Characterization of Stress-Induced Sudden Death in Cardiomyopathic Hamsters
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0022-3565/98/2841-0125$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 284, No. 1
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 284:125–135, 1998
Characterization of Stress-Induced Sudden Death in
Cardiomyopathic Hamsters
NOBUYA MATSUOKA, HIROYUKI ARAKAWA, HIROSHI KODAMA and ISAMU YAMAGUCHI
Basic Research Group, Tsukuba Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, 300 –26 Japan
Accepted for publication September 10, 1997 This paper is available online at http://www.jpet.org
ABSTRACT
Stress is known clinically and experimentally to contribute to significantly prevented the lethal effects of the stress. Further-
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the development or exacerbation of cardiovascular dysfunc- more, it was demonstrated that the drug significantly reduced
tion. In an attempt to construct an animal model of stress- the increase in the weights of the heart, adrenal, liver and
induced cardiovascular dysfunction and to understand its kidney observed in the stressed cardiomyopathic hamsters,
mechanisms, the effects of cold-immobilization stress and its whereas phentolamine (0.1–10 mg/kg) and atropine (0.1–10
cardiovascular consequences were investigated in cardiomyo- mg/kg) did not prevent the stress-induced sudden death. The
pathic Syrian hamsters (BIO 14.6) and age-matched healthy series of acute experiments using single exposure of this stress
control hamsters. Repeated exposure (5 days) to cold-immo- revealed that the stress evoked severe arrhythmia in some of
bilization in the supine position induced no detectable ill effects the cardiomyopathic hamsters and increased the levels of cir-
in the healthy control hamsters but had a lethal effect in the culating catecholamines in both healthy and cardiomyopathic
cardiomyopathic hamsters: more than half of the animals died hamsters. These results taken together suggest that stress
suddenly during or after the stress sessions. Autopsy study of accelerates the cardiovascular dysfunction in cardiomyopathic
these animals showed significant increases in the weights of hamsters and provide the first evidence that excitation of the
the heart, adrenal, liver and kidney and in the serum levels of sympathetic nerves, in which b-adrenoceptors appear to be
alkaline phosphatase, urea nitrogen, creatinine and glucose in involved, but not the parasympathetic nerves, has an important
the cardiomyopathic hamsters subjected to the stress. Pro- role in the etiology of stress-induced cardiac sudden death of
pranolol (0.1–10 mg/kg i.p.) administered just before each cold- cardiomyopathic hamsters.
immobilization for 5 consecutive days dose-dependently and
A growing body of evidence suggests that behavioral stress ous stressors that can augment sympathetic neural traffic to
has a part in the precipitation of life-threatening cardiovas- the heart reportedly lower the vulnerable period threshold
cular dysfunction. Indeed, stress has been known clinically for ventricular fibrillation, resulting in sudden death in dogs
and experimentally to contribute to the development or ex- or pigs (Kolman et al., 1976; Parker et al., 1987).
acerbation of cardiovascular dysfunction, and it is identified On the other hand, the cardiomyopathic Syrian hamster is
as a risk factor of hypertension, cardiac dysrhythmia, or even known to develop a genetically determined cardiomyopathy,
sudden cardiac death (Eliot, 1987; Galosy et al., 1981). with progressive development of congestive heart failure,
Among such cardiovascular disorders, sudden cardiac death resembling human congestive cardiomyopathies (Bajusz et
is the leading mode of death in adults in the industrial world al., 1966; Bajusz and Lossnitzer, 1968; Strobeck et al., 1979).
(Manolio and Furberg, 1994). Although the precise mecha- The cardiomyopathy is characterized by multifocal myocar-
nisms of cardiac sudden death are not yet fully understood, dial necrosis that begins at 40 to 50 days of age and causes
increasing evidence has indicated that sudden death result- premature death from congestive heart failure or arrhyth-
ing from ventricular fibrillation may be triggered by behav- mia, usually within 1 year. Although the pathogenesis of the
ioral and neural factors (Engel, 1971; Kamarck and Jen- disease is imperfectly understood, a body of evidence has
nings, 1991; Lown, 1987). For instance, several physiological
suggested there are defects in the myocytes that are suscep-
precursors of sudden death are promoted by psychological
tible to the effect of stressful stimuli such as transient is-
stress, especially in persons with coronary heart disease (Ka-
chemia (Lossnitzer et al., 1975). It has recently been reported
marck and Jennings, 1991). In experimental animals, vari-
that cold-restraint stress had lethal consequences in cardio-
myopathic hamsters (Tapp et al., 1989a, 1989b). These find-
Received for publication November 15, 1996. ings taken together prompted us to construct an animal
ABBREVIATIONS: BCF, body cavity fluid; CPK, creatine phosphokinase; GOT, glutamic oxalacetic transaminase; GPT, glutamic pyruvic
transaminase; NE, norepinephrine; E, epinephrine.
