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205
Alterations in Calcium Antagonist Receptors
and Sodium-Calcium Exchange in
Cardiomyopathic Hamster Tissues
John A. Wagner, Harlan F. Weisman, Adele M. Snowman, Ian J. Reynolds,
Myron L. Weisfeldt, and Solomon H. Snyder
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The Syrian cardiomyopathic (CM) hamster (BIO 14.6) develops a progressive cardiomyopathy
characterized by cellular necrosis, hypertrophy, and, eventually, cardiac dilatation and
congestive heart failure. Several lines of evidence implicate cellular calcium overload as an
Important etiologic factor. We previously reported an increased number of receptors for
calcium antagonist drugs, which block voltage-dependent calcium channels, in heart, skeletal
muscle, and brain tissue of these hamsters in the early necrotic stage of the disease. To better
characterize the pathophysiological significance of this abnormality we evaluated calcium
antagonist receptor binding and Na + -Ca 2+ exchange in CM and control hamsters at different
stages of disease as documented by quantitative histopathologic assessment. In CM hamsters as
young as 10 days, an age previously thought to be before the onset of disease, we identified
cardiac myocyte hypertrophy, a twofold increase in calcium antagonist receptor binding in
heart and brain, and a 50% increase in skeletal muscle. Overt histological lesions were present
in skeletal muscle at 25 days and in heart between 28-30 days. The size of cardiac lesions
increased over time and changed from necrotic foci with cellular infiltration to fibrotic or
calcined lesions by 360 days. Myocardial cellular hypertrophy persisted through the late stages
of the disease (360 days), but increased calcium antagonist binding was present in heart only to
6 months of age, in skeletal muscle to 90 days, and in brain to 30 days. Na + -Ca 2+ exchange in
heart was normal until 15 days and then increased by 400% at 30 days suggesting that this
augmentation might be a secondary response to the earlier increase in calcium antagonist
receptors. At 360 days cardiac Na + -Ca 2+ exchange was decreased by 50%, likely reflecting
progressive cardiac damage. The increase in calcium antagonist receptors in CM animals as
young as 10 days supports the hypothesis that abnormalities in voltage-dependent calcium
channels play a role in the pathophysiology of CM hamsters. (Circulation Research 1989;65:
205-214)
A
n inbred strain of hamsters, the Syrian cardiomyopathic (CM) hamster (BIO 14.6,
Bio Breeders, Fitchburg, Massachusetts),
displays hereditary abnormalities in cardiac and
skeletal muscle. Heart involvement results in myocardial hypertrophy followed by progressive cardiac dilatation and premature death from congestive
From the Departments of Neuroscience, Pharmacology and
Molecular Sciences, Psychiatry and Behavioral Sciences and
Division of Cardiology, Department of Medicine, The Johns
Hopkins Medical Institutions, Baltimore, Maryland.
Supported by US Public Health Service grants MH-18501,
RSA award DA-00074 to S.H.S., and HL-17655 to M.L.W.
H.F.W. is the recipient of a Oinician-Scientist Award from The
Johns Hopkins School of Medicine.
Address for correspondence: Dr. Solomon H. Snyder, The
Johns Hopkins University School of Medicine, Department of
Neuroscience, 725 North Wolfe Street, Baltimore, MD 21205.
Received August 23, 1988; accepted December 28, 1988.
heart failure.1-3 Calcium overload of myocytes has
been implicated in the etiology of abnormalities in
CM hamsters; since calcium uptake in the myocardium is increased,4-5 cardiac action potentials display prolongation compatible with augmented
voltage-dependent calcium channels, 6 and calcium
antagonist drugs such as verapamil are uniquely
efficacious in ameliorating the manifestations of the
disease. 4 ' 7 - 9 CM hamsters may provide a useful
model of human cardiac diseases such as ischemia
and reperfusion and hypertrophic cardiomyopathy
that share common pathophysiological mechanisms
as well as a selective efficacy of verapamil.10
Previously, we reported enhanced numbers of
calcium antagonist receptors in heart, skeletal muscle, and brain tissue of CM hamsters,11 observations confirmed by others. 12 - 16 In the present study,
we characterize in greater detail the alterations in
206
Circulation Research
Vol 65, No 1, July 1989
calcium antagonist receptor binding in several tissues of CM hamsters at different stages of disease
as determined by quantitative histopathology and
also describe alterations in Na+-Ca2+ exchange.
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Materials and Methods
Histopathologic Studies
Male hamsters (BIO 14.6 and age-matched FIB
controls; Bio Breeders) were killed at various ages to
document the presence and extent of disease. At
sacrifice, the animals received sodium heparin (100
units i.p.) and were anesthetized with intraperitoneal
sodium methohexital (35 mg/kg). They were then
weighed, and their body lengths were measured from
nose tip to the beginning Of the tail. The hearts were
excised, placed in iced 30 mM KC1 to achieve
diastolic arrest, blotted dry, weighed, and fixed by
immersion in a 4% buffered formaldehyde solution
for 24 hours. The brains were also removed, weighed
after blot drying, and fixed whole by immersion in
the formaldehyde solution. Small samples of skeletal
muscle were obtained from the tongue, acromiotrapezius, biceps femoris, rectus abdominis, and external oblique and also fixed in formaldehyde.
