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LABORATORY INVESTIGATION
MYOCARDIAL METABOLISM
Quantitative x-ray microanalysis of the elemental
composition of individual myocytes in hypoxic
rabbit myocardium
L. MAXIMILIAN BUJA, M.D., KAREN P. BURTON, PH.D., HERBERT K. HAGLER, PH.D.,
AND JAMES T. WILLERSON, M.D.
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ABSTRACT The purpose of this study was to use energy dispersive x-ray microanalysis to test the
following hypotheses: (1) that individual myocytes may exhibit important variation in the severity of
alterations in intracellular ionic homeostasis in response to hypoxia and (2) that hypoxic myocytes may
accumulate certain elements in quantities sufficient to impair organellar function and structure. A rabbit
interventricular septal preparation with attached small right ventricular papillary muscles was used to
obtain control oxygenated myocardium (six papillary muscles) and myocardium rendered hypoxic for 1
to 11/2 hr (n
8). Myocardium not perfused in vitro was also obtained (n = 4). Microanalysis was
performed on freeze-dried thin sections of unfixed papillary muscles. Elemental concentrations were
determined by suitable cryostandards of elements of interest. Sarcoplasm and mitochondria of most
hypoxic myocytes exhibited significant alterations of diffusible elements, including increases in sodium and chloride and decreases in potassium, phosphorus, and magnesium, without major change in
calcium. The most severely altered myocytes showed evidence of calcium overloading manifested by
markedly increased levels of mitochondrial calcium and phosphorus associated with formation of
electron-dense mitochondrial inclusions. Levels of mitochondrial calcium and phosphorus exceeded
those previously found to markedly impair the function and structure of isolated mitochondria. Thus xray microanalysis of unfixed cryosections provides direct measurements of subcellular alterations in
elemental composition of individual myocytes in injured myocardium and demonstrates that both
calcium and phosphorus accumulate in mitochondria of severely injured myocytes in concentrations
sufficient to exert deleterious effects on these organelles.
Circulation 68, No. 4, 872-882, 1983.
=
MEMBRANE DYSFUNCTION, impaired volume
regulation, and alterations in intracellular distribution
of ions have been implicated as factors involved in the
evolution of irreversible myocardial injury induced by
ischemia or hypoxia. '-9 Several mechanisms, including perturbation of membrane phospholipids,'l'2 reduction in high-energy phosphates,'3 and release of
lysosomal enzymes,14, 15 have been implicated in the
pathogenesis of the membrane, volume, and electrolyte alterations. These alterations, once initiated, may
induce degradative reactions and functional and structural defects in mitochondria and other organelles.1'26
Nevertheless, uncertainty remains regarding the exact
From the Departments of Pathology, Physiology, and Internal Medicine (Cardiology Division), The University of Texas Health Science
Center at Dallas.
Supported by NIH Ischemic Heart Disease Specialized Center of
Research Grant P50-HL- 17669 and by the Harry S. Moss Heart Center.
Address for correspondence: L. Max Buja, M.D., Department of
Pathology, The University of Texas Health Science Center at Dallas,
5323 Harry Hines Blvd., Dallas, TX 75235.
Received Jan. 25, 1983; revision accepted June 9, 1983.
872
levels of various elements in subcellular compartments
of normal and injured cardiac muscle cells, since much
of the evidence for altered elemental composition of
injured cells has been obtained indirectly from measurements of the elemental content of whole tissues. It
also is known that individual myocytes show variation
in the severity of damage in response to regional ischemia in vivo6, 27 and to in vitro global hypoxia3 4 and
ischemia.5 These considerations emphasize the important need for in situ measurements of elemental concentrations in individual myocytes.