125126 Matsuoka et al. Vol. 284
model of cardiac sudden death associated with stress by nol and 277 mM 1-octanesulfonate (Nacalai Tesque, Kyoto, Japan)
investigating the effect of cold-immobilization stress and its and 10 mM disodium EDTA (Nacalai Tesque); flow rate, 1 ml/min.
cardiovascular consequences in cardiomyopathic hamsters. ECG telemetry. To examine the acute effect of the cold-immobi-
The purpose of this study was to characterize the sudden lization stress on electrocardiographic responses, telemetry ECG
death of these animals and to clarify the role of the auto- was recorded before and after the stress session for 60 min using the
Cardiotel telemetry system (Data Sciences, St. Paul, MN) in a sep-
nomic nervous systems in the genesis of the sudden death.
arate series of acute experiments. Two healthy hamsters and two
cardiomyopathic hamsters were used in the experiment. Briefly, the
animals were anesthetized with pentobarbital (40 mg/kg i.p.), and
Materials and Methods dorsal celiotomy was performed. Each transmitter (model TA11CTA-
Animals. The animals used were 2-month-old BIO 14.6 cardio- F40) consisted of a small body (volume, 4 ml; weight, 9 g; 15 3 9 3
myopathic hamsters and age-matched F1B healthy hamsters pur- 25 mm) with two coiled wires protruding from one end of the cylin-
chased from Canadian Hybrid Farms (Nova Scotia, Canada). The der. The body of the transmitter was placed in the dorsal cavity.
hamsters were individually housed in plastic cages and given unlim- Subsequently, the two recording leads were pulled along two subcu-
ited access to Purina mouse chow and tap water. Both groups were taneous tunnels toward the left and right clavicular regions, where
placed in a temperature-controlled environment (22 6 1°C) under a the tips of the leads were sutured to the pectoral muscles. The
12-hr light/dark schedule with lights off at 3 p.m. and were allowed receiver (model CTR85-SA) measured 350 3 220 3 30 mm and was
to adapt to these conditions for 4 weeks before the experiment. All placed outside the refrigerator during the recording sessions. Before
animals procedures were carried out as approved by the Animal Care the beginning of immobilization stress, a 10-sec ECG recording was
Downloaded from jpet.aspetjournals.org at ASPET Journals on October 29, 2015
and Use Committee at Fujisawa Pharmaceutical Co. Ltd. performed every 20 min while the animal was alone and undisturbed
Stress procedures. Hamsters of each group were further subdi- in its home cage (base-line condition). The recording was not per-
vided into stress and nonstress groups. The stress protocol was formed during the stress session for 60 min, but immediately after
carried out on 5 consecutive days and began at 3 p.m. The stressed the animals were taken out of the refrigerator, ECGs were recorded
hamsters including the healthy controls were subjected to daily 1-hr again, and the recordings continued until either the animals died or
periods of supine immobilization at 4°C. They were immobilized by 3 days after conclusion of the stress sessions. The ECG was moni-
extending their four limbs and taping them onto the corners of a tored on a cathode-ray tube, registered on paper and fed into a
small board; they were then left in a refrigerator for 1 hr. The dedicated instrument that automatically performed amplification,
nonstressed healthy and cardiomyopathic hamsters were not immo- sampling (500 Hz) and analog-to-digital conversion of the signal. The
bilized and were just left in their housing cages outside a refrigerator digitized data were stored on IBM-PC for offline processing and
for 1 hr. Immediately before and after each stress session as well as analyzed by using a software package.
the next morning after each session, the hamsters were checked to Drugs. The drugs used here were DL-propranolol hydrochloride,
measure their body weights and to see if they were still alive. There- phentolamine hydrochloride and atropine hydrochloride (Sigma
after, we checked the hamsters twice daily until 7 days after the last Chemical, St. Louis, MO). All drugs were prepared just before the
stress session. tests. They were dissolved in physiological saline and given intra-
Autopsy. Hamsters were autopsied after either being found dead peritoneally in a volume of 2 ml/kg just before the immobilization
or after decapitation on the final day of the experiment (day 12). In stress for 5 consecutive days.