The fixed hearts were serially sliced into 2-mm
rings parallel to the atrioventricular groove from
base to apex. Each of the slices was embedded in
glycol methacrylate. The plastic-embedded blocks
were then cut in 3 jxm thick serial sections with a
glass knife and Sorvall JB-4 microtome. The serial
sections were cut in groups of four with 200 fim
between groups. The sections were mounted on
glass slides, and the first section from each group
was stained with toluidine blue, the second with
hematoxylin-eosin, the third with Masson's trichrome for connective tissue, and the fourth with
the Von Kossa method for calcium deposits and
Kernechtrot counterstain.17
The brains were sliced serially into 2-mm coronal
sections, which were embedded in paraffin. The
skeletal muscle specimens were also embedded in
paraffin. The embedded brain slices were cut into
5-^tm-thick sections, mounted on slides, and stained
with hematoxylin-eosin. Three sections of 5-^rn
thickness from each skeletal muscle block were cut
and mounted and stained with hematoxyMn-eosin,
Masson trichrome, or Von Kossa/Kernechtrot
stains.
The image of each histological heart section was
projected at a magnification of 7.5-10.5 depending
on the heart size and traced onto paper. Intramyocardial lesions were identified and also traced. The
lesions were classified into four subtypes: 1) cellular: myocytolysis and contraction band necrosis
with cellular infiltration; 2) mixed: cellular infiltration with fibrosis; 3) fibrotic: collagen fibers and
fibroblasts without other cell types; and 4) calcified:
lesions with calcification with or without cellular
infiltration or fibrosis. The presence, type, and
extent of cardiac lesions were confirmed by micros-
copy. The areas encompassed by the epicardial
contour, the endocardial contour and lesions, were
digitized using a Videoplan image analysis microcomputer (Carl Zeiss, Thornwood, New York).
Cross-sectional left ventricular cavity diameter was
measured along a line connecting the centers of
mass of the right ventricular and left ventricular
chambers. The myocardial area of each section was
calculated by subtracting the endocardial area from
the epicardial area. For each heart, total left ventricular myocardial volume, left ventricular cavity
volume, and lesion volume (total and by each
subtype) were estimated by serial reconstruction of
integrated cross-sectional areas of the histological
heart rings. The amount of each type of myocardial
injury as well as total amount of tissue injury were
expressed as a percentage of total calculated ventricular volume.
To assess the development and extent of myocyte
hypertrophy, maximal myocyte diameter was determined from the serial sections. Cross-sectional profiles of cells were outlined at a magnification of
x500 by projecting the cursor of the Videoplan
computer through a camera lucida attached to a
light microscope. The maximal diameter of the
digitized profile was then automatically measured
and recorded. To assure that measurements were
made near the cell centers, only cell profiles containing nuclei were measured. Between 25 and 50
cells per serial section were measurable with these
criteria. Because the 3-^.m sections from which the
measurements were made were taken at 200 -fim
intervals—a distance two to three times the length
of myocytes—no cell was measured more than
once. At least 200 cells from each of the original
heart rings were measured, resulting in a minimum
of 600 measured cells for the smallest hearts to over
1,000 cells for the largest hearts. Differences in cell
diameter between CM and control hamsters at each
age were tested for significance by Student's t tests.
Tissue Preparation for Binding Assays
Littermates of the CM and control hamsters used
in the pathological studies were killed at the same
ages by cervical dislocation and decapitation, and
their brains, hearts, and hindleg skeletal muscle
were rapidly removed and placed on ice. All further
steps took place at 4° C.
Brains were homogenized in 50 mM HEPES, pH
7.4, 50 mg wet wt tissue/ml buffer, using a Brinkmann potytron (setting on half-maximal for 30 seconds; Brinkmann Instruments, Westbury, New
York). Homogenates were centrifuged for 15 minutes at 40,000g. The pellet was resuspended in 50
mM HEPES, pH 7.4, and centrtfuged for 15 minutes at 40,0G0#. This pellet was resuspended in 50
mM HEPES, pH 7.4, 100 mg wet wt brain/ml
buffer, and stored at -20° C until use.
Hearts were homogenized in 260 mM sucrose, 50
mM HEPES, pH 7.4, 50 mg wet wt tissue/ml buffer
using a Brinkmann polytron (setting on half-
Wagner et al Ca2+ Antagonist Receptors and Na+-Ca2+ Exchange in Hamster Cardiomyopathy
maximal for 30 seconds). Homogenates were centrifuged at l.OOQg for 10 minutes, and the resulting
supematants were filtered through four layers of
gauze and centrifuged at 10,000g for 20 minutes.
Supematants were diluted with enough 3 M KC1, 50
mM HEPES, pH 7.4, to bring the final KC1 concentration to 1 M, and the suspensions were incubated
on ice for 30 minutes followed by centrifugation at
150,000g for 60 minutes. Resulting pellets were
resuspended in 50 mM HEPES, pH 7.4, 5 mg
protein/ml buffer, and stored at -20° C until use.
Skeletal muscle was prepared similarly. Calcium
antagonist binding activity remains unaltered in
samples frozen up to 6 months.
207
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100-150 fig protein/1 ml assay, were incubated with
0.1-0.3 nM [3H]nitrendipine and varying concentrations of cold nitrendipine. Assays were terminated
after 1 hour at room temperature (22-24° C) by
filtration over glass fiber filters (Schleicher and
Schuell, Keene, New Hampshire) using a Brandel
cell Harvester (Brandel, Gaithersburg, Maryland).
