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1001
Subcellular Calcium Content in
Cardiomyopathic Hamster Hearts In Vivo:
An Electron Probe Study
Meredith Bond, Abdul-Rahman Jaraki, Candis H. Disch, and Bemadine P. Healy
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In the Syrian cardiomyopathic hamster heart, abnormal cellular calcium regulation, resulting
in cellular calcium overload, is believed to play a role in the pathogenesis of cardiac hypertrophy
and failure. Alternatively, the primary abnormality may be coronary vasospasm, resulting in
reperfusion-induced necrosis. According to the latter hypothesis, only those cells that suffer an
ischemic insult would contain elevated calcium levels. To determine whether a generalized
elevation in myocytic calcium exists in myopathic hamster hearts, we measured cellular and
subcellular calcium concentrations by electron probe microanalysis in cryosections of 50-day
and 96-day myopathic and control hearts, rapidly frozen in vivo. Total calcium content of
ventricular homogenates from each group was also measured by atomic absorption spectrophotometry. No significant differences hi subcellular calcium were found by electron probe
microanalysis among 50-day and 96-day myopathics and their age-matched controls. In 50-day
myopathic and control hearts, mitochondria! calcium was 0.7±0.2 and 0.9±0.2, respectively,
and A-band calcium was 3.0±0.4 and 2.6±0.4 nunol calcium/kg dry wt(±SEM). Results from
96-day animals were similar. Localized regions of elevated calcium were found only at sites of
necrotic foci: in Na+-loaded cells (mitochondria: 4.7±1.3 (SEM) mmol/kg dry wt), in dying cells
(mitochondria: 72±22 (SEM) mmol/kg dry wt) or as extracellular deposits (7-10 mol/kg dry
wt). Total calcium content of hearts from myopathic hamsters, as determined by atomic
absorption spectrophotometry, was also 13 times (50-day) and 50 times (96-day) higher than
controls. These results demonstrate that there is a marked heterogeneity in cellular calcium
content in myopathic hamster hearts, but the data do not support the hypothesis of a generalized
cellular calcium overload. (Circulation Research 1989;64:1001-1012)
N
ormal cardiac function depends on the cell's
ability to maintain cytosolic calcium (Ca2+)
within narrow limits; cardiac dysfunction,
on the other hand, is frequently associated with
cellular Ca2+ abnormalities.12 The Syrian cardiomyopathic (myo) hamster is a well-studied animal model
of idiopathic cardiomyopathy, in which the heart
hypertrophies and subsequently develops a dilated
type of cardiomyopathy, associated with focal areas
of myocardial necrosis, fibrosis, and calcification.*-3
A variety of studies have demonstrated Ca2+ abnormalities to be present from an early, presymptomatic age in myo hamster hearts. Reported changes
From the Department of Heart and Hypertension, Research
Institute of the Cleveland Clinic Foundation, Cleveland, Ohio.
Supported by a Grant-in-Aid from the American Heart Association, Northeast Ohio Affiliate, to M.B. and by the Research
Institute of the Cleveland Clinic Foundation.
Address for correspondence: Meredith Bond, PhD, Department of Heart and Hypertension, Cleveland Clinic Research
Institute, FFb-43, One Clinic Center, 9500 Euclid Avenue,
Cleveland, OH 44195-5069.
Received July 6, 1988; accepted October 28, 1988.
include a decreased activity of the sarcolemmal
Ca2+-ATPase67 and decreased Na + ,K + -ATPase
activity,7 as well as increased density of voltage
sensitive Ca2+ channels,8 although the latter observation is controversial.9 In view of the early onset
of many of these changes in Ca2+ metabolism, it has
been proposed that a general elevation of intracellular Ca2+ may be an important initiating or aggravating factor in the etiology of the disease. 10 "
Alternatively, it has been proposed by Sonnenblick and coworkers12 that the primary defect responsible for development of heart failure in myo hamsters may not be an abnormality in Ca2+ handling by
the myocytes themselves but an increased reactivity of the coronary arteries and arterioles. They and
others have provided evidence for the existence, in
myo hamsters, of coronary vasospasm in situ13 and
coronary flow heterogeneity,14 as well as increased
reactivity of the microvasculature.13 These investigators predicted that transient ischemia and the
reperfusion-induced necrosis that follows would
more readily explain the localized, discrete nature
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Circulation Research
Vol 64, No 5, May 1989
of the observed pathological lesions in myo hearts
than a generalized defect in myocytic Ca2+
regulation.12 In contrast to the alternative hypothesis of a generalized Ca2+ overload, reperfusion of
populations of ischemic myocytes could result in
elevations in cell Ca2+ concentration16-'8 only in
localized regions of the heart. Based on this vascular hypothesis, which would result in a focal type of
lesion, one would predict the existence of a marked
heterogeneity in cellular Ca2+ content in myo hamster hearts, as opposed to a more generalized cellular Ca2+ overload.