the animals that died from sudden death, 10 hours was the maxi- Statistical analysis. All results were expressed as mean 6
mum time that was allowed to elapse between death and autopsy. S.E.M. Statistical significance of differences was calculated using
Autopsy consisted of removal of the organs and absorption of pleural Student’s t test (two-tailed) for the changes in organ weights and
and peritoneal fluid on a preweighted gauze pad. Organs and BCF serum parameters. Mortality results were analyzed by Fisher’s exact
were then weighed quickly. In a experiment, organs were dried at probability test. Cumulative surviving percents of hamsters during
90°C for 6 hr, and their dry weights were determined. The surviving the course of the experiments were analyzed using a generalized
animals were killed at the end of the experiment, 7 days after the Wilcoxon test.
last stress session, and autopsied as above, including the collection of
blood. Trunk blood was collected into tubes and centrifuged, and the
serum was separated. Serum concentrations of CPK, GOT, GPT, Results
ALP, urea nitrogen, creatinine, total cholesterol, total bilirubin, trig- Effects of cold-immobilization stress on cardiomyo-
lycerides and glucose were measured using an autobiochemical an- pathic hamsters. Figure 1 shows the mortality. No healthy
alyzer (TBA-20R; Toshiba, Tokyo, Japan). Corticosterone levels in
hamsters with or without stress or nonstressed cardiomyo-
the serum were measured with a commercial corticosterone 125I-RIA
kit (ICN Biochemicals, Costa Mesa, CA).
Catecholamine determinations. To examine the effect of single
exposure to cold-immobilization stress on circulating cat-
echolamines, plasma NE and E levels were investigated in separate
series of acute experiments. Blood of the hamsters was obtained from
the abdominal aorta through a heparinized catheter with the animal
under halothane anesthesia and was collected at the time points of 0,
15, 30 and 60 min after the beginning of stress. The blood taken from
the hamsters without stress 20 min after pentobarbital anesthesia
(40 mg/kg i.p.) served as base-line data. Four or five healthy ham-
sters and cardiomyopathic hamsters were used in each group of
different time points. NE and E in the plasma were immediately
extracted by the method of Hallman et al. (1978) and assayed elec-
trochemically by HPLC as described by Watson (1981). The analyt-
Fig. 1. Percentage of surviving cardiomyopathic and age-matched con-
ical conditions were as follows: HPLC pump, model EP-10 (Eicom,
trol healthy hamsters in stress treatment. ppP , .05, statistically signif-
Kyoto, Japan); electrochemical detector, model ECD-100 (Eicom); icant compared with nonstressed cardiomyopathic hamsters (by Fisher’s
HPLC column, CA-5ODS, 4.6 3 150 mm (Eicom); mobile phase, 0.1 exact probability test). Numbers in parentheses indicate the number of
M sodium phosphate buffer solution (pH 6.0) with 10% (v/v) metha- animals in each group.1998 Stress and Cardiomyopathic Hamsters 127
pathic hamsters died during the course of the experiment. In
82.3 6 6.3b
146.8 6 3.2
139.4 6 2.7
103.4 6 1.5
contrast, 8 of 13 stressed cardiomyopathic hamsters died;
Day 12
there was a statistically significant difference between the
mortality of the two groups (P , .01 by Fisher’s exact prob-
ability test). As shown in figure 2, which represents the
cumulative surviving percent, 5 animals among the stressed
82.4 6 5.5b
145.6 6 3.4
138.2 6 2.7
102.6 6 1.3
cardiomyopathic hamsters died during the 5 days of stress
Day 11
sessions, and 3 animals died after the stress termination.
The difference between the two groups was statistically sig-
nificant (P , .01 by generalized Wilcoxon’s test).
Table 1 summarizes the daily changes in the body weight
83.0 6 5.2b
145.9 6 3.3
137.9 6 2.7
102.8 6 1.4
of the animals during the experiment. Cold-immobilization
Day 10
had a minimal effect on the body weights of the healthy
control hamsters, with a statistically significant (P , .05 by
Student’s t test) reduction only on days 6 and 8; thereafter,
the body weights recovered. In the cardiomyopathic ham-
82.7 6 4.7b
145.2 6 3.3
136.9 6 2.6
100.4 6 1.2
sters, however, the stress significantly (P , .01) decreased
Day 9
body weight, and the mean body weight loss persisted during
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the 1 week of observation after the termination of the stress
sessions.
Effects of cold-immobilization stress on the organ
136.4 6 2.6a
80.1 6 4.1c
145.3 6 3.3
102.0 6 1.2
weights of cardiomyopathic hamsters. The effects of
P , .05, b P , .01, c P , .001; statistically significant compared with the value of each corresponding nonstressed group (by Student’s t test).