Filters were washed with three 4 ml aliquots of
ice-cold 50 mM NaCl. Filters were placed in 4 ml of
Formula 963 liquid scintillation cocktail (NENDuPont, Boston, Massachusetts), and trapped radioactivity was counted in a Beckman LS2800 scintillation counter (Beckman Instruments, Fullerton,
California) at an efficiency of 55%. Specific binding
was defined as total binding less binding in the
presence of 1 yuM nitrendipine. Saturation plots
were analyzed with the UGAND computer program.20
Protein was measured with the BCA assay (Pierce,
Rockford, Illinois) with BSA fraction V (Sigma
Chemical, St. Louis, Missouri) as protein standard.
Tissue Preparation for Na+-Ca2+ Exchange Assay
Hamsters were killed, and brains and hearts were
rapidly removed and placed on ice. All further steps
took place at 4° C. Synaptosomes were prepared
according to the method of Hajos18 with the following modifications. Brains were homogenized in 260
mM sucrose and 50 mM HEPES, pH 7.4, using a
motor-driven, loose-fitting Teflon-glass homogenizer (12 up-and-down strokes). The homogenate
was centrifuged at l,00Qg for 10 minutes, and the
pellet was discarded. The supernatant was diluted
with an equal volume of basal buffer (mM: NaCl
160, Tris 20, pH 7.4) and centrifuged at 9,000g for
15 minutes. The resulting pellet was gently resuspended in 3 ml of 260 mM sucrose and 50 mM
HEPES, pH 7.4, using the Teflon-glass homogenizer, and the suspension was layered over 20 ml of
800 mM sucrose. This two-step gradient was centrifuged at 10,000g for 25 minutes. The interface
between the low-density supernatant and the pellet
was aspirated, diluted with basal buffer, and centrifuged at ll,000g for 15 minutes. This pellet was
resuspended in 2 ml basal buffer. The synaptosomes
were loaded with Na by overnight incubation in
basal buffer at 4° C.
Hearts were prepared as described.19 Briefly,
hearts were rinsed with ice-cold 300 mM sucrose, 5
mM MgSO4,10 mM imidazole, pH 7.0, minced with
scissors, and combined with 4 volumes of 300 mM
sucrose, 5 mM MgSO4, 10 mM imidazole, pH 7.0.
All further steps took place at 4° C. This mixture
was subjected to four 5-second bursts with a Brinkmann polytron at setting 8. The mixture was homogenized using a motor-driven Teflon-glass homogenizer (two up-and-down strokes). The sucrose
concentration was adjusted to 600 mM with a 1 M
sucrose solution and centrifuged at 28,00Qg for 30
minutes. The supernatant was diluted 1.5-fold with
basal buffer and centrifuged at 43,6O0g for 30 minutes. The resulting pellet was resuspended in basal
buffer and incubated overnight at 4° C.
Materials
[3H]Nitrendipine (specific activity 78 Ci/mmol)
and ^CaC^ were obtained from NEN-DuPont.
Nitrendipine was obtained from sources described
previously.11 Choline chloride was obtained from
Calbiochem, La Jolla, California; choline used for
Na+-Ca2+ exchange incubation buffers was recrystallized from ethanol and stored under high vacuum
until use. All other chemicals were reagent grade
from commercial sources.
Calcium Antagonist Binding
Dihydropyridine (DHP) binding using pHJnitrendipine was assayed as described.11 Membranes, diluted
with 50 mM HEPES, pH 7.4, to a concentration of
Results
Gross Pathological Findings
Animal weight, body length, heart weight, and
brain weight increased progressively with age.
45Ca2+ Flux Measurements
45
Ca2+ uptake through N-type Ca2+ channels into
hamster brain synaptosomes was measured as
described.21 Measurement of Na+-Ca2+ exchange
was essentially as described.19 Briefly, Na+-loaded
synaptosomes and cardiac membrane vesicles were
briefly preincubated at 30° C; 25 fil aliquots were
injected into 975 (JL\ of basal buffer or Na+-free
buffer (160 mM choline chloride recrystallized from
ethanol, 20 mM Tris HC1, pH 7.4) and incubated for
1 second at 30° C. Reactions also contained appropriate concentrations of 45CaCl2. Reactions were
terminated by the addition of 3 ml ice-cold stop
buffer (160 mM choline chloride, 1 mM CaCl2, 20
mM Tris HC1, pH 7.7,1.8 mM LaCl3) timed with the
use of an electronic metronome. Vesicles were
collected on glass fiber filters, as above, using a
Brandel cell harvester and rinsed twice with 4 ml
ice-cold stop buffer without LaCl3. Retained radioactivity was determined as above. Na + -Ca 2+
exchange was defined as the difference between
uptake in Na-free and basal buffers. Data were
analyzed by Lineweaver-Burke transformation and
simple linear regression.
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Circulation Research
Vol 65, No 1, July 1989
TABLE 1. Morphologic Features of Cardiomyopathic Hearts
A. Extent of hypertrophy
Heart wt/body wt(x10~ 3 )
Age
14.6
FIB
(days)
4.6±0.1
10
4.6±1.8
4.9±0.1
4.9±0.1
15
4.1±0.1
4.1±0.1
30
3.9±0.1
5.4±0.2*
180
3.8±0.2
6.2±0.6*
240
360
7.4±1.5*
4.1±0.1
Heart wt/brain wt
FIB
14.6
0.07±0.03
0.14±0.02
0.24±0.02
0.52±0.02
0.56±0.01
0.48±0.02
0.08±0.01
0.14±0.02
0.25±0.01
0.61+0.05*
0.79±0.0S*
O.70±O.O3*
B. Percent of total myocardium occupied by lesions (14.6 only)
Cellular
Days
Mixed
0
0
10
Fibrotic
Maximum cell diameter (/un)
FIB
14.6
10.0±0.1
11.4+0.2'
12.5 ±0.1*
11.5+0.1
16.3 ±0.2*
14.5±0.1
24.5±0.4f
17.3±0.2
18.7±0.3
27.4 ±0.4f
28.8 ±0.4f
21.2±0.6
Total
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0
Calcified
0
15
0
0
0
0
0
30
0.1±0.05
0
180
240
0
0
0.1 ±0.05
3.1±0.1
0.8±0.4
0.2±0.2
1.2±0.2
0.5±0.1
360
0
0
0.4+0.3
8.0+0.01
12.6±0.5
14.7
3.6±0.2
11.2±1.0
13.2
1.4
0
Results are expressed as mean±SEM. Four to 11 animals for each group were studied except that the volume fraction of lesions was
measured in only one 360-day-old CM (BIO 14.6) hamster. Comparisons between cardiomyopathic hearts and control (FIB) hamsters
were made with Student's t tests.