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To date, there have been no direct measurements
of cellular or subcellular Ca2+ concentration in myo
hamster hearts in situ. Some investigators have
measured the total Ca2+ content of ventricular
homogenates from myo hearts by atomic absorption
spectrophotometry (AA) and found it to be markedly elevated compared with the Ca2+ content of
control hearts. 31920 Ca2+ content of isolated mitochondrial fractions has also been shown to be
significantly elevated in myo hearts. 1120 The
approaches used in these studies are, however,
limited in their ability to identify whether the
observed elevation in cardiac and/or mitochondrial
Ca2+ is a homogeneous phenomenon throughout the
heart. An alternative methodology, which avoids
the problems associated with measurements of total
tissue Ca2+ by AA, is electron probe microanalysis
(EPMA) of intracellular and subcellular concentrations of Ca2+ and other elements in cryosections of
heart rapidly frozen in situ.2122
In this study, we investigated the question of
intracellular Ca2+ overload in myo hamster hearts by
directly measuring subcellular Ca2+ content in freezedried cryosections obtained from hearts of myo
hamsters and hearts of normal, age-matched controls
rapidly frozen in vivo. Using this approach, it has
been possible to obtain for the first time direct
measurements of subcellular elemental content and
elemental distribution in the heart in vivo. Specifically, we sought to determine whether a general
elevation in total cytosolic Ca2+ is found in myo
hamster hearts 1) during relatively early stages of
disease development (50 days) and 2) at a later stage,
when significant progression of the disease, including
cardiac hypertrophy, has occurred (96 days).4-5 In
addition, we investigated whether intracellular Ca2+
overload, if it occurs, is reflected in enhanced mitochondrial Ca2+ uptake. Alternatively, in the absence
of a general elevation in basal (diastolic) Ca2+, our
objective has been to identify specific localized sites
of elevated Ca2+ within myo hamster hearts.
Materials and Methods
Animals Studied
Myo hamsters of the BIO 14.6 strain and agematched control golden hamsters were obtained from
Canadian Hybrid Farms, Centerville, Nova Scotia,
Canada. Two different age-groups were studied:
44-57 days (mean age, 50 days) and 93-100 days
(mean age, 96 days).
Rapid Freezing of Hearts In Vivo
To rapidly freeze the hamster heart in vivo, a pair
of hand-held, mechanically triggered, spring-loaded
clamps with freezing surfaces of frozen Freon 22
was used. Their use and operation has previously
been described in detail.2223 Briefly, freezing involves
filling Al cups, which detach from the ends of the
jaws of the clamps, with liquid Freon 22. The Freon
is then frozen (freezing point, -156° C) by immersion of the cups, to below the level of the rim, with
liquid N2. Just before freezing of the heart in vivo,
the cups containing frozen Freon are attached to the
ends of the clamps and submerged in liquid N2.
Hamsters were anesthetized by intraperitoneal
injection of Inactin (80-90 mg/kg body wt) and then
taped, dorsal side down, to a board supported over
a water bath. Animals were tracheotomized and
ventilated with room air at 80-90 breaths/min, with
a tidal volume of 1-1.2 ml. To ensure that the
ventilation rate and tidal volume used was optimal,
in the first few animals, the femoral artery was
cannulated, 200-/A1 blood samples were collected in
heparinized Clinitubes, and blood pH, Po?, and
PCO2 were measured in a Gas Analyzer (Radiometer, Copenhagen, Denmark). The right carotid
artery was cannulated with PE-10 polyethylene
tubing, and the cannula was then gently inserted
past the aortic valve into the left ventricle. Femoral
arterial pressure (when measured), left ventricular
pressure (LVP), and the first derivative of LVP, dP/
dt, were recorded throughout the experiment, up to
the time of freezing of the heart, on a Gould
four-channel recorder. (DP/dt was recorded only as
an aid to help identify the point in the cardiac cycle
at which the heart was frozen.)
Once a stable recording of LVP was obtained, the
abdomen was opened, the diaphragm cut, and the
animal ventilated against positive pressure. A medial
incision was made through the sternum, and the ribs
were drawn back toward the head to expose the
heart. In the earlier hearts that were frozen, the
temperature of the water bath was increased to
provide a warm and moist environment in the
vicinity of the exposed heart. In others, warmed
saline (approximately 37° C) was dripped slowly
over the surface of the heart. A suture was then
placed superficially in the apex of the heart, and a
curved piece of Teflon, with a circular hole cut in its
center, was gently placed over the heart, so that the
heart protruded through the hole; the use of such a
"lung pressor" greatly facilitates rapid freezing of
the heart without inadvertent freezing of part of the
lungs.
Hearts were frozen at a specific time point in the
cardiac cycle by running the chart recorder at a high
speed (50 or 100 mm/sec). The beating heart was
then lifted away from the body by the apical suture.
The clamps, with attached Al cups containing fro-
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zen Freon 22, were held out of the liquid N2 for
about 5 seconds to allow a melted layer of Freon to
form over the frozen surface.20 The heart was then
rapidly frozen by triggering the clamps by hand to
rapidly close. The precise time-point of the freeze,
within the cardiac cycle, was indicated by an artifact on the LVP pressure trace (Figure 1). Only
hearts frozen during diastole were used in this
study.
The frozen heart, sandwiched between the frozen Freon surfaces of the closed clamps, was
quickly cut away from the thorax and rapidly
transferred into liquid N2. After removal from the
clamps under liquid N2, the atria were chipped
away, and the region of the apex at the site of the
suture was also removed.