We have used the analytical electron microscopic
technique of energy-dispersive x-ray microanalysis2>30
to measure elemental concentrations in sarcoplasm and
mitochondria in individual myocytes of isolated perfused rabbit myocardium under oxygenated and hypoxic conditions. This technique has the unique capability of yielding quantitative data for several elements
within discrete subcellular regions. Diffusible elements were preserved by means of suitable methods of
CIRCULATION
LABORATORY INVESTIGATION-MYOCARDIAL METABOLISM
tissue freezing and cryosectioning.3' 36 Electron probe
x-ray microanalysis has been applied previously to
study physiologic phenomena and cell damage in
smooth, skeletal, and cardiac muscle and other tissues.37-56 In this study, diffusible elements in myocytes
of control and hypoxic myocardium were quantified by
means of separately analyzed cryosections of fresh frozen tissues and gelatin-glycerol elemental standards.32, 36
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The specific hypotheses tested in this study were (1)
that individual myocytes may exhibit important variation in the severity of alterations in intracellular ionic
homeostasis in response to hypoxia and (2) that hypoxic myocytes may accumulate certain elements in
quantities sufficient to impair organellar function and
structure. Quantitative microanalytical data are presented which indicate that mitochondria of severely
injured myocytes are exposed in situ to levels of calcium and phosphate that have previously been shown
to induce severe damage to the isolated organelles.
Materials and methods
Model system. The isolated perfused rabbit interventricular
septal preparation was used as described previously.5 57 The
standard preparation was modified by leaving the small right
ventricular papillary muscles attached to the septum.35 36 The
chamber was sealed to maintain a nitrogen environment, constant humidity, and temperature of 300 + 10 C. The septa were
paced at a rate of 42 beats/min. The standard perfusion solution
was a modified, Krebs-Henseleit medium containing 139 mM
sodium, 102 mM chloride, 28.6 mM bicarbonate, 5.4 mM
potassium, 2.5 mM calcium, 1.2 mM phosphate, 1.0 mM magnesium, 1.0 mM sulfate, 10 mM glucose, 5 mM fumarate, S
mM glutamate, and 5 mM pyruvate; the solution was bubbled
with 95% 02 and 5% CO2.5
All septa were stabilized for 1 hr in the oxygenated medium
and accepted for study if at least 15 g of developed tension was
produced with a resting tension of up to 10 g. Eight muscles
were subjected to hypoxic perfusion for 1 to 11/2 hr by perfusion
with medium bubbled with 95% N2 and 5% CO2. Six control
muscles were perfused with oxygenated medium for comparable periods. The functional status of the septa was determined
by recording standard physiologic contractile parameters
throughout the experimental protocols.57 Four nonperfused papillary muscles were obtained when the rabbits were killed for
comparison with the perfused preparations.
Papillary muscle sampling and freezing. After the desired
period of in vitro perfusion, the hood was removed, a papillary
muscle was clamped in a muscle holder, and the base of the
papillary muscle was cut away from the septum.35 36, 55 A tissue-mounting pin was placed between the muscle and a small
retaining spring. The whole assembly was then plunged into a
double-freezing Dewar flask of propane slush cooled with liquid
nitrogen. A similar sampling and freezing procedure was used
for papillary muscles from nonperfused hearts. The muscle
sampling assembly was transferred from the propane to liquid
nitrogen, and the pin and attached central portion of papillary
muscle were detached from the clamp. Liquid nitrogen-cooled
propane was chosen as the initial freezing medium because it
affords more rapid freezing rates compared with most cryogenic
agents.35' 58, 59 Freezing of the muscles by contact with a liquid
Vol. 68, No. 4, October 1983
nitrogen- or liquid helium-cooled copper block31 6(63 was not
feasible because of the use of the muscle holder-mounting pin
apparatus.
Cryoultramicrotomy. Cryoultramicrotomy of myocardium
and elemental standards was performed with a cryosystem interfaced with an ultramicrotome.35' 36 The specimen temperature
was maintained at - 1200 C and the knife temperature at -1000
C. Sections in the range of 100 nm were cut with a glass knife
and dry mounted onto 50 mesh, Formvar-coated copper grids. A
top layer of Formvar was applied to the sections. The sections
were then freeze-dried in the cryochamber.
Freeze substitution of frozen tissue. Some papillary muscles were used for freeze substitution rather than cryosectioning. The frozen muscles were transferred to a vial containing
10% osmium in acetone that had been frozen in liquid nitrogen.63 The vial was placed in a liquid nitrogen reservoir and
allowed to warm to room temperature over 16 to 20 hr as the
liquid nitrogen evaporated. The tissue was embedded in Eponaraldite, thin sectioned, and stained with uranyl acetate and lead
citrate for routine transmission electron microscopy.