•p<0.05.
Between 10 and 30 days of age, there were no
significant differences in these values between control and CM hamsters at each age (data not shown).
At 180 days, control hamsters had significantly
greater body weight (141±1 g vs. 114±2g) and
greater body length (18.8±0.8 cm vs. 15.9±0.1 cm).
By contrast, heart weight at 180 days of age was
less in the control hamsters (0.56±0.02 g vs.
0.65 ±0.04 g), but there continued to be no difference in brain weight (1.06+0.02 g vs. 1.02+0.02 g).
This pattern of greater body weight and length, near
equal brain weight, but lower heart weight in controls compared with CM animals was also seen at
240 and 360 days (data not shown). Left ventricular
cavity size was not significantly different between
the younger control and CM hamsters (e.g., 30 day
mean cavity diameter was 1.9±0.1 mm in the controls and 2.1 ±0.2 mm in the CM hamsters). At 180
days, mean cavity diameter was 2.9 ±0.1 mm in
controls and 3.8±0.2 mm in CM animals (p<0.05).
Thereafter, the CM hamsters exhibited progressively greater cavity dilatation.
Histological Findings
Cardiac myocyte hypertrophy was apparent in
CM hamsters as early as 10 days, the youngest age
we examined (Table 1). This is striking because it is
generally felt that the animals are free of disease at
this early age and by gross measurement there is no
significant increase in heart mass. 1 - 3 As in previous
studies, we did not identify any other histological
abnormalities at this age. CM hamster myocytes
were significantly larger at all other ages, but grosser
indexes of hypertrophy, such as the ratios of heart
weight to body weight and heart weight to brain
weight, did not show significant differences between
the control and CM hamsters until 180 days of age.
Although the ratio of heart weight to body weight is
more often used as an index of gross hypertrophy,
the ratio of heart weight to brain weight may be
preferable, because the CM hamsters have slower
increases in their body weight than controls, which
alters the usual relation between heart and body
weight.
Heart lesions were not usually seen until the
animals were 30 days of age, but a few animals
showed small myocytolytic necrotic lesions at 28
days (Table IB). Although some CM animals at 30
days showed myocytolysis without cellular infiltration, these lesions comprised less than 0.1% of the
myocardium and thus are not shown separately in
the table. The typical lesion at 30 days showed
necrosis including the presence of contraction bands
and a dense cellular infiltrate composed of cells
resembling histiocytes, lymphocytes, and monocytes (Figure 1A, Table IB). There was evidence of
early healing in some lesions with fibroblasts and
collagen deposition. A large proportion of the cellular and mixed lesions showed at least some calcification on the Von Kossa stained sections (Figure
IB). Aside from a small dip in the amount of
calcium deposition between 180 and 240 days, which
likely was due to aberrant sampling, the amount of
calcium deposition became more prevalent with
time (Table IB). Widespread fibrosis with no exclusively cellular lesions was evident at 180 days and
more pronounced at 240 and 360 days. These quantitative morphometric findings are in agreement
with earlier qualitative descriptions of the histological features of the cardiomyopathy.'
Wagner et al Ca2+ Antagonist Receptors and Na+-CaJ+ Exchange in Hamster CardJomyopathy
209
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FIGURE 1. A: Cardiac histopathology of
a 30-day-oid cardiomyopathic hamster. The
central lesion is characterized by a dense
cellular infiltrate with myocyte necrosis.
The upper margin of the lesion consists of
contraction band necrosis, a finding typical of injury related to cellular calcium
overload (hemataxylin-eosin, original magnification, x64). B: A more advanced
lesion in a 90-day-old hamster is characterized by calcium deposition and fibrosis
with minimal cellular
components
(hemataxylin-eosin, original magnification, x64).
B
Focal myocytolysis was seen in skeletal muscle
as early as 25 days (Figure 2). In general, the
progression of skeletal muscle lesions paralleled
those of the heart but was somewhat accelerated.
No brain lesions were apparent.
Ontogeny of Altered Calcium Antagonist
Receptor Binding Sites in Cardiomyopathic
Hamsters
In our initial studies, we showed increases in
numbers of dihydropyridine calcium antagonist
receptors as well as phenylalkylamine calcium antagonist receptors at 30, 60, and 90 days in several CM
hamster tissues.11 An important concern is whether
alterations in receptor binding sites are secondary
to cardiac pathology. Accordingly, in the present
study we have examined [3H]nitrendipine binding to
dihydropyridine receptors at ages ranging from 10
to 360 days (Table 2; Figures 3 and 4). At 10 days,
the earliest time examined, [3H]nitrendipine binding
was enhanced about twofold in membranes of heart
and brain and almost 50% in skeletal muscle. Sta-
tistically significant increases continued to be apparent for [3H]nitrendipine binding to 180 days in
heart, 90 days in skeletal muscle, and 30 days in
brain. To ascertain whether alterations in [3H]
nitrendipine binding derived from abnormalities in
the affinity of the ligand for binding sites or in the
numbers of binding sites, we conducted saturation
analysis monitoring binding of different concentrations of the [3Ff]nitrendipine. The [3H]nitrendipine
increases in binding can be attributed solely to
augmented B ^ values with no change in KD levels
in the three tissues (Figures 3 and 4; data for
skeletal muscle not depicted).