Day 8
cold-immobilization stress on the organ wet weights of the
cardiomyopathic hamsters were evaluated, and the results
are summarized in figure 3. Seven days after the conclusion
of the stress sessions, BCF was increased in both the control
135.7 6 2.3a
Body weight
82.6 6 3.1c
143.9 6 3.2
99.4 6 1.4
and cardiomyopathic hamsters, although the change was not
Day 6
statistically significant. Also, the wet weights of the adrenal,
kidney, liver and heart were significantly (P , .05 by Stu-
dent’s t test) increased in the stressed cardiomyopathic ham-
sters compared with the nonstressed cardiomyopathic ani-
g
83.3 6 2.5c
143.8 6 3.1
136.8 6 2.4
99.9 6 1.5
mals, whereas the spleen was not affected by the stress (fig.
Day 5
3). The stress produced no detectable changes in the healthy
animals except that it significantly but slightly increased the
weights of the heart and liver. To clarify the nature of the
increase in the organ weights, the dry weights of the organs
85.2 6 1.9c
142.6 6 3.2
137.8 6 2.5
99.4 6 1.5
were determined. Table 2 shows that the stress significantly
Day 4
increased the dry weight of the adrenal, kidney, liver and
heart in a comparable magnitude with the measurement of
wet weight of each organ, suggesting the presence of hyper-
trophy or remodeling of each organ.
Changes in the body weights of stressed cardiomyopathic hamsters
87.8 6 1.9c
142.5 6 3.2
138.9 6 2.6
99.9 6 1.6
The pooled data from our previous experiments were re
Day 3
analyzed to clarify the time course changes in the organ
weights of the stressed cardiomyopathic hamsters without
any drug dosing, and the results were summarized in figure
H, healthy control hamsters; CM, cardiomyopathic hamster.
90.5 6 1.9b
141.8 6 3.1
140.9 6 2.7
98.5 6 1.6
Day 2
143.4 6 2.9
143.9 6 2.6
97.1 6 2.1
97.0 6 1.9
Day 1
8
8
8
13
n
CM 1 stress
Treatment
H 1 stress
TABLE 1
Fig. 2. Cumulative survival curves of unstressed and stressed cardio-
CM
myopathic hamsters. P value was calculated by generalized Wilcoxon
a
H
test.128 Matsuoka et al. Vol. 284
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Fig. 3. Changes in hamster organ weights and BCF weights caused by cold-immobilization stress. Each column and bar represent the mean 6 S.E.M.
pP , .05, ppP , .01, pppP , .001, significantly different from each corresponding nonstressed group (by unpaired Student’s t test). †P , .05, ††P ,
.01, †††P , .001, significantly different from nonstressed healthy hamsters (by unpaired Student’s t test). H; healthy hamsters, CM; cardiomyopathic
hamsters.
TABLE 2
Changes in the organ dry weights of stressed cardiomyopathic hamsters
Organ weight
Treatment n
Adrenal Heart Kidney Liver Spleen
mg/g b.wt.
H 6 0.053 6 0.002 0.670 6 0.10 1.57 6 0.02 13.9 6 0.25 0.196 6 0.008
H 1 stress 6 0.060 6 0.002a 0.704 6 0.015 1.63 6 0.01a 14.3 6 0.31 0.195 6 0.007
CM 6 0.052 6 0.002 0.687 6 0.009 1.86 6 0.02e 12.5 6 0.22d 0.266 6 0.010e
CM 1 stress 7 0.076 6 0.005b 0.894 6 0.055b 3.20 6 0.25c 15.0 6 0.87a 0.249 6 0.018
H, healthy control hamsters; CM, cardiomyopathic hamsters.
a
P , .05, b P , .01, c P , .001, statistically significant compared with the value of each corresponding nonstressed group (by Student’s t test).
d
P , .01, e P , .001, statistically significant compared with the value of control healthy hamsters (by Student’s t test).
4. All data were extracted and arranged from the stressed serum parameters were determined from the blood of surviv-
cardiomyopathic hamsters (96 animals) that died from sud- ing hamsters on day 12. The data obtained are summarized
den death on each time point during the experimental period. in figure 5. Serum levels of CPK in the nonstressed cardio-
As shown in figure 4, BCF weight was slightly increased myopathic hamsters were found to be significantly higher
during the stress sessions and then returned toward the than in the nonstressed healthy control hamsters, but cold-
levels of the nonstressed animals when the stress sessions immobilization stress hardly affected the levels in either
were stopped. On the other hand, the stress quickly and group. Similar results were obtained in GPT (fig. 5) and GOT
progressively elevated the weight of the adrenal, and the (data not shown). On the other hand, the stress markedly
increases were sustained even after the termination of the elevated serum levels of urea nitrogen and creatinine in the
stress. A similar time course change was observed in the cardiomyopathic hamsters, as shown in figure 5, but pro-
weights of the kidneys. Spleen weight increased transiently, duced no changes in these parameters in the healthy ham-
followed by a marked reduction caused by the stress, whereas sters. Similarly, significant increases were observed in the
the heart and liver increased in their weight at a slow onset serum levels of alkaline phosphatase and total protein in the
starting from day 3 or 4, and the increases persisted after the stressed cardiomyopathic hamsters (data not shown). Blood
conclusion of the stress sessions. glucose, a sensitive indicator of stress response (De Boer et
Effects of cold-immobilization stress on the serum al., 1990), also was markedly elevated only in the stressed
parameters in cardiomyopathic hamsters. Changes in cardiomyopathic hamsters. Serum levels of total bilirubin,1998 Stress and Cardiomyopathic Hamsters 129
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Fig. 4. Time course changes in hamster organ weights and BCF weights caused by cold-immobilization stress. Each symbol and bar represent the
mean 6 S.E.M. Broken line in each graph represents the level of nonstressed cardiomyopathic hamsters that were killed and autopsied on day 12.