Cryoultramicrotomy
Two to three pieces of frozen myocardium
(approximately 2-3 mm across) were cut from the
epicardial surface of the left ventricle of the frozen
heart with a liquid Nj-chilled chisel. In some of the
hearts from 96-day myo hearts, the piece of myocardium used for cryoultramicrotomy was specifically chosen from regions adjacent to, and including, scarred or fibrotic areas, which have a
transmural distribution7 and can be readily identified in the frozen hearts under liquid N2, with the aid
of a dissecting microscope. The presence of fibrotic
lesions in these areas was later confirmed, at the
light microscopic level, using histological sections
cut from the same area that had been used for
cryoultramicrotomy and EPMA (see below). The
small pieces of the frozen heart were then transferred, under liquid N2, to the precooled chamber of
a Reichert-Jung FC4D cryoultramicrotome (Cambridge Instrument Co, Deerfield, Illinois) (ambient
temperature, -125° C; knife and specimen temperature, -100° C) and glued to Al pins, with the
epicardial surface facing up, using toluene (freezing
point, -93° C) as a low-temperature cement.24 One
FIGURE 1. Examples of left ventricular pressure traces (LVP) and the first derivative ofLVP
(dPIdt) from two hamster hearts rapidly frozen
with Freon clamps in vivo: in early systole (A)
and in early diastole (B). The instant that the
heart is frozen is indicated by an artifact (arrow)
on the pressure traces.
pin with attached frozen myocardium, was then
inserted into the microtome chuck and ultrathin
cryosections (100-150 nm thick) cut from the surface with a glass knife, with the plane of the
sections parallel to the longitudinal axis of the
superficial cardiac muscle fibers.
Cryosections were transferred dry, within the
cooled chamber, using a chilled eyelash probe, onto
Ca2+-free carbon support films, prepared as previously described22 on nickel or copper 400-mesh thin
bar grids, which were clamped in a Reichert grid
holder. The cryosections were then gently pressed
flat onto the carbon-coated grids, using a liquid Nrchilled copper rod, and transferred, within the chamber of the cryomicrotome, to a liquid Nr-chilled
brass block. The brass block containing the grids
was then transferred into an Edwards vacuum evaporator (Rochester, New York) for freeze-drying at
or below 10"6 torr overnight. The following day, the
freeze-dried cryosections were immediately carboncoated to minimize any possible rehydration from
room air and stored under vacuum in a dessicator.
Electron Probe Microanalysis
Standards and quantitation. Analytical computer
programs written in the Somlyos' laboratory at the
University of Pennsylvania were adapted for use
with an Edax x-ray detector and multichannel analyzer (Edax International Inc, Schaumburg, Illinois). Primary and binary reference standards,
together with an absolute sulfur standard (from
dried films of 3% bovine serum albumin), were
collected. Together with weighting factors obtained
from the binary elemental standards, the x-ray
spectra collected from albumin allowed the determination of the absolute concentrations of the elements (Na+, Mg2+, P, S, C r , K + , and Ca2+) measured in the cryosections (in miilimoles per kilogram
dry weight), as previously described.26 The first and
second derivatives of the K+ peaks in the reference
spectra were included in the multiple least-squares
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Circulation Research
Vol 64, No 5, May 1989
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fit to correct for undetected shifts in detector
calibration.27
Absence of systemic errors in the quantitation of
intracellular Ca2+ concentrations, was validated by
the results of x-ray measurements of the total Ca2+
content of red blood cells (0.13±0.3 [SEM] mmol/
kg dry wt; n=5) in intramyocardial capillaries of
control hamster hearts rapidly frozen in vivo. This
value is consistent with the very low values previously measured by AA28 and by EPMA.23
Data collection. Samples were transferred into a
Philips 400T electron microscope (Pittsburgh, Pennsylvania) using a Gatan temperature-regulated specimen holder (Pleasonton, California). The specimen
was then cooled to —100° C for spectral collection.
Energy dispersive x-ray spectra were collected from
the freeze-dried cryosections in spot mode, at 80
kV, using an Edax 30 mm2 Si(Li) x-ray detector and
Edax multichannel analyzer. Spectra were subsequently analyzed on a pdp 11/34 computer (Digital
Equipment Corporation, Marlboro, Massachusetts)
by a minimum least-squares fit to stored reference
spectra.26 Elemental quantitation was carried out
using the Hall thin film quantitation procedure.29
X-ray spectra were collected to a standard deviation for Ca2+ of 0.9-1.1 mmol/kg dry wt. Spectra
were collected from three cells from each of three to
six animals per experimental group and from three
separate areas: whole cell (one spectrum per cell),
which was an area over several sarcomeres, but not
including mitochondria; from mitochondria (two
per cell); and from the A band (two per cell). A fifth
group consisted of x-ray spectra collected from
cells found to be Na+-loaded (K/Na<2) in the
vicinity of necrotic foci in cryosections from 96-day
myo hearts.
Statistical Analysis
To distinguish treatment differences from interanimal or cell-to-cell variability as well as variability due to the x-ray counting process itself, x-ray
spectra collected from mitochondria, A band, and
cell were separately analyzed by nested one-way
analysis of variance as previously described.30 If a
significant F statistic were obtained, the group or
groups showing a significantly different mean Ca2+
content was then identified using a pairwise linear
contrast of means. The x-ray data were downloaded
from the pdp 11/34 computer onto an AT personal
computer and statistical analyses performed using
the SAS statistical package (SAS Institute Inc,
Cary, North Carolina).
Atomic Absorption Spectrophotometry
The portion of ventricles not used for cryoultramicrotomy was thawed and wet weight determined.
The tissue was dried overnight on tared Al foil in an
oven at 105° C and the dry weight then measured.
The dried ventricles were then homogenized in a
mortar and pestle in a small volume of IN HN0 3
containing 7.2 mM LaCl3. The Ca2+ and Mg2* con-
centrations in diluted aliquots of the supernatant
(obtained after centrifugation of the HN0 3 acid
extract at 700g for 5 minutes) were then measured
with a Perkin-Elmer 2380 atomic absorption spectrophotometer (Eden Prairie, Minnesota) using an
air acetylene flame. Differences in Ca2+ and Mg*+
content were determined for each age group with an
unpaired two-tailed Student's t test.