Na+-Ca2+ Exchange in Heart and Brain
of Cardiomyopathic Hamsters
Previously we reported augmented 45Ca2+ accumulation in nerve ending preparations (synaptosomes) of CM hamster brain.11 Calcium accumulation can be mediated either by voltage-dependent
calcium channels or by Na + -Ca 2+ exchange.
Recently, we21-22 and others23-25 have observed
210
Circulation Research Vol 65, No 1, July 1989
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FrGURE 2. A: Skeletal muscle from a 28-day-old control (FIB) animal demonstrating normal histological findings
(hematoxylin-eosin, original magnification, x64). B: Skeletal muscle from a 28-day-old cardiomyopathic (BIO 14.6)
hamster showing myocytofysis and cellular infiltration (hematoxylin-eosin, original magnification, x64).
that, when substantial levels of sodium are included
in the incubation buffer, synaptosomal 45Ca2+ accumulation is attributable primarily to Na + -Ca 2+
exchange rather than to voltage-dependent calcium
channels.21 Since sodium was included in incubation buffers employed for our studies of synaptosomal calcium uptake in CM hamsters,11 the resultant calcium accumulation presumably involved
Na+-Ca2+ exchange. To examine this question directly,
we compared synaptosomal 45Ca2+ accumulation with
sodium in the buffer as in earlier experiments11 and
with choline in the buffer under conditions in which
we have shown that calcium uptake involves Only
N-type voltage-dependent calcium channels.21 As
observed previously, when sodium was included in
the incubation buffer, synaptosomes from CM hamsters displayed augmented Ca2+ accumulation (control = 1.22±0.23 and CM- = 1.78±0.19 nmol/mg;
p<0.05). Nitrendipine (1 /*M) did not alter control or
CM 45Ca2+ accumulation in the presence of sodium.
However, with choline in the buffer, no abnormality
in 45Ca2J|" uptake was detected (control = 3.60±0.41
and CM = 3.73±0.62 nmol/mg). Therefore, the
increase in Ca2+ uptake by synaptosomes is not mediated through N-type Ca channels.
To directly examine Na+-Ca2+ exchange, we measured ^Ca2* uptake into membrane vesicles prepared from brain and heart (Figures 5 and 6).
Vesicles were loaded for 16 hours with 160 mM
NaCl and 45Ca2+ uptake then monitored in a 1second incubation in buffers containing either 160
mM sodium or 160 mM choline. In the presence of
choline, the large inside-outside gradient for sodium
will result in sodium efflux associated with 45Ca2+
influx via the Na + -Ca 2+ exchange mechanism
whereas the Na+-Ca2+ exchange mechanism should
not be operative with identical concentrations of
sodium on the inside and outside of the vesicles.
Accordingly, Na+-Ca2+ exchange can be monitored
as the difference between 45Ca uptake in buffers
containing choline in the medium minus uptake in
buffers containing sodium in the medium. In these
experiments, we evaluated Na+-Ca2+ exchange in
the presence of different concentrations of calcium
to ascertain whether any alterations would be determined by changes in affinity (Km) or maximal velocity of uptake (V,^).
Na+-Ca2+ exchange was increased in both brain
and heart of CM hamsters. However, unlike calcium
antagonist receptor binding, which was most elevated in young hamsters, no changes were evident in
Na+-Ca2+ exchange in 10- and 15-day-old hamsters,
while V ^ (nmoles per milligram protein ±SEM)
was doubled at 30 days in CM brain (control 25±3;
CM 53±6) and increased fivefold at 30 days in CM
heart (control 22±3; CM 110±15). At 360 days
Wagner et al Ca1+ Antagonist Receptors and Na+-Ca2+ Exchange in Hamster Cardiomyopathy
211
TABLE 2. [3H]NItiTndiplne Binding In the Heart, Brain, and
Skeletal Muscle of Cardiomyopathic Hamsters
(fmol/mg protein)
(days)
FIB
14.6
Heart
10
15
30
180
360
120±35
140±35
210±22
220±32
2O0±25
234±38*
410±120
370±37*
320±28*
26O±35
E
gaoa
O
0.2
Brain
10
15
30
90
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180
240
360
150±35
134+32
110±10
120±22
125±15
138±21
135 ±27
31O±61*
250+40*
210±ll f
150±17
126+11
13O±29
147±32
1,210±100
l,260±80
1,410±140
1,350+180
1,430±210
l,4OO±230
1,490+200
1,7^160 *
l,920±130t
l,870±190'
l,880±210
l,840±240
Skeletal Muscle
10
15
30
90
180
240
360
0.4
[•«] NITRENDIPINE SOUND (»mol/ag)
<MMn
B
SOLOS-
l,780±190'
0.2
CL4
[•«] N1TMNDIPINE SOUND (»«ol/m«>
1,620±230
Results are expressed as mean±SD for groups of six to 10
animals of each strain for each age. KD values did not differ
significantly between groups and were similar to previously
reported values.11 In brain, KD values ranged from 0.22 to 0.42
nM. In heart, KD values varied from 0.15 to 0.33 DM. In skeletal
muscle, KD values ranged from 2.9 to 4.3 nM. There was a small,
but not statistically significant, tendency for KD values to increase
in all three tissues in older animals (180-360 days). Comparisons
between CM (BIO 14.6) and control (FIB) hamsters were made
with Student's t tests.