Numbers in parentheses indicate the number of animals in each group that died on each day.
calcium and triglycerides were minimally affected by the before the stress, as seen in row C of figure 7. However, the
stress (data not shown), and the stress tended to increase the animal that received immobilization stress for 1 hr a day
serum corticosterone level in the cardiomyopathic hamsters, showed significant arrhythmia; there was obvious heart rate
although the change was not statistically significant. slowing and an occasional atrial fibrillation (row D). As
Changes in plasma catecholamine levels. As shown in shown in row E of figure 7, one of two animals showed an
figure 6, single exposure to cold-immobilization stress in- abnormal auriculo-ventricular block before its death.
creased the plasma NE and E levels significantly in healthy Pharmacological analysis on the stress-induced sud-
control hamsters. Plasma NE level in the stressed healthy den death in cardiomyopathic hamsters. Propranolol, a
hamsters peaked immediately after the beginning of stress beta blocker, was evaluated for its effect on the sudden death
and declined gradually during the 1-hr stress period to re- of stressed cardiomyopathic hamsters in an attempt to elu-
cover to the base-line levels. Plasma E level in the stressed cidate the role of the sympathetic nerves. Figure 8 shows the
hamsters reached to the plateau at 15 min, and this increase mortality results. No healthy hamsters with or without
was sustained throughout the stress period. However, there stress and no nonstressed cardiomyopathic hamsters died
were no significant changes in the time course of these cat- during the course of the experiment. In contrast, 5 of 6
echolamine levels between the cardiomyopathic and control stressed cardiomyopathic hamsters died. Administration of
healthy hamsters. propranolol (0.1–10 mg/kg i.p.) dose-dependently reduced the
ECG telemetry. The acute effect of the cold-immobiliza- mortality seen in the stressed cardiomyopathic hamsters
tion stress on electrocardiographic responses was investi- with statistically significant changes in the mortality for the
gated by telemetry system in two healthy and two cardiomyo- group dosed with 10 mg/kg propranolol compared with the
pathic hamsters before and after the stress. As shown in saline-treated stressed cardiomyopathic hamsters (P , .05
figure 7, the nonstressed healthy hamsters showed no signif- by Fisher’s exact probability test). As shown in figure 9, no
icant changes in their sinus rhythm after cold-immobiliza- cardiomyopathic animals receiving 10 mg/kg propranolol
tion stress (row A, prestress; row B, poststress), whereas the died during the experiment.
cardiomyopathic hamsters showed a regular sinus rhythm Six days after the completion of the stress sessions, BCF
with shorter RR interval compared with the healthy controls and organ weights were measured; the results are presented130 Matsuoka et al. Vol. 284
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Fig. 5. Changes in hamster serum parameters caused by immobilization stress. Each column and bar represent the mean 6 S.E.M. pP , .05,
significantly different from each corresponding nonstressed group (by unpaired Student’s t test). †††P , .001, significantly different from nonstressed
healthy hamsters (by unpaired Student’s t test). H; healthy hamsters, CM; cardiomyopathic hamsters.
Fig. 6. Time-course changes in the levels of plasma NE
and E in healthy control and cardiomyopathic hamsters
after cold-immobilization stress. Each value represents
the mean 6 S.E.M. from four or five animals at different
time points. H; healthy hamsters, CM; cardiomyopathic
hamsters.
in figure 10. The heart, adrenal and kidney weights were of phentolamine (0.1–10 mg/kg i.p.) hardly affected the mor-
significantly (P , .05) increased in the stressed cardiomyo- tality seen in the stressed cardiomyopathic hamsters.
pathic hamsters compared with the nonstressed cardiomyo- When atropine, an antimuscarinic, was investigated for its
pathic animals. However, the stress caused no detectable action in this model, atropine (0.1–10 mg/kg i.p.) minimally
changes in healthy animals. The administration of propran- affected the sudden death seen in the stressed cardiomyo-
olol significantly and dose-dependently prevented the in- pathic hamsters, as shown in figure 12. Although doses of ,1
crease of weight of any organ in the stressed cardiomyopathic mg/kg atropine tended to accelerate the lethal effect of the
hamsters, with statistically significant (P , .05) difference in stress, in turn the larger dose (10 mg/kg) showed rather a
the group dosed with 10 mg/kg of propranolol (fig. 10). BCF tendency to delay death in the cardiomyopathic hamsters.
was minimally affected by the drug (data not shown).