Histology
The samples of ventricle that had been mounted
on pins for cryoultramicrotomy were saved for
histological analysis. The frozen pieces of myocardium on the AJ pins were thawed at room temperature in 2.5% glutaraldehyde in 0.1 M cacodylate
buffer, postfixed in OsO4, dehydrated in alcohol
and embedded in Epon. Sections 0.5 fim thick
were cut on an LKB Ultracut IV microtome
(Cambridge Instruments, Ijamsville, Maryland) and
stained with a polychromatic stain containing toluidine blue plus basic fuschin.31
Results
Rapid Freezing of the Heart In Vivo
Figure 1 shows two examples of traces of LVP
and dP/dt just before in vivo freezing of hamster
hearts in early systole (A) and in diastole (B),
respectively. The instantaneous freezing of each
heart is clearly indicated by an artifact on the
pressure traces. While the heart rate tends to be
somewhat slower than in the closed-chested animals, the use of hearts rapidly frozen in vivo, for
the determination of subcellular elemental content
by EPMA, more closely approximates the natural
state of the beating heart in the living animal than
does an isolated preparation.
The type of ultrastructural preservation achieved
by rapidly freezing hamster hearts in vivo with
Freon 22 clamps is exemplified in the ultrathin,
freeze-dried cryosection shown in Figure 2. Subcellular structures, such as mitochondria, A band, I
band, and nucleus are readily identified. The quality
of freezing is comparable to that obtained by other
investigators in cardiac preparations, such as papillary muscle, rapidly frozen in vitro.32-34
Physiological Parameters
Throughout the period of experimental manipulations and surgery, both myo and control hamsters
were maintained under optimally oxygenated conditions, with no significant differences in blood gas
values between the two strains: blood pH averaged
7.38±0.06(SD), mean PCO2 and PO2 were 38±8
mm Hg and 84±5 mm Hg, respectively (pooling
results from both groups).
Both 50-day and 96-day myo hamsters weighed
approximately 15% less than their age-matched
control (Table 1). Both systolic and diastolic blood
pressure as well as heart rate, measured in Inactinanesthetized animals, was also significantly lower
(p<0.01) in the myo strain at both ages.
Bond et al Calcium in Myopathic Hearts
1005
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—
. ^ _ ;
FIGURE 2. An example of an ultrathin, freeze-dried cryosection cut from the epicardial surface of a hamster heart
rapidly frozen in vivo. M, mitochondria; A, A band; N, nucleus.
Total Ca and Mg Content of Ventricles ofMyo
and Control Hearts Measured by Atomic
Absorption Spectrophotometry
The total ventricular Ca2+ content measured by AA
in nitric acid digests of 50-day and 96-day myo and
control hearts is shown in Figure 3. Ca2+ was found to
be markedly elevated in the myo hearts, particularly in the2+96-day-old animals. In contrast, myocardial Mg content did not differ significantly
between 96-day myo and control hearts (myo:
38.4±2.1 [SEM] mmol/kg dry wt [n=T\; controls:
39.2±1.3 [SEM] mmol/kg dry wt [«=7]). Among
hearts of 50-day myo and control hamsters, Mg2+
content was somewhat higher in the myo group
(p<0.02) than in the controls (myo: 41.6±1.2 mmol/
kg dry wt [n=10]; controls: 36.5±1.6 mmol/kg dry
wt[n=10]).
Cellular and Subcellular Elemental Composition
In Vivo
Total cellular and subcellular concentrations of
Na + , Mg2+, P, S, Cl", K \ and Ca2+ in 50-day and
TABLE 1.
96-day myo and control hearts, measured in myocytes of normal ultrastructural appearance away
from fibrotic areas are presented in Tables 2-4
(groups 1-4). Group 4 (96-day myo) also includes
x-ray spectra from low Na+ cells cut from regions of
the ventricle in the vicinity of necrotic foci, as
determined by eye under the dissecting microscope,
but found not to be immediately adjacent to the
pathological lesion by visual inspection of both the
cryosections and the plastic embedded sections
later obtained from the cutting face of the frozen
block. The elemental composition of Na+-loaded
cells, in the immediate vicinity of necrotic foci is
also shown in Tables 2-4 (group 5). Average values
for cellular and subcellular Ca2+ content are summarized graphically in Figure 4. No significant
differences in either total cell, A band, or mitochondrial elemental composition were revealed by nested
analysis of variance among groups 1-4. In particular, it should be noted that the mean Ca2+ content in
all of these four groups, for each of the subcellular
Physiological Parameters From 50- or 96-Day Control and Myopathic Hamsters
96±1 day
50±lday
Body weight (grams)
Systolic BP (mm Hg)
Diastolic BP (mm Hg)
Heart rate (beats/min)
Myopathic
(34)
Control
(26)
Myopathic
(17)
Control
(25)
70.9±l.l
126.7+6.9
89.1±5.4
276.0±10.9
84.8±2.3*
168.6+5.9*
112.0+4.5*
349.6+11.7"*
IO1.3±2.4
114.8±8.0
8O.4±7.3
273.8±18.1
124.2+2.1*
149.5±5.6*
103.4+4.5*
363.1 + 15.1
BP, blood pressure. Values are mean±SEM.
*p<0.01 by two-tailed Student's t test vs. age-matched control.
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Circulation Research
Vol 64, No 5, May 1989
near fibrotic scars is clearly evident from a comparison of the size of the Ca2+ x-ray peak in an
averaged mitochondrial spectrum (A and B) with
the Ca2+ peak in an averaged spectrum from mitochondria in myocytes distant from regions of necrosis in 96-day myo hearts (C and D).