Analytical electron microscopy. A JEOL lOOC electron microscope with a high-resolution scanning attachment and a Kevex 30 mm2, 158 eV energy-dispersive x-ray detector was
used.35 36 X-ray spectra were collected and analyzed with a
Tracor Northern (TN) 2000 multichannel analyzer. For analysis, the microscope was operated in the scanning transmission
mode at 80 kV, 50 mA emission current, 30 degree specimen
tilt, and beam diameter of 20 to 60 nm.
Before data collection, the TN-2000 multichannel analyzer
was calibrated for gain and zero shifts with copper and aluminum grids sandwiched together. The count rate was adjusted to
1000 cps over the range of 0 to 10 KeV and gain and zero
adjustments were made ± 2 eV with the aluminum K alpha and
copper K alpha x-ray lines. The x-ray spectra were collected
over the range of 0 to 10 KeV for 50 sec at a resolution of 20 eV
per channel. The selected area raster mode was used to collect
spectra from a 0.75 x 0.75 izm area at 30,000 x . Deconvolution of the test spectra from tissue sections was obtained by the
multiple least squares fitting routine available on the TN
2000.36. 64 This program uses elemental reference spectra (primary standards) previously collected from thin crystals of various salts composed of elements of interest and permits separation of counts contributed by two elements with close or
partially overlapping peaks such as the sodium K and copper L
peaks and the potassium K beta and calcium K alpha peaks.36'64
The contribution from copper L x-rays was small and constant
in all spectra.
The quantitative analysis of spectra from thin biological
specimens was based on the peak-to-continuum ratio being proportional to concentration as described by Hall et al.28 and
Shuman et al.29, 30 The continuum used for the calculations was
from 5.5 to 6.5 KeV. Peak-to-continuum ratios were converted
to elemental concentrations by the weighting factors derived
from elemental secondary standards prepared according to the
method of Roomans and Seveus.32 This method involved preparation of different concentrations of various salts (NaCl,
CaCl2, K2P04, etc.) in a gelatin and glycerol solution. Freezedried sections of the gelatin standards were analyzed, and standard curves for each element were obtained.36
Several approaches were tested for the quantitative analysis
of elements present in relatively low concentration that generate
low-intensity peaks in x-ray spectra. An initial approach (method 1) was to set a one or two sigma (standard deviation) criterion
for detection of peaks during the deconvolution procedure. This
method yielded undetectable calcium and magnesium levels for
some spectra (with more undetectable values for two sigma than
one sigma criterion) because normal intracytoplasmic levels of
873
BUJA et al.
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these elements approach the limits of detectability of the method.30' 3 The calcium and magnesium values for these spectra
were assigned values of 0 for purposes of calculation of mean
calcium and magnesium levels. As an alternative approach
(method 2), single summed spectra were derived from 10 individual analyses from the same cell before deconvolution and
quantitative analysis.4 Because of the increased counts and
improved statistics, undetectable elemental peaks were avoided
by this method.48 Another approach (method 3) was to set sigma
at 0 during the deconvolution of the individual spectra. This
method yielded negative as well as positive peak values and
eliminated the need to assign a 0 value to any peaks. The
resulting peak values with no missing numbers may then be
processed statistically, as described previously.37 Similar mean
values (tables 1 and 2) were obtained by method 1 with a one
sigma criterion, method 2, and method 3, whereas significantly
lower values were obtained for calcium and magnesium by
method I with a two sigma criterion. It should be noted that
methods 2 and 3 avoid the statistical problem of dealing with 0
values.
With regard to these x-ray microanalytic methods, further
evaluation is needed of the potential effects of radiation damage
and the mass contribution of the support and carbon films on
absolute elemental quantification.36 It also should be pointed
out that, for some applications, x-ray microanalysis can be
performed with the conventional transmission mode rather than,
as in the present study, the scanning transmission mode of the
electron microscope.
Statistical analysis. For the contractile data, a group t test
was used.65 For the microanalytical data, an analysis of variance
was used.65 Multiple group comparisons were made with the
Newman-Keuls test and differences were considered to be significant at the p < .05 level.