Phentolamine, a nonselective alpha adrenoceptor blocker,
was evaluated for its effect on the sudden death of the Discussion
stressed cardiomyopathic hamsters. Figure 11 shows the The first important finding of the present study is that
mortality results. No healthy hamsters with or without cold-immobilization stress exerted a lethal effect in cardio-
stress and no nonstressed cardiomyopathic hamsters died myopathic Syrian hamsters but not in the control hamsters,
during the course of the experiment. In contrast, 5 of 6 of the suggesting that the stress aggravated the cardiovascular
stressed cardiomyopathic hamsters died, and administration dysfunction seen in cardiomyopathic hamsters with a covert1998 Stress and Cardiomyopathic Hamsters 131
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Fig. 7. Electrocardiographic traces from healthy and cardiomyopathic hamsters measured by telemetry ECG recording system before and after
cold-immobilization stress. A, Data from healthy hamsters 2 hr before the administration of the stress. Regular sinus rhythm is seen. B, Data from
healthy hamsters 2 hr after the administration of the stress. No changes in sinus rhythm are seen. C, Data from cardiomyopathic hamsters 2 hr before
the administration of the stress. Regular sinus rhythm is seen, although the heart rate is faster than that os control healthy hamsters. D, Data from
cardiomyopathic hamsters 2 hr after the administration of the stress. There is obvious heart rate slowing and an occasional atrial fibrillation. E, Data
from a cardiomyopathic hamster just before its death (24 hr after the stress). This animal showed an auriculo-ventricular block just before its death.
heart disease. The evidence is principally in line with the the stress could accelerate the cardiac hypertrophy in cardio-
findings of Ottenweller et al. (1987). Extending their find- myopathic hamsters as revealed by the dry organ weights. It
ings, however, the present study for the first time adds evi- was also supported by the result that stress elevated BCF in
dence of the possible cause of death. The fact that only the the animals, an index that is known to reflect the cardiovas-
animals with covert heart disease died suggests that stress cular age of each animal (Ottenweller et al., 1987, 1988),
has serious and even lethal consequences in cardiovascular suggesting the presence of fluid retention in the body cavity
function. Other, more direct evidence is derived from the commonly seen in animals with congestive heart failure. The
present telemetry recording data, which showed the presence absence of an additional increase in the activity of CPK, one
of severe arrhythmia in some of the cardiomyopathic ham- of the reliable measures of cardiac myolysis (Van Der Veen
sters after cold-immobilization stress. Acute heart failure and Willebrands, 1966), in stressed cardiomyopathic ham-
and/or lethal arrhythmia might be responsible for the death sters may be explained by the fact that the enzyme activity in
of the stressed cardiomyopathic hamsters because heart and the nonstressed cardiomyopathic animals was already much
adrenal weights were markedly increased in the animals. higher and more saturated than that in the healthy ones.
Given the fact that cardiomyopathic hamsters are known to In the present study, stress produced a marked increase in
develop hypertrophy of the heart spontaneously (Bajusz et the kidney weight and the serum levels of ALP, urea nitrogen
al., 1966; Bajusz and Lossnitzer, 1968; Strobeck et al., 1979), and creatinine in the cardiomyopathic hamsters, indicating a132 Matsuoka et al. Vol. 284
Fig. 8. Effects of propranolol (0.1–10 mg/kg) on sudden death caused by
immobilization stress in cardiomyopathic hamsters. Each value repre-
sents the percentage of surviving hamsters. pP , .05, statistically signif-
icant compared with saline-treated stressed cardiomyopathic hamsters
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(by Fisher’s exact probability test). Number in parentheses indicates the
number of animals in each group. Propranolol was administered intra-
peritoneally just before the stress for 5 consecutive days. C; nonstress
control group, S; stress group.