Nor 50 d
•H
Myo SO d
Nor 9 6 d
Myo 96 d
200
150
[C]
mmol/kg dry wt • / - SEM
2
100
50
250
300
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FIGURE 3. Total ventricular Ca * content measured by
atomic absorption spectrophotometry (AA) in nitric acid
digests of nine 50-day control (nor) and ten 50-day
myopathic (myo) hearts and from seven 96-day nor and
seven 96-day myo hearts. *p<0.05 vs. 50-day nor; +p<0.01
vs. 96-day nor.
regions analyzed, is very low. Thus, we find no
evidence of elevated subcellular Ca2+ content in
myocytes of normal ultrastructural appearance, away
from necrotic foci, in hearts of myo hamsters at
either 50 or % days of age.
To pursue the cause of the marked elevation in
total ventricular Ca content, measured by AA, in
myo hearts versus controls (Figure 3), 619 the subcellular composition of myocardium in the immediate vicinity of fibrotic scars (Figure 5) was examined in cryosections from 96-day myo hearts. It was
found that cells of normal ultrastructural appearance in the vicinity of these scarred regions were
frequently Na+.-loaded (group 5 in Tables 2-4 and
Figure 4); the subcellular Ca2+ content of this population of cells (identified by a K+/Na+ ratio of
<2:1) was significantly elevated compared with
every other group analyzed from myo and control
hearts. When compared with group 4, the increase
in mitochondrial Ca2+ content in this population of
cells was proportionally even greater than the
observed elevations in cellular or A band Ca2+,
increasing almost fivefold.
In Figure 6, the marked elevation in mitochondrial Ca2+ content measured in the Na+-loaded cells
TABLE 2.
Subcellular Ca Content at Sites of
Pathological Lesions
Cells within necrotic regions that were clearly
irreversibly injured were also identified, as shown
in the cryosection in Figure 7, where remnants of Z
lines are still evident but crystalline deposits are
present in the mitochondria. Mitochondrial Ca2+ in
such cells ranged from 21 to 123 mmol/kg dry wt, up
to 140 times higher than in normal myocytes, away
from fibrotic lesions, in the 96-day myo hearts. An
example of an x-ray spectrum from such a Ca2+loaded mitochondrion, showing a marked Ca2+ and
P elevation and loss of K + , compared with the
spectra shown in Figure 6, is shown in the inset to
Figure 7. Other localized sites of highly elevated
Ca2+, again found exclusively within necrotic zones,
were extracellular Ca2+ phosphate deposits containing 7-10 mol Ca2+/kg dry wt.
Discussion
Numerous studies have focused on abnormalities
of Ca2+ handling as a cause of, or aggravating factor
in, the development of a dilated cardiomyopathy in
the Syrian cardiomyopathic hamster. Most studies
to date, which have shown such abnormalities,
have used methods that fail to distinguish focal or
regional abnormalities from those that are homogeneous phenomena throughout the heart. For example, a global measure of ventricular Ca2+ content by
AA can be significantly affected by the existence of
localized regions containing a very high Ca2+ content. Similarly, the mean Ca2+ concentration of a
population of isolated mitochondria can be artificially elevated by the contribution of a subpopulation of mitochondria that are Ca2+-loaded.35 In
addition, during mitochondrial isolation, Ca2+ uptake
can readily occur unless special precautions are
taken, such as inclusion of ruthenium red in the
isolation medium.36
Total Cell Elemental Content (mmol/kg dry wt ± SEM)
Age/Strain
50-day Nor
50-day Myo
96-day Nor
96-day Myo
96-day Myo
K/Na<2
No. of
Hamsters
Total
No. of
Spectra
-4
6
3
4
4
12
14
10
15
8
Na+
' 169.4±14.7
. 161.2±17.7
132.0±19.9
159.8+16.8
280.5+24.0*
Nor, normal (control); Myo, myopathic.
*p<0.01 vs. each of the other groups.
Mg»
P
S
57.0±2.4
419.3±23.4
60.7±2.3
65.4±1.8
60.4±3.6
57.3+3.2
461.5±18.8
496.5±21.9
363.9±24.4
388.3±13.4
435.7+13.6
422.1+20.1
391.4±25.0
393.8±14.9
384.9+16.2
cr
K+
Ca2+
119.8+8.7
471.1 ±17.3
561.7±46.2
569.3±38.6
3.4±0.7
2.9±0.5
2.5±0.7
498.5±20.3
3.3±0.5
7.2±1.6*
115.6±14.0
91.7±6.6
110.4±10.1
144.2±11.4
397.4±21.4
Bond et al
TABLE 3.
Calcium in Myopathic Hearts
1007
Elemental Content of A Band (mmol/kg dry wt ± SEM)
Age/Strain
50-day Nor
50-day Myo
%-day Nor
96-day Myo
96-day Myo
No. of
Hamsters
Total
No. of
Spectra
4
6
3
4
4
20
31
22
28
19
S
cr
K+
Ca2+
351.8±20.8
368.0+17.3
378.5+22.2
429.2±17.1
428.8+15.0
423.7±15.1
436.1 ±10.6
407.8±13.2
113.5+6.5
131.6±8.7
2.6±0.4
3.0±0.4
97.7±5.3
106.9+6.3
567.5±21.0
688.2±30.5
689.7±43.3
590.9±16.3
3.7±0.8
3.6±0.4
169.5±6.3t
472.3+20.7*
5.3±0.6§
Na+
Mg*+
P
181.5+14.3
181.9±10.7
67.3±2.8
75.6±2.2
159.2±15.4
I68.2±11.9
82.5+3.3
72.2±2.6
68.8±3.2
334.8+16.8*
373.7+17.6
338.5+22.4
Nor, normal (control); Myo, myopathic.