Results
Contractile parameters. Control muscles were maintained with oxygenated perfusate. Other muscles were
subjected to 1 to 11/2 hr of hypoxia after an initial 1 hr
of perfusion with oxygenated medium. At the end of
the initial 1 hr stabilization period, the control muscles
and the test muscles (those subsequently made hypoxic) had a resting tension (RT) of 8.2 ± 1.5 (SD)
and 7.2 + 1.5 g, a developed tension (DT) of 22. 1 ±
8.9 and 24.4 + 6.9 g, and maximal rate of tension
development (+dT/dt) of 118.8 ± 50 and 130.8 ± 38
g/sec. (The means were not significantly different.)
At the end of the experiments, control and hypoxic
muscles showed the following changes (expressed as
percent of control values): RT of 74.1 + 12.9 and
308.0 ± 122.6(p< .001), DTof 102.9 ± 6.3and5.6
± 5.9 (p < .001), and + dT/dt of 102.7 ± 14.2 and
5.1 + 6.4 (p < .001), respectively. There were no
significant differences between the muscles subjected
to either 1 or 1 /2 hr of hypoxia.
Ultrastructure. Transmission electron microscopy of
papillary muscles freeze-substituted in osmium-acetone was performed to evaluate the general quality of
tissue preservation after rapid freezing. As shown in
figure 1, the muscle cells immediately adjacent to the
endocardium of the papillary muscles showed virtually
874
no ice crystal formation. Slightly deeper myocytes had
a finely vacuolated appearance in the sarcoplasm and
myofibrils caused by slight ice crystal damage. Mitochondria did not show this alteration. In this region,
the size of the ice crystal disruption was considerably
smaller than the raster area used for x-ray microanalysis (figure 1). For x-ray microanalysis, unfixed, unstained cryosections were examined by scanning transmission electron microscopy. Although scanning
transmission electron micrographs of unfixed, unstained cryosections have lower contrast and resolution than transmission electron micrographs of freezesubstituted tissue, they are presented in figures 2 to 4
because they are representative of the image quality
under actual conditions of x-ray microanalysis in the
present study.
Scanning transmission electron microscopy of unfixed, unstained cryosections of nonperfused papillary
muscles showed general structural features of myocardium (figure 2, A). Muscle cells were separated by
capillaries containing erythrocytes, which had a fine
lattice structure indicative of mild ice crystal damage.
Muscle cells exhibited nuclei and myofibrils with Z, I,
and A bands. The muscle cells also had dense ovoid
structures with the size and distribution of mitochondria. T tubules and cistemae of sarcoplasmic reticulum
could not be identified readily. In many sections the
myocytes had fine holes or vacuoles. Although some
of these profiles may have represented elements of the
T system or sarcoplasmic reticulum, most of them
probably represented defects produced by mild ice crystal damage. 31,33
Scanning transmission electron microscopy of cryosections of papillary muscles from perfused oxygenated control septa showed features of myocardium similar to those observed in sections of fresh frozen tissue
(figure 2, B). Scanning transmission electron microscopy of cryosections of perfused hypoxic myocardium
revealed that most muscle cells also had ultrastructural
features similar to those of control myocardium, although these sections frequently had low contrast with
poorly defined myofibrils (figure 3). However, approximately one-third of the myocytes examined in
four of the eight papillary muscles had multiple, very
electron-dense inclusions within the ovoid mitochondrial profiles (figure 4). These cells also had generally
low contrast with poorly defined cytoplasmic elements.
X-ray microanalysis. Representative x-ray spectra
from control and hypoxic papillary muscles are shown
in figure 5. Quantitative data for sodium, chloride,
potassium, sulfur, calcium, phosphorus, and magneCIRCULATION
_;s}~ ~ A'
LABORATORY INVESTIGATION-MYOCARDIAL METABOLISM
4t4Arr.
Z
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FIGURE 1. Transmission electron micrograph of a stained and osmicated epoxy thin section from freeze-substituted rabbit
papillary muscle. Ultrastructural features of cardiac muscle cells are well preserved. The myocyte (top) more superficial to the
endocardium shows virtually no ice crystal formation, whereas the deeper myocyte has a finely vacuolated appearance caused by
slight ice crystal damage. Bar = 2 ,im.
sium are presented in tables 1 and 2. The data for
sarcoplasm were derived from analyses of regions of
interest containing myofibrils as the predominant element. The data for mitochondria were derived from
analyses of the ovoid dense structures. For the hypoxic
papillary muscles, data are presented separately for
muscle cells with and without mitochondrial inclusions.
