats—
Dose 0.2 cc. per 100 Gm. of a 10 Per cent Solution
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Number of
Animals
Procedure
Positive
Results
Per cent
Positive
Results
20
Control (standardized
dose)
Tetraethylammonium
chloride (5 mg./lOO
Gm.)
Hexamethonium bromide (2.5 mg./lOO
19
95
10
10
0
0*
0
0*
Gm.)
10
10
10
10
5
15
15
10
5
10
Bilateral vagotomy
Bilateral
adrenalectomy
Hydergine (0.9 mg./rat)
Medullo-spinal section
Medullo-spinal section
plus transfusion
Intracranial nervous
destruction
Upper spinal destruction
Intracranial and upper
spinal nervous destruction
Intracranial and upper
spinal nervous destruction plus transfusion
Lower spinal destruction
9
9
90
90
1
1
10*
30*
20*
4
26*
4
26*
2
10*
0
0*
3
9
90
* Statistically significant differences from control
results.
later induced ventricular arrhythmias in only
one animal.
Influence of the Central Nervous System
The importance of the central nervous system in the production of these arrhythmias
* Laboratories Sandoz generously provided us with
the Hydergine used in these experiments. Each cubic
centimeter of Hyclerginc contains 0.3 mg. of dehydrogenated alkaloids of ergot.
was evidenced with experiments destroying
localized regions of the brain stem. In order to
overcome apnea, the trachea was dissected
and canalized with a 1.5 mm. needle and
intermittent positive pressure respiration was
given. A wide median posterior incision visualized the spine and the skull. Sections were
made at fixed bony landmarks: for example,
through the first cervical vertebra, easily identified immediately below the skull, and through
the first dorsal vertebra, easily identified in the
rat by the presence of a large cartilaginous
process. Histologic sections showed that the
first procedure severed the medulla from the
spine, and that those performed below the
first dorsal vertebra allowed the destruction of
dorsolumbar spinal tissue.
(a) Medullospinal Section. In 10 rats, a
specially designed cutting hook was introduced
above the first cervical vertebra and the
medulla was severed from the spinal cord.
Arrhythmias followed the injection of CaCl2 in
only three rats. As it was felt that hemorrhage
secondary to operative procedures might have
changed the results, in five other rats the section was performed after the transfusion of
4 cc. of homologous freshly heparinized blood
(obtained from other rats through the abdominal aorta). Results were positive in only one
rat.
(6) Encephalic Destruction. In 15 rats, sharp
scissors were introduced into the skull through
the upper cervical vertebra, and the nervous
tissue was destroyed by divulsion. Four rats
showed ventricular arrhythmias after the injection of CaCl>.
(c) Upper Spinal Destruction. In 15 animals
the osseous dorsal aspect of the spine was
lifted and the underlying nervous tissue destroyed with scissors. Positive results were obtained in only four rats after the injection of
CaCl2.
(d) Encephalic and Upper Spinal Destruction. In 19 rats the two destructive procedures
were both performed. When CaCl> was injected, in only two instances did ventricular
arrhythmias appear. It was observed that in
these animals the veins were markedly collapsed. It was also felt that operative procedures might modify circulating blood volume
MALINOW, BATLLE AND MALAMUD
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and thereby increase the effective CaCU levels
in the blood; experiments then would not be
comparable with our controls. Consequently,
hypovolemia was overcorrected with homologous transfusions. In five rats, the abovementioned procedures were repeated and 4 cc.
of freshly heparinized homologous blood was
administered two minutes before the CaCla
injection. 'Ventricular arrhythmias appeared in
none of these animals.
(e) Lower Spinal, Destruction. In order to
rule out the nonspecific effects of nervous
destruction, the spine was destroyed down to
the tail in 10 rats. In nine of these animals,
the injection of CaCl2 induced ventricular
fibrillation.
DISCUSSION
In the interpretation of our experiments, it
is important to point out that negative results
after the injection of CaClo were not the effect
of hypotension due to drugs or operative procedures, since acute hypotension alone, induced
by massive bleeding, was not able to protect
rats from the arrhythmic effects of CaCl>.
The above-mentioned experiments show that
standard doses of CaCIa induce ventricular
arrhythmias in the rat only when an intact
central nervous system, nonvagal conduction
pathways, and adrenergic mediators exist.