*p<0.01 vs. each of the other groups.
tp<0.01 vs. %-day Nor.
p<0.05 vs. 96-day Myo, 50-day Nor.
tp<0.0\ vs. 50-day Myo, 96-day Nor.
§p<0.0l vs. 50-day Nor and Myo, p<0.05 vs. %-day Nor and Myo.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
EPMA, in contrast, allows cellular and subcellular
measurements of Ca2+ in rapidly frozen myocardium
in situ. Using this approach, the total concentration
of Ca2+, and other elements, in subcellular compartments, can be quantified in individual cells with a
spatial resolution of up to 10 nm.21 Using EPMA thin
film quantitation routines,26-27>» the sensitivity of this
technique to small differences in total Ca2+ content,
within defined microvolumes of the cell, is of the
order of 0.5-0.7 mmol Ca2+/kg dry wt.2*•*>
The particular advantages of EPMA over other
methods are that 1) subcellular fractionation of the
tissue is avoided, and 2) provided that tissue samples are prepared by rapid freezing rather than by
conventional aldehyde fixation, the possibility of
redistribution of Ca2+ and other diffusible elements
is avoided.37 Furthermore, and of particular relevance to this study, 3) regional and cellular variations in the concentration of Ca2+ and other elements can be readily identified by EPMA, since
spectra are obtained on a cell-by-cell basis.
In this study, a technique has been developed to
rapidly freeze hamster hearts in vivo, thus permitting measurement of subcellular elemental content
in the intact, blood-perfused heart, without recourse
to either tissue isolation or subcellular fractionation
and without the use of liquid fixatives, in other
words, under conditions that most closely approximate the physiological state of the animal.
TABLE 4.
Subcellular Elemental Content in Normal
Hamster In Vivo
The EPMA results obtained from control hamster
hearts in the current study show a somewhat higher
intracellular Na+ concentration and Ca2+ concentration than EPMA measurements obtained by some
other groups from rapidly frozen in vitro preparations. 3233 It is likely that these differences are due to
species differences as well as to differences, alluded
to previously, between in vitro and in vivo preparations. Specifically, the in vivo heart rate in hamsters (Table 1) is 250-350 beats/min or 4-6 Hz,
while in vitro cardiac preparations such as papillary
muscle, are generally stimulated at the much lower
rates of 0.2-0.3 Hz. Increased rates of beating have
been shown to result in intracellular Na + accumulation.38^39 An elevation of cytoplasmic free Ca2+ in
diastole, as a direct function of an increased rate of
contraction, has also been demonstrated in cardiac
myocytes loaded with the Ca2+ sensitive dye, indo
I.*0 Since free and bound Ca2+ should be in equilibrium, an elevation in the cytosolic free Ca2+ concentration would result in a higher proportion of
cytoplasmic Ca2+ binding sites (e.g., on troponin
and calmodulin) being occupied by Ca2+ at any
moment. Thus, one might predict that total cytoplasmic Ca2+, as measured by EPMA, would also
increase as a function of an increased rate of
contraction.
Elemental Content of Mitochondria (mmol/kg dry wt ± SEM)
Age/Strain
50-day Nor
50-day Myo
%-day Nor
%-day Myo
%-day Myo
K/Na<2
No. of
Hamsters
Total
No. of
Spectra
4
6
3
4
4
22
37
21
28
16
Na+
%.6+9.8
109.0+9.2
109.9+12.8
104.6±9.2
210.0+14.9*
Nor, normal (control); Myo, myopathic.
*p<0.01 vs. each of the other groups.
tp<0.001 vs. each of the other groups.
P
43.6±1.5
42.4+1.3
51.5+2.4
40.8+1.6
44.1+2.7
538.7+17.5
583.5+14.4
571.9+18.4
535.0+9.2
543.5±20.3
S
413.0+13.2
439.8±7.2
443.5±8.4
453.6±8.5
427.0+9.8
cr
++
Ca2+
76.2+5.6
85.3+5.3
344.6±10.4
440.0+33.0
77.1+5.2
81.1±5.7
125.3+14.0
452.2+28.1
367.6±9.4
348.9±26.9
0.9+0.2
0.7±0.2
1.2+0.4
0.9±0.3
4.7±1.3t
1008
Circulation Research Vol 64, No 5, May 1989
Nor 50 d
Myo 50 d
Nor 96 d
4
-\,
JMITO
JABAND
1-
1
lCELL
-i
H
—1
4
Myo 96 d
'—u
Myo 96 d (K/Na<2)
-p
0
2
4
+•
6
1— —1
8
*
4. Average EPMA measurements of
the total Ca2* concentration of cell, A band, and
mitochondria from 50-day and 96-day myo and
control (nor) hamster hearts rapidly frozen in
vivo. Only cells with an elevated Na+ concentration "(KlNa <2)" but with normal ultrastructure, in the vicinity of pathological lesions,
demonstrated significant elevations in A-band
Ca2+, reflected in a marked elevation of mitochondrial Ca2*. *p<0.01 vs. every other group
°p<0.0l vs. 50-day myo, nor; +p<0.05 vs. 96-day
myo, nor.