Sarcoplasm and mitochondria of myocytes from the
six control perfused muscles had a similar elemental
composition compared with myocytes from the four
control nonperfused muscles (tables 1 and 2). In the
eight hypoxic papillary muscles, myocytes without
mitochondrial inclusions exhibited increased sodium
and chloride, decreased potassium, phosphorus, and
Vol. 68, No. 4, October 1983
magnesium, and slightly increased calcium in the sarcoplasm. Mitochondria in these cells had increased
sodium and chloride, decreased potassium, phosphorus, and magnesium, but calcium levels were similar to
control levels. Hypoxic myocytes with mitochondrial
inclusions exhibited an increase in sodium and chloride, decrease in potassium, phosphorus, and magnesium and a slight further increase in calcium in the
sarcoplasm. Mitochondria in these cells exhibited
markedly increased levels of calcium and phosphorus,
greatly increased levels of sodium, and low levels of
potassium. Relatively few spectra were collected from
the sarcoplasm of these cells with mitochondrial inclusions because morphologic detail and section contrast
were particularly poor in these areas. Myocytes with
875
BUJA et al.
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FIGURE 2. Scanning transmission electron micrograph (STEM) of unstained, frozen-dried sections of control papillary
muscles. A, Nonperfused control muscle exhibits erythrocytes in vascular spaces between the myocytes. The pattern of
myofibrillar
cross-striations is faint. The dark ovoid structures
in
the myocytes have the distribution of mitochondria. Bar
gim. B, Perfused control muscle exhibits myofibrils with cross-striations and dense ovoid mitochrondria. Bar
mitochondrial inclusions constituted approximately
one-third of the cells examined in four of the eight
hypoxic muscles.
Discussion
In this study, the values for concentrations of most
elements in mitochondria and sarcoplasm of control
myocardium, measured directly by analytical electron
microscopy, were of the same order of magnitude as
the estimated intracellular values calculated from measurements of total tissue water, total tissue electrolytes, and intracellular and extracellular spaces by
means of various tracer molecules.61t These values
also were comparable to values obtained by analytical
electron microscopy by other investigators for cardiac,
skeletal, and smooth muscle cells.37'8, 40. 4, 43 In these
studies, mean calculated calcium values were I to 7
mM/kg dry weight for cytoplasm, 0.38 to 4 mM/kg dry
weight for mitochondria, and 66 to 77 mM/kg dry
weight for the relatively large terminal cistemnae of
the sarcoplasmic reticulum of skeletal muscle and certain intermitochondrial regions (presumed to be ele876
=
2
2 gim.
ments of sarcoplasmic reticulum) of cardiac mus-
cle.37' 38, 40. 41. 43 The sodium values in the present study
were high and exhibited relatively large variability.
These findings may be related to several factors, including the poor sensitivity of x-ray microanalysis for
quantification of sodium29 30 37 and, possibly, partial
depolarization of cardiac myocytes during sampling or
other factors related to the particular experimental
model. X-ray microanalysis measures total elemental
concentrations, of which only a fraction exists as free
ions.69, 70
Our results demonstrated that hypoxic papillary
muscles had striking differences in elemental concentrations compared with both the nonperfused and perfused control groups. The most likely explanation for
these elemental changes is that they occurred as part of
progressive alterations in various membrane ionic
transport systems, in membrane permeability, and in
cell volume regulation, which develop with hypoxic
and ischemic myocardial injury.1 9The majority of the
hypoxic muscle cells exhibited increased sodium levels and reduced potassium, phosphorus, and magneCIRCULATION
LABORATORY INVESTIGATION-MYOCARDIAL METABOLISM
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FIGURE 3. STEM of unstained, frozen-dried sections of hypoxic papillary muscle. A, Hypoxic myocyte exhibits myofibrils
with cross-striations and dense ovoid mitochondria. Bar
gim. B, This area shows prominent mitochrondria without
inclusions. Bar = 2 im.