*p<0.05.
•aoi-
V<0.01.
Na+-Ca2+ exchange was not altered in brains and
reduced 50% in hearts of CM hamsters (control 18 ±4;
CM 9±3). The alterations in Na+-Ca2+ exchange
involved V , ^ with no change in the Km values.
Discussion
In the present study we have confirmed and
extended our previous observations that calcium
antagonist receptor binding levels are augmented in
tissues of CM hamsters. Using the same BIO 14.6
strain of CM hamsters, Finkel et al12-13 and Kobayashi et al14 have confirmed the increase in cardiac
[3H]nitrendipine binding. Some investigators have
been unable to demonstrate this increase in dihydropyridine binding sites in related cardiomyopathic strains. Howlett and Gordon26 did notfindan
increase in the number of dihydropyridine binding
sites in cardiac muscle homogenates from 50- to
60-day-old CHF 146 CM hamsters compared with
matched controls (CHF 148). However, this CM
strain is not as well characterized as the BIO 14.6
0.1
0.4
[*H] NITRCNOIPINE I0UND (»Ml/*g)
FIGURE 3. Saturation analysis of [3H]nitrendipine binding in heart of 10- (A), 30- (B), and 360-day-old (C) control
(FIB) and cardiomyopathic (CM) (BIO 14.6) hamsters.
The concentration of nitrendipine varied between 0.05 and
100 nM. Data shown are from typical experiments performed in duplicate. Experiments were repeated six to 10
times. The total number of binding sites (B^ is increased
in CM hamsters, with no change in binding affinity.
strain, and it is possible that the progression of
disease differs between the two strains. Because we
found that the presence and extent of the abnormality in dihydropyridine binding sites changes as the
disease progresses, differences in disease progression between the two strains may explain the differences in our results. In another study, no increase
in [3H]PN 200-110 binding was observed in crude
heart homogenates from 35- to 41-day-old 53.58 CM
hamsters.27 However, when Kuo et al15 examined
212
Circulation Research Vol 65, No 1, July 1989
A
A
o CONTROL
ZOO-
o
•
1
CONTROL
O2
O.I
0.2
[>H] H|T*CNDIPINE BOUND
,T
./
CM
0.4
0.5
IpwM/n«)
B
B
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i
o CONTROL
• CM
^ /
o/
•
> 100-
0.2
0.2
ai
0.4
ai
[>H] NITTCNDIPINE BOUNP (pro
200
o CONTROL
• CM
o CONTROL
• CU
-o.t
[ * H ] NITRCNDIPINC BOUND(paol/ag)
4. Saturation analysis of [3H]nitrendipine binding in brain of 10- (A), 30- (B), and 360-day-old (C)
control (FIB) and cardiomyopathic (CM) (BIO 14.6)
hamsters using the same techniques as described for
heart (Figure 3). The total number of binding sites (B^J
is significantly increased in CM hamsters at 10 and 30
days of age but similar to controls at 360 days. As with
heart tissue the increase in B^a is seen with no change in
binding affinity.
FIGURE
[3H]PN 200-110 binding to a purified cardiac sar-
colemmal preparation from 2-month-old hamsters
of the same 53.58 strain, they found an augmentation in calcium antagonist receptor number compared with matched controls. In addition, in another
study, Kuo et al16 observed an increase in dihydropyridine calcium antagonist receptors in cardiac
sarcolemma from 30-day-old BIO 14.6 CM hamsters using the reversible ligand [JH]PN 200-110 as
well as photoaffinity techniques with the dihydro-
0.4
FIGURE 5. Lineweaver-Burke plots of brain Na+-Ca2+
exchange in 10- (A), 30- (B), and 360-day-old (C) control
and cardiomyopathic (CM) hamsters. Typical plots are
depicted. Experiments were repeated seven or eight times.
pyridine [3H]aziodopine. The two studies that failed
to demonstrate an increase in calcium antagonist
receptor sites used crude muscle homogenates rather
than purified membrane preparations employed in
studies showing an increase in dihydropyridine binding. Other tissue components in the homogenates
might have masked increases in dihydropyridine
binding.
One striking finding of the present study was that
the augmented number of calcium antagonist binding sites was most apparent in younger hamsters
and was no longer detectable in older animals with
more extensive cardiac hypertrophy and congestive
heart failure. The fact that the largest increases
occur in the very young hamsters before the development of necrotic lesions or heart failure suggests
Wagner et al Ca1+ Antagonist Receptors and Na+-Ca2+ Exchange in Hamster Cardiomyopathy
•
am.
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az
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• en
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-0.2
yf
y^
/
0.2
0,4
/fco'+l iy M- )
40CH
200-
-0.2
6. Lineweaver-Burke plots of heart Na+-Ca2+
exchange in 10- (A), 30- (B), and 360-day-old (C) conW
and cardiomyopathic (CM) hamsters. Typical plots are
depicted of experiments repeated on seven or eight
occasions.
FIGURE
that the abnormalities are not secondary to cardiac
pathology. Moreover, the increased numbers of
receptors in brain and skeletal muscle support this
conclusion.