FIGURE
10
mmol/kg dry wt • / - SEM
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
We believe that it is unlikely that the Ca2+ content
measured over the A band includes a contribution
from Ca2+ stores in the junctional sarcoplasmic
reticulum, as it has been shown, in ultrastructural
studies, that the Ca2+ sequestering stores of junctional and corbular sarcoplasmic reticulum are found
principally in the regions of the Z line and I band.41
Cardiovascular Function in Cardiomyopathic
versus Control Hamsters
The lower blood pressure measured in Inactinanesthetized myo hamsters might suggest that
cardiovascular function is depressed in prefailure
myo hamsters, while the absence of a reflex
increase in heart rate, with decreased blood pres-
FIGURE 5. Epon embedded sections (0.5 fim) from the epicardial surface of a 96-day myo heart, cut from a region
including afibrotic scar. Macrophage infiltration (black arrow) and necrotic myocytes (white arrow; enlarged in the inset)
are evident. BV, blood vessel..Magnification, *270; inset, X540.
Bond et al
n
A
4 -
si
*• 2
c
III
KKa
3 --
• 0
Jfir
f
B
400 300
200
:
100
Jl\ lm
No
}
1
400
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
•
1
2
]
.
j
300
200 100 -
s
Co
[Co] •4.7tO3
[Co] •09103
0
JI
\
Co
1
0
A
sure, may be indicative of an altered baroreflex
sensitivity.42 However, no significant differences
in blood pressure were noted in conscious myo
and age-matched control hamsters at 45 or 150
days (Pierre Wicker, personal communication). It
is thus possible that the depressed cardiovascular
function observed in 50-day and 96-day anesthetized myo hamsters may be due to different sensitivities of the two strains to anesthesia.
Subcellular Elemental Content in
Cardiomyopathic Hearts
In the majority of myocytes of normal ultrastructural appearance in myo hamster hearts, the intracellular and subcellular elemental content (Na+,
Mg2+, P, S, Cl", and Ca2+), measured by EPMA,
was not different from that measured in hearts of
control hamsters of the same age. Our findings for
Na + , Mg2+, P, Cl", and K+ are very similar to the
results of Smith and coworkers43 in cryosections
from myo and control hearts, rapidly frozen in
vitro. However, contrary to their results, we found
no evidence for an elevated S content in myo
hearts. Since S is much more labile under the
electron beam at room temperature than when the
specimen is cooled,26 these differences may be due
to the fact that our analyses were performed at
-100° C while those of Smith and colleagues were
carried out at room temperature. In addition, our
methods and quantitation routines provide us with
the added sensitivity to measure subcellular Ca2+
concentrations, which have not previously been
reported in myo hamster hearts prepared by rapid
freezing.
Contrary to conclusions from previous measurements of either 1) total ventricular Ca2+ content,
2) the Ca2+ content of mitochondrial fractions, and
3) the marked and generalized elevation in the Ca2+
i
1009
KKa
D
j
1
/
1
Calcium In Myopathic Hearts
1
FIGURE 6. A: Average of 28 x-ray
spectra from mitochondria from four
96-day myo hearts rapidly frozen in
vivo. B: Averaged x-ray spectrum
from A shown at 10 times higher
gain and with potassium Ka and Kb
peaks stripped by computer. C: Average of 16 spectra from mitochondria
in cryosections of 96-day myo hearts
from regions of the myocardium
immediately adjacent to scarred or
necrotic foci. D: Spectrum from
panel C with potassium peaks
stripped (gain x.10). Mitochondrial
Ca1* concentrations are in millimoles
per kilogram dry weight±SEM.
1
2
k«V
concentration, previously measured by EPMA, in
aldehyde-fixed myo hamster hearts,44 our EPMA
measurements in cryosections of hearts rapidly
frozen in vivo, revealed no evidence of a generalized, inherent intracellular Ca2+ overload at either
50 or 96 days of age. The similarity of the total
cytosolic Ca2+ concentration in myo and control
hamster hearts is reflected in the equally low mitochondrial Ca2+ content. If, in myo hearts, the free
cytosolic Ca2+ concentration were significantly elevated above normal physiological levels, the mitochondrial Ca2+ uptake pathway would be activated,45
resulting in a rapid elevation of mitochondrial Ca2+.
Only in myocytes containing an elevated Na + content, in the vicinity of necrotic foci, was an increase
in mitochondrial Ca2+ observed.
A distinct heterogeneity in cellular Ca2+ content
throughout myo hamster hearts, as exemplified by
96-day hearts, was identified in this study. This
heterogeneity is compatible with the long-recognized
focal nature of myocardial necrosis, fibrosis, and
calcification that develop during the course of the
disease.46 Our pathological studies confirm the focal
nature of the morphological lesions and suggest that
Ca2+ overload of myocytes is also focal and closely
related to areas of necrosis. This conclusion is
consistent with previous EPMA measurements performed on ethanol-fixed tissue from 90-day myo
hamster hearts of calcium phosphate-containing mitochondrial granules in myocytes adjacent to interstitial calcified plaques.46
We have also demonstrated in this study that
cells, in the vicinity of necrotic or fibrotic lesions,
which show no evidence of structural abnormalities, tend to be both Na+- and Ca2+-loaded. Since
the cell membrane is normally relatively impermeant to Na + , one possible explanation for these
findings may be an increased leakiness of the cell
1010
Circulation Research
Vol 64, No 5, May 1989
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FIGURE 7. Freeze-dried cryosection of cardiac myocyte from necrotic area at epicardial surface of 96-day myopathic
heart. Parallel arrows indicate remnants of Z lines in this damaged cell; thick arrow, mitochondria containing an
elevated Co3* phosphate content. The x-ray spectrum from this mitochondrion is shown in the inset.
membrane. These cells may have suffered a transient ischemic insult or else have an inherent defect
in their ability to handle Ca2+, a defect that is
present only in some myocytes. Mitochondria, in
particular, contained a markedly elevated Ca2+ content in these abnormal cells. Evidence of a heterogeneous Ca2+ distribution has also previously been
found in mitochondrial fractions from skeletal muscle of myo hamsters,47 which, like the heart, is
characterized by localized regions of cell necrosis.