sium levels without a major change in calcium. Other
hypoxic muscle cells also showed evidence of calcium
overloading, with formation of multiple electrondense granules in the mitochondria. These mitochondria exhibited increased calcium and phosphorus levels, suggesting that the deposits were composed
primarily of calcium phosphate. Such calcific deposits
form in severely damaged cells with access to a source
of extracellular calcium.2' 27, 40V42, 50. 53 The calcium
phosphate inclusions observed in cryosections in the
present study probably correspond to the granular electron-dense inclusions and not the amorphous matrix
(flocculent) densities observed in fixed, epoxy-embedded preparations of severely damaged myocytes, since
only the former type of inclusion contains calcium and
exhibits inherent electron density. 17 50' 53
Previous studies using other techniques have shown
that net loss of intracellular potassium is an early response to hypoxic and ischemic injury.9'71'72 More severe injury is associated with altered distribution of
polyvalent cations, including net intracellular accumulation of Ca2^2, 8, 18, 50, 73 Using ionic lanthanum as an
ultrastructurally identifiable membrane probe, we
have previously shown that isolated cat papillary musVol. 68, No. 4, October 1983
cles develop severe alterations in permneability of plasma membranes and mitochondrial membranes after 90
min of hypoxia in vitro.3'4 Similar abnormal lanthanum permeability has been demonstrated after 60 to 90
min of ischemia and reflow in the rabbit perfused septal preparation.5 Widespread abnormal intracellular
lanthanum accumulation occurred at a transitional
stage in the progression from reversible to irreversible
contractile depression in these models."
Compared with other mitochondria in hypoxic cells,
mitochondria with dense granules showed a mean increase of 369 mM/kg (26-fold increase) in calcium and
21 1 mM/kg (56% increase) in phosphorus. The large
standard deviations for the measurements of calcium
and phosphorus in mitochondrial inclusions probably
were related to variation in the size and elemental
content of the inclusions. With regard to the relatively
small change in phosphorus concentration between hypoxic mitochondria with and without inclusions, simultaneous depletion of organic phosphorus (including high-energy phosphates) and accumulation of
inorganic phosphorus could result in a large increase in
inorganic phosphorus with relatively little net increase
in total phosphorus concentration. Hagler et al.53 found
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BUJA et al.
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FIGURE 4. STEM of unstained, frozen-dried sections of hypoxic papillary muscle. A, Some of the mitochondria in these
myocytes have electron-dense inclusions. Bar 2 gim. B, A higher magnification view of mitochondria with electron-dense
inclusions. Bar = I ,um.
that initial mitochondrial inclusions formed in temporarily ischemic myocardium and preserved by anhydrous fixation had calculated calcium-to-phosphorus
molar ratios of 0.99 + 0. 11. Thus it is reasonable to
conclude that the excess amounts of calcium and phosphorus observed in mitochondrial inclusions in cryosections of hypoxic myocardium represent inorganic
ions that accumulated in the hypoxic cells and were
then concentrated in the mitochondria before formation of the granules.
We have previously shown that incubation of isolated mitochondria in vitro with either micromolar
amounts of Ca+2 (200 ng/mg protein or 200 gM) or
millimolar amounts of H2PO4 (5 to 50 mM) results in
severe depression of mitochondrial function and, in the
case of Ca+2, severe structural damage to the mitochondrial membranes.24 Similar results have been
reported by others.22-28 These levels of calcium and
phosphorus were within the range of levels estimated
to accumulate in damaged myocytes from various
measurements of whole tissues or isolated mitochondria. 120 27 The present study provides direct quantitative measurements to show that calcium and phospho878
rus can accumulate in intact cells to the toxic levels
observed to be deleterious to organellar function and
structure in studies of isolated mitochondria.