We found evidence of myocardial cell hypertrophy in CM animals at the same early age that the
increase in calcium antagonist receptors was identified. Other investigators have suggested that myocardial hypertrophy is a secondary phenomenon in
the CM hamster, serving as a compensatory response
to myocardial tissue injury and resultant loss of
ventricular function.28 However, in the present
study, myocyte hypertrophy was present at a time
before either structural or functional impairment
had been documented.2-4 The discrepancy between
the quantitative morphometric data indicating an
early increase in cardiac myocyte diameter and the
213
grosser estimates of cardiac hypertrophy (heart
weight-to-body weight and heart weight-to-brain
weight ratios) is probably seen because these latter
techniques have less sensitivity in detecting modest
degrees of hypertrophy, especially in the extremely
small hearts of the younger hamsters. For example,
the approximate 10% greater cell diameter seen in
the CM animals 30 days old or younger would
account for an approximate 20% increase in cell
mass, using a cylindrical model for myocytes. This
difference was detected by several thousand cell
diameter determinations in each group of animals.
Assuming a<0.05 and jS<0.2, we would have had to
weigh about 100 hearts in each group to be certain
that we could detect the same degree of hypertrophy, given the variance present in our heart weight
determinations. Nevertheless, we cannot exclude
the possibility that the CM hamsters have fewer,
but larger, left ventricular myocytes than the control hamsters, accounting for the increased cell
mass with little or no change in overall heart mass.
In the present study, we provide evidence that
the increased synaptosomal accumulation of calcium in brains of CM hamsters is attributable to
augmented Na+-Ca2+ exchange rather than to an
abnormality in voltage-dependent calcium channels
in nerve terminals. The calcium antagonist receptor
binding sites we have monitored are associated with
voltage-dependent calcium channels that mediate
calcium influx. At least three subtypes of voltagedependent, calcium channels have been differentiated,29-30 and the calcium antagonist receptors are
only associated with one of these, the L subtype.
N-type calcium channel activity is readily observed
in buffers containing choline in place of sodium.21
L-type calcium channel activity is not reliably
detected in synaptosomal preparations. We monitored voltage-dependent ^Ca accumulation into
brain synaptosomes mediated by the N-type calcium
channel and detected no abnormality in CM hamsters. Accordingly, it appears that the genetically
determined defect in CM hamsters is restricted to
the L-type calcium channel.
The increased Na+-Ca2+ exchange occurs subsequent to the alteration in calcium antagonist receptors and so may reflect a secondary response to
increased calcium influx following the increased numbers of L-type voltage-dependent channels. The
notion that the increased Na+-Ca2+ exchange is a
secondary compensatory phenomenon may also
explain why more pronounced increases in Na+-Ca2+
exchange are seen in heart, which displays pathological alterations, than in the brain, where no alterations are apparent. Increases in the Na+-Ca2+
exchange system, which physiologically extrudes
Ca2+ from cells, may occur in response to the intracellular Ca2+ overload characteristic of CM hearts.
In summary, the present study supports conclusions from our earlier work11 suggesting that abnormalities in calcium antagonist receptors associated
with voltage-dependent calcium channels play a
214
Circulation Research
Vol 65, No 1, July 1989
major role in the pathophysiology of CM hamsters.
The CM hamsters may provide a model for human
hypertrophic cardiomyopathy, which shares some
pathophysiological features as well as the selective
therapeutic response to calcium antagonist drugs.
Supporting this possibility, Ferry and Kaumann31 as
well as ourselves32 have observed increased calcium antagonist receptor binding in cardiac tissue
from these patients comparable in degree to the
alterations in CM hamsters.
References
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1. Homburger F, Baker JR, Nixon CW, Whitney R: Primary
generalized polymyopathy and cardiac necrosis in an inbred
line of Syrian hamsters. Med Exp 1962;6:339-345
2. Bajuz E, Baker JR, Nixon CW, Homburger F: Spontaneous
hereditary myocardial degeneration and congestive heart
failure in a strain of Syrian hamsters. Ann NYAcad Sci 1969;
156:105-129
3. Gertz EW: Cardiomyopathic Syrian hamster: A possible model
of human disease. Prog Exp Tumor Res 1972;16:242-260
4. Lossnitzer K, Janke J, Hein B, Stauch M, Fleckenstein A:
Disturbed myocardial metabolism: A possible pathogenctic
factor in the hereditary cardiomyopathy of the Syrian hamster, in Fleckenstein A, Rona G (eds): Recent Advances in
Studies on Cardiac Structure and Metabolism, Vol 6: Pathophysiology and Morphology of Myocardial Cell Alteration.
Baltimore, Md, University Park Press, 1975, pp 207-217
5. Wrogemann K, Nylen EG: Mitochondria] calcium overloading in cardiomyopathic hamsters. / Mol Cell Cardiol 1978;
10:185-195
6. Rossner KL, Sachs HG: Electrophysiological study of SJTian hamster hereditary caidiomyopathy. Cardiovasc Res
1978;12:436-443
7. Jasmin G, Solymoss B: Prevention of hereditary cardiomyopathy in the hamster by verapamil and other agents. Proc
Soc Exp Biol Med 1975;149:193-198
8. Rouleau JL, Chuck LHS, Hollosi G, Kidd P, Sievers RE,
Wikman-Coffelt J, Parmley WW: Verapamil preserves myocardial contractility in the hereditary cardiomyopathy of the
Syrian hamster. Ore Res 1982;50:405-412
9. Markiewicz W, Wu SS, Parmley WW, Higgins CB, Sievers
R, James TL, Wikman-Coffelt J, Jasmin G: Evaluation of the
hereditary Syrian hamster cardiomyopathy by 31P nuclear
magnetic resonance spectroscopy: Improvement after verapamil therapy. Ore Res 1986;59:597-604
10. Weisman HF, Weisfeldt ML: Toward an understanding of
the molecular basis of cardiomyopathies. J Am Coll Cardiol
1987;19:1135-1138
11. Wagner JA, Reynolds U, Weisman HF, Dudeck P, Weisfeldt
ML, Snyder SH: Calcium antagonist receptors in cardiomyopathic hamster: Selective increases in heart, muscle, brain.