Fig. 9. Survival curves of unstressed and stressed cardiomyopathic
hamsters receiving propranolol treatment. Propranolol was administered
intraperitoneally just before the stress for 5 consecutive days in the first
week.
reduction in glomerular filtration rate possibly related to a
decrease in myocardial performance indicative of sudden
ventricular dysfunction. These changes were suggestive of
the involvement of acute failure in the kidney as well as in
the heart for the cause of sudden death in the stressed
cardiomyopathic hamsters. This view is compatible with ear- Fig. 10. Effects of propranolol on organ weights in stressed cardiomyo-
lier findings by others that certain forms of stress can pro- pathic hamsters. Each column and bar represent the mean 6 S.E.M. pP ,
duce abnormalities in the kidney (Altland and Highman, .05, ppP , .01, significantly different compared with the values of saline-
treated stressed cardiomyopathic hamsters (by unpaired Student’s t test).
1961; Knocker, 1955). Increased activity of the sympathetic †P , .05, ††P , .01, significantly different from the value of each corre-
nerves is one of the most important factors responsible for sponding nonstressed group (by unpaired Student’s t test). ### P , .001,
the increased afterload in cardiac dysfunction. Efferent sym- significantly different from nonstressed healthy animals (by unpaired
Student’s t test). C; nonstress control group, S; stress group.
pathetic activity is known to be distributed in a nonuniform
way, with significant increases to the heart and kidney, but
normal activity to some other organs, such as the liver or role of activated sympathetic drive in renal failure that could
lung (Esler et al., 1985). Increased renal sympathetic activity have resulted in the sudden deaths of the stressed cardio-
is known to contribute significantly to altered renal hemody- myopathic hamsters. Multiple organ failure might further be
namics, sodium and water retention and modulation of the involved in the mechanism underlying the vulnerability of
actions of other vasoactive hormones. For example, renal the cardiomyopathic hamsters to stress because the liver
blood flow is reduced in animals with heart failure (Millard et weight also was increased in the cardiomyopathic animals.
al., 1972). In humans, the sympathetic outflow to the kidney Another important finding of the present study is that a
is significantly increased in patients with heart failure beta blocker, propranolol, dose-dependently prevented the
(Hasking et al., 1986; Zelis and Flaim, 1982). Our present lethal effect of the stress in the cardiomyopathic hamsters.
results coincide with this evidence and suggest an important Taken together with the fact that the drug significantly re-1998 Stress and Cardiomyopathic Hamsters 133
present results determining plasma NE levels strongly sug-
gest the acceleration of peripheral sympathetic activity after
the stress. Catecholamines released from the adrenal me-
dulla are generally supportive of the actions of the rest of the
sympathetic nerves, which depend mainly on the release of
NE. In the present study, plasma E concentration increased
more slowly after the onset of stress than NE, and the in-
crease was more prolonged than that of NE. The changes in
the time course of plasma NE and E are in accordance with
well known differences in the actions of E released from the
adrenal and NE released from sympathetic postganglionic
nerve terminals. Given the evidence that E is usually the
predominant amine released from the adrenal and it has a
much greater affinity for beta adrenoceptors than NE, circu-
lating E could be involved in the sudden death of stressed
Fig. 11. Effects of phentolamine on sudden death caused by immobili- cardiomyopathic hamsters. Interestingly, however, there
zation stress in cardiomyopathic hamsters. Each value represents the
percentage of surviving hamsters. Numbers in parentheses indicate the were no significant changes in the time course of NE levels
number of animals in each group. Phentolamine was administered intra- between the cardiomyopathic and healthy control hamsters.
Downloaded from jpet.aspetjournals.org at ASPET Journals on October 29, 2015
peritoneally just before the stress for 5 consecutive days. C; nonstress This finding suggests that circulating NE is an important
control group, S; stress group.
factor but not a sole mechanism of sudden death of stressed
cardiomyopathic hamsters. More detailed studies using car-
diomyopathic hamsters with adrenalectomy or sympathec-
tomy would be required to better address the qualitative
difference of the contributions of sympathetic nerve dis-
charge and circulating E to the sudden death of cardiomyo-
pathic hamsters by stress.