In this latter study, two populations of mitochondria were identified, one fraction with a 50-fold
increase in Ca2+ over normal and another larger
fraction with Ca2+ concentrations close to normal,
indicating that only some areas of the tissue contained Ca2+-loaded cells. It is also of interest to note
that, similar to ourfindingsin myocytes near necrotic
foci, an increase in intracellular Na+ and Ca2+ and,
in particular, an elevated mitochondrial Ca2+ content was measured by EPMA in rabbit papillary
muscle that had been reperfused after a period of
hypoxia.18
The observation of mitochondrial Ca2+ loading in
Na+-loaded cells near necrotic foci in myo hamster
hearts indicates that the cytoplasmic free Ca2+
concentration must have increased well above physiological levels, possibly due to an increased permeability of the plasma membrane. The mitochondria! Ca uptake pathway normally exhibits a low
affinity for Ca2+ (in cardiac mitochondria the Km is
approximately 30 /xM in the presence of 1 mM
Mg2*48); however, in the presence of physiological
Mg2+ concentrations, the rate of Ca2+ uptake by
mitochondria is a sigmoidal function of cytoplasmic
Ca2+,49 and thus Ca2+ uptake increases rapidly at
supraphysiological Ca2+ levels.45
In 96-day myo hearts, the very high Ca2+ found in
dying cells, as well as extracellular Ca2+ phosphate
precipitates at sites of pathological lesions, could
readily account for the 50-fold higher Ca2+ content
of 96-day myo versus control hearts measured by
AA. Furthermore, a variability in the number of
calcified lesions in myopathic hearts, and thus in the
number of regions containing extremely high Ca2+
concentrations, could readily explain the large variability (standard deviation) of AA measurements of
total ventricular Ca2+ content in this and previous
studies. Since lesions were much less widespread in
50-day than in 96-day myo hearts, as also noted by
previous investigators,6-8 we did not attempt to cut
cryosections from regions adjacent to necrotic foci
in the 50-day hearts. However, confirming previous
studies,6-8 we did note in histological sections from
50-day hearts that pathological lesions were present. Thus, we propose that in 50-day, as in 96-day,
myo hearts, the large increase in Ca2+ content over
age-matched controls, measured by AA, can also
account for by the presence of localized calcified
lesions. The fact that at 50 days, the Ca2+ content
was only 78±24 (SEM) mmol/kg dry wt compared
with 251 ±39 (SEM) mmol/kg dry wt in 96-day myo
Bond et al
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
hearts confirms the previous findings that lesions
are more common in the older animals.
In contrast to the elevated Ca2+ content measured
by AA in myo hamster hearts, AA measurements of
total tissue Mg2+ showed little (50-day) or no (96day) variability between myo and control hearts, as
also found by other investigators.19-20 The 14%
higher Mg2* content of 50-day myo versus 50-day
control hearts is unlikely to be due to differences
in the Mg2+ content of the cardiac myocytes
themselves, as it was not reflected in significant
differences in the EPMA measurements (Tables
2-4). Since AA Mg2+ measurements reflect total
Mg2"1" content of a homogenate of the whole ventricle, the differences observed are more likely to arise
from differences in Mg2+ content of some other
cellular or noncellular component (e.g., red blood
cells, vascular smooth muscle cells, or fibroblasts).
The fact that AA measurements include a contribution from red blood cells and plasma, both of which
contain a relatively low Mg2+ concentration, may
also explain the fact that the absolute values of the
Mg2+ data obtained by AA were somewhat lower
than the Mg2+ concentrations measured by EPMA.
In summary, we conclude from this study that
there is no evidence for a generalized abnormality
in myocytic Ca2+ regulation, leading to cellular Ca2+
overload in prefailure myopathic hamster hearts in
diastole. In homogenates of whole ventricle, the
elevated Ca2+ content measured in previous studies
by AA was confirmed and shown to be attributable
to discrete, localized sites of elevated Ca2+ in the
regions of necrotic lesions. These findings of a
heterogeneous distribution of a Ca2+ abnormality
are consistent with the morphological studies that
have revealed focal myocardial necrosis, fibrosis,
and calcification. Such a focal abnormality could
reflect a defect in cellular handling of Ca2+ that
affects only some cells of the heart. Alternatively it
could support the notion of microvascular spasm
leading to focal injury.
Acknowledgments
The authors wish to thank Dr Andrew Somlyo,
University of Pennsylvania, for generous provision
of the analytical programs for electron probe
microanalysis; Dr James McMahon, Division of Laboratory Medicine, CCF, for use of the analytical
electron microscope; Mr Kevin Waters, Department
of Musculoskeletal Research, CCF, for computer
programming and for computer resource management; Mr Kirk Easely, Department of Biostatistics
and Epidemiology, CCF, for help with statistics and
Mr Sherwin Parikh for technical assistance.
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KEYWORDS • cardiomyopathy • electron probe microanalysis
• calcium • rapid freezing
Subcellular calcium content in cardiomyopathic hamster hearts in vivo: an electron probe
study.
M Bond, A R Jaraki, C H Disch and B P Healy
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Circ Res. 1989;64:1001-1012
doi: 10.1161/01.RES.64.5.1001
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1989 American Heart Association, Inc. All rights reserved.
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