Science 1986;232:515-518
12. Finkel MS, Marks ES, Patterson RE, Spier EH, Steadman
K, Keiser HR: Increased cardiac calcium channels in hamster cardiomyopathy. Am J Cardiol 1986^7:1205-1206
13. Finkel MS, Marks ES, Patterson RE, Speir EH, Steadman
KA, Keiser HR: Correlation of changes in cardiac calcium
channels with hemodynamics in Syrian hamster cardiomyopathy and heart failure. Life Sci 1987;41:153-159
14. Kobayashi A, Yamashita T, Kaneko M, Nishiyama T,
Hayashi H, Yamazaki N: Effects of verapamil on experi-
mental cardiomyopathy in the Bio 14.6 Syrian hamster. JAm
Coll Cardiol 1987;10:1128-1134
15. Kuo TH, Tsang W, Wiener J: Defective Ca2+-pumping
ATPase of heart sarcolerama from cardiomyopathic hamster. Biochim Biophys Acta 1987;900:10-16
16. Kuo TH, Johnson DF, Tsang W, Wiener J: Photoaffinity
labeling of the calcium channel antagonist receptor in the
heart of the cardiomyopathic hamster. Biochem Biophys Res
Commun 1987;148:926-933
17. Culling CFA, Allison RT, Barr WT: Cellular Pathology
Technique, ed 4. London, Butterworths, 1985, pp 155-160,
167-169, 294-295
18. Hajos F: An improved method for the preparation of synaptosomal fraction in high purity. Brain Res 1975;93:485-489
19. Reeves JP, Sutko JL: Sodium-calcium ion exchange in
cardiac membrane vesicles. Proc NatlAcad Sci USA 1979;
76:590-594
20. McPherson GA: A practical computer-based approach to the
analysis of radioligand binding experiments. Comput Methods Programs Biomed 1983;17:107-114
21. Reynolds U, Wagner JA, Snyder SH, Thayer SA, Olivera
BM, Miller RJ: Brain voltage-sensitive calcium channels
subtypes differentiated by omega-conotoxin fraction GVIA.
Proc NatlAcad Sci USA 1986;83:8804-8807
22. Wagner JA, Guggino SE, Reynolds D, Snowman AM,
Biswas A, Olivera BM, Snyder SH: Calcium antagonist
receptors. Clinical and physiological relevance. Ann NY
AcadSci 1988;522:116-133
23. Turner TJ, Goldin SM: Calcium channels in rat brain synaptosomes. Identification and pharmacological characterization. JNeurosci 1985;5:841-849
24. Suszhiw JB, O'Leary ME, Murawsky MM, Wang T: Presynaptic calcium channels in rat cortical synaptosomes: Fast
kinetics of phasic calcium influx, channel inactivation, and
relationship to nitrendipine receptors. / Neurosci 1986;
6:1349-1357
25. Coutinho OP, Carvalho CAM, Carvalho AP: Calcium uptake
related to K+-depolarization and Na+/Ca2+ exchange in
sheep brain synaptosomes. Brain Res 1984;290:261-271
26. Howlett SE, Gordon T: Calcium channels in normal and
dystrophic hamster cardiac muscle: [^HJNitrendipine binding studies. Biochem Pharmacol 1987;36:2653-2659
27. Bazan E, Schwartz A, Gardner S, Wells JW, Sole MJ,
Johnson CL: Receptors for calcium channel antagonists in
cardiomyopathy (abstract). Fed Proc 1987;46:852
28. Sonnenblick EH, Fein F, Capasso JM, Factor SM: Microvascular spasm as a cause of cardiomyopathies and the
calcium-blocking agent verapamil as potential primary therapy. Am J Cardiol 1985;55:179B-184B
29. Nowycky MC, Fox AP, Tsien RW: Three types of neuronal
calcium channels with different calcium agonist sensitivity.
Nature 1985;316:440-443
30. Nilius B, Hess P, Lansman JB, Tsien RW: A novel type of
cardiac calcium channel in ventricular cells. Nature 1985;
316:443-446
31. Ferry DR, Kaumann AJ: Relationship between betaadrenoceptors and calcium channels in human ventricular
myocardium. Br J Pharmacol 1987;90:447-457
32. Wagner JA, Sax FL, Weisman HF, Porterfield J, Mclntosh
C, Weisfeldt ML, Snyder SH, Epstein SE: Calciumantagonist receptors in the atrial tissue of patients with
hypertrophic cardiorayopathy. N Engl J Med 1989;
320:755-761
KEY WORDS • hamster cardiomyopathy • calcium antagonist
receptors • sodium-calcium exchange • hypertrophy
Alterations in calcium antagonist receptors and sodium-calcium exchange in
cardiomyopathic hamster tissues.
J A Wagner, H F Weisman, A M Snowman, I J Reynolds, M L Weisfeldt and S H Snyder
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Circ Res. 1989;65:205-214
doi: 10.1161/01.RES.65.1.205
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