The present finding that phentolamine failed to prevent
the sudden death of the animals subjected to cold-immobili-
zation stress would suggest that alpha adrenergic receptors
had little or no part in the stress-induced cardiac sudden
death. This finding may rule out the possibility of the in-
volvement of increased resistance of the peripheral vascula-
tures in the etiology of the stress-induced death in cardio-
myopathic hamsters. It has been well documented that both
divisions of the autonomic nervous system are usually toni-
cally active and the inhibitory effects of the tonic vagal ac-
Fig. 12. Effects of atropine on sudden death caused by immobilization tivity on the heart oppose the facilitatory influences of the
stress in cardiomyopathic hamsters. Each value represents the percent-
age of surviving hamsters. Numbers in parentheses indicate the number
tonic sympathetic activity. Thus, these antagonistic interac-
of animals in each group. Atropine was administered intraperitoneally tions of the autonomic nervous systems are supposed to be
just before the stress for 5 consecutive days. C; nonstress control group, involved in the cardiac dysfunction that leads to the sudden
S; stress group. death observed in stressed cardiomyopathic hamsters (Ver-
rier and Hagestad, 1985). However, the fact that atropine
failed to prevent the lethal effects of cold-immobilization
stored the increases in the weights of the heart, adrenal and stress or to affect the increase of weights of the heart, liver,
kidney caused by the stress, these results suggest that sym- adrenal and kidney (data not shown) in the stressed cardio-
pathetic beta adrenoceptors play an important role in the myopathic animals suggests that the parasympathetic
stress-induced cardiac sudden death of the cardiomyopathic nerves, including even the brain cholinergic systems, had
hamsters. An activation of the sympathetic nerves triggered little part in the stress-induced cardiac sudden death of the
by stress might participate in the sudden death as a conse- cardiomyopathic hamsters. To address in greater detail the
quence of increased incidence of heart failure and/or cardiac role of functional cross-talk between the sympathetic beta
arrhythmia. Furthermore, it is possible that the protective adrenoceptors and alpha adrenergic receptors or the para-
action of propranolol against sudden death might involve not sympathetic nerves, however, future studies will be required
only the inhibitory action of propranolol on the reduced tone to investigate the effects of the combination of phentolamine
of sympathetic nerve discharge that was excited by stress but or atropine with small dosages of propranolol on sudden
also the direct antiarrhythmic and anti-ischemic effects of death in the present model. It would be interesting and
the drug. important to see whether such a drug combination is more
Because the sympathetic nervous system and circulating effective in preventing survival.
catecholamine levels are major factors involved in the mech- In light of the validity of the present experimental para-
anisms of cardiac sudden death, increases in the circulating digm as an animal model of stress-associated cardiac sudden
levels of catecholamines might have caused or exacerbated death, the present results so far are in good accord with
potentially lethal arrhythmia and resulted in myocardial experimental and clinical findings that have shown benefi-
ischemia in the stressed cardiomyopathic hamsters. The cial effects of the blockade of beta adrenoceptors on cardiac134 Matsuoka et al. Vol. 284
sudden death (Beta-blocker Heart Attack Trial Research Altland PD and Highman B (1961) Effects of exercise on serum enzyme values and
tissues of rats. Am J Physiol 201:393–395.
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activity, such as propranolol, timolol or metoprolol, on car- hamsters with hereditary myocardiopathy: chemical and histochemical studies,
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diac sudden death (Beta-blocker Heart Attack Trial Research myopathies (Bajusz E and Rona G, eds) pp 519 –531, University Park Press,
Group, 1982; Widerhorn and Rahimtoola, 1994). In dogs, Baltimore.
Verrier (1986, 1987) found that beta blockade by propranolol Bajusz E, Baker JR, Nixon CW and Homburger F (1966) Spontaneous, hereditary
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in agreement with previous results by others suggesting the pranolol in patients with acute myocardial infarction. JAMA 247:1707–1714.
Bloom S and Cancilla PA (1969) Myocytolysis and mitochondrial calcification in rat
important role of the sympathetic nervous system in the myocardium after low doses of isoproterenol. Am J Pathol 54:373–391.
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Ann Clin Res 19:88 –95.
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aline release and sympathetic nervous system activity. J Hypertens 3:117–129.
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and Cancilla, 1969; Rona, 1985). In addition, a positive cor- neuropharmacology of cardiovascular regulation and stress. Neurosci Biobehav
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relation has been found between levels of myocardial NE and Hallman H, Farnebo LO, Hamberger B and Jonsson G (1978) A sensitive method for
the degree of cardiomyopathy in hamsters (Angelakos et al., the determination of plasma catecholamines using liquid chromatography with
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It is tempting to further speculate that beta adrenoceptors Norepinephrine spillover to plasma in patients with congestive heart failure:
Evidence of increased overall and cardiorenal sympathetic nervous activity. Cir-
in the brain might be involved in the response. The blockade culation 73:615– 621.
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that the stress produces lethal changes in the heart and/or Ottenweller JE, Tapp WN, Creighton D and Natelson BH (1988) Aging, stress, and
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drive and results in sudden death, although further studies Parker GW, Michael LH and Entman ML (1987) An animal model to examine the
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terms of its mechanisms and suggests therapeutic implica- Alprazolam reduces stress-induced mortality in cardiomyopathic hamsters. Phar-
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sudden cardiac death, in Sudden Cardiac Death: Prevalence, Mechanisms, and
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