These results consequently suggest the neurogenic origin of the induced arrhythmias, with
probable centers within the brain stem (hypothalamus?); impulse conduction occurs through
the upper spinal cord and then to the heart
via sympathetic pathways.
The mechanism of CaCl2 arrhythmias in the
rat is apparently similar to those operative in
the arrhythmias brought about by stimulation
of the central nervous system9' 10 or by action
chloroform11' 12' 13 and cyclopropane.14' 15 These
experiments showed a sensitization to epinephrine during cyclopropane anesthesia in dogs,
which depended on stimulation of an encephalic
center situated above the pons, which sends
impulses to the heart through the sympathetic
system.11 The heart effects are initiated by
the stimulation of intra-abdominal receptors by
cyclopropane. Impulses are then sent to
the hypothalamus through the splanchnic
557
nerves.14' 15 This explains why the destruction
of the central nervous system16 or the injection
of sympatholytic agents, such as dibenamine,17'18 prevents the appearance of arrhythmias.
Ventricular arrhythmias have also been described in rabbits under benzene inhalation,
and the importance of the nervous system was
demonstrated when the arrythmias were prevented by stellate ganglionectomy. The same
protection was evidenced by upper thoracic
root excision and by section of lateral spinal
tracts down to C-7. No protection was seen
when sections were performed below D-3, nor
by mesencephalic sections, bilateral vagotomy
or lower dorsal sympathectomy.19'20 These
experiments, performed several weeks after
surgery, when no spinal shock was present,
also showed no correlation between protection
from the arrhythmias and blood pressure
changes. They also demonstrated that trigeminal stimulation in the rabbit sent cliencephalic impulses down through the spinal
lateral tracts, the spinal roots, and the upper
dorsal sympathetic nerves, and was thus producing bigeminal ventricular premature systoles
Neurologic mechanisms in human ventricular arrhythmias are generally accepted21; they
have been reported during anxiety22 and emotional disturbances,23 as well as a result of
reflexes.24'26 We have found that ventricular
arrhythmias may disappear in patients following the injection of autonomic blocking drugs;
moreover, patients with organic lesions of the
central nervous system may show ventricular
arrhythmias26'21-2J that can be partially abolished with autonomic blocking drugs. It is believed that ventricular arrhythmias experimentally induced in rats are probably similar
in origin to some persistent or paroxysmal
cardiac disturbances found in man.
The demonstrated neurogenic mechanisms
in the rat do not rule out the possibility that
CaCl2 may be able to produce arrhythmias by
acting directly upon the myocardium. Such a
direct effect has been demonstrated by us,
using higher concentrations of CaCl229 in a rat
heart-lung preparation.
558
NERVOUS MECHANISMS IN CALCIUM CHLORIDE ARRHYTHMIAS
AND MOE, G. K.: The role of thoracic sympathetic-pathways in the induction of ventricular
ectopic rhythms by epinephrine and cyclopropane. J. Pharmacol. & Exper. Therap. 101:
176, 1951.
SUMMARY
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Ventricular flutter and fibrillation, induced
by standard doses of CaCl2 in rats, are prevented by the injection of tetraethylammonium
chloride, hexamethonium bromide or Hydergine, as well as by localized destructions of the
central nervous system. Bilateral vagotomy,
bilateral adrenalectomy, and lower spinal cord
destruction did not prevent these arrhythmias.
The neurogenic mechanisms and the involved
pathways have been thus demonstrated, but
direct arrhythmic effects of higher concentrations of CaCl2 upon the myocardium were also
noted. The pathogenic and pharmacologic
similarities with other ectopic rhythms experimentally induced in different species or spontaneously arising in man are discussed. Proper
caution should be used in extrapolations from
experimental arrhythmias to pathologic conditions in man.
8
B.: Prevention of neurogenic ventricular arrhythmias in the rat by autonomic blockingdrugs. Influence of blood pressure changes.
(Submitted for publication).
9
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Nervous Mechanisms in Ventricular Arrhythmias Induced by Calcium Chloride in Rats
MANUEL RENÉ MALINOW, FERNANDO F. BATLLE and BERNARDO MALAMUD
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Circ Res. 1953;1:554-559
doi: 10.1161/01.RES.1.6.554
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