The reasons for the selective accumulation of calcium and phosphorus in mitochondria of a subset of
myocytes in hypoxic myocardium is unclear. Marked
accumulation of calcium in mitochondria is generally
considered to be an energy-dependent phenomenon,
which also requires the presence of a receptor anion
such as phosphate.21 22 Mitochondria with massive calcium phosphate deposits are typically observed in severely damaged myocytes during reperfusion after ischemic injury 7 17, 50 53 or reoxygenation after anoxic
injury.7475 Mitochondrial calcium phosphate loading
also has been observed in isolated cardiac myocytes
with hyperpermeable plasma membranes when the
Ca2l content of the medium exceeds 10-6M41' A similar phenomenon occurs when myocardium is perfused
with calcium-free medium fullowed by calcium-containing medium ("calcium paradox").76 77 In the present study, accumulation of mitochondrial calcium
phosphate deposits in some myocytes of hypoxic myocardium could have been related to specific features of
CIRCULATION
LABORATORY INVESTIGATION-MYOCARDIAL METABOLISM
GROUP 2 MITO
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FIGURE 5. X-ray spectra from mitochondria of control and hypoxic myocytes. Group 1 mito, Spectrum from control
mitochondrion has sodium (Na), magnesium (Mg), silicon (Si), phosphorus (P), sulfur (S), chlorine (Cl), and potassium (K)
peaks. Group 2 mito, Spectrum from perfused control mitochondrion has elemental peak-to-continuum ratios similar to those for
the nonperfused control. Group 3 mito, Spectrum from hypoxic mitochondrion without inclusions shows prominent sodium peak
and decreased phosphorus and potassium peaks. Group 4 mito, Spectrum from hypoxic mitochondrion with inclusions shows
prominent calcium and phosphorus peaks, high sodium peak, and very low potassium peak.
findings.4 Isolated myocardium subjected to
global hypoxia or ischemia exhibits a nonuniform response in the severity of injury exhibited by individual
myocytes,?5 and a similar phenomenon occurs with
ischemia in vivo.6 Regardless of the mechanism, the
accumulation of calcium and phosphorus in mitochondrial inclusions may identify myocytes, which develop
the most severe alterations of mitochondrial and plasma membranes. Further studies are underway to estab-
the experimental model. Our measurements were performed on myocardium that was perfused for 60 to 90
min with hypoxic medium that also provided a ready
source of extracellular calcium and phosphate. The
muscles may have been exposed to
previous
low level of 02
a
during the experiments, since a low Po2 (in the range of
40 to 60 mm Hg) was measured in the N2-bubbled
perfusate collected in the chamber after its circulation
through the experimental apparatus, in agreement with
TABLE 1
Elemental concentrations (mM/g dry weight,
microanalysis
mean +
SD) in sarcoplasm of control and hypoxic cardiac myocytes determined by x-ray
Element
Na
Muscle groups
352.2A
Control, nonperfused
(4 muscles, 56 spectra)
Control, perfused
(6 muscles, 68 spectra)
Hypoxic, perfused
(8 muscles)
No mitochondrial inclusions
(107 spectra)
+ 113.8
±99.7
632.OB
416.9B
+208.7
±289.0
(16 spectra)
of
246.8A
160.5
+328.7
546.2B
Mitochondrial inclusions
Superscripts indicate results
278.8A,C
+78.5
3434A
+
K
Cl
359.6B.C
+
305.6
S
Ca
P
383.5A
+87.6
+ 154.4
463 2B287 3B
±66.7
+ 132.3
+97.2
±
260.8c
+81.7
228.Oc
±15.1
±79.2
t 19.3
516.OA
+
105.8
283. lc
±98.0
186. 1D
+33.1
236.Oc
+60.8
198.5D
t 84.3
10.3A
495.9A
+ 10.5
IO4A
12.1
WOB
3205C
Mg
75.OA
+37.4
71.1A
+40.9
47,8B
±38.3
14*8c
±21.3
statistical analysis (analysis of variance and Newman-Keuls test) (A * B * C + D).
Vol. 68, No. 4, October 1983
879
BUJA et al.
TABLE 2
Elemental concentrations (mM/kg dry weight, mean + SD) in mitochondria of control and hypoxic cardiac myocytes determined by x-ray
microanalysis
Element
Muscle groups
Control, nonperfused
(4muscles, 55 spectra)
Control, perfused
(6muscles, 75 spectra)
Hypoxic, perfused
(8 muscles)
No mitochondrial inclusions
(96 spectra)
Mitochondrial inclusions
(52 spectra)
Na
Cl
K
S
282.8A
+115.9
273.9A
+115.9
253.OA
+78.0
232.2A
+88.7
408.3A
±100.6
360.6B
+120.5
384.6A
+84.4
571.4B
±300.9
701l.c
+336.2
422.4B
±231.9
424 OB
±306.2
275.8C
+73.9
147.2D
+52.4
Ca
P
Mg
309.1B
510.2A
±89.4
506.1A
+7.5
9.2A
±48.1
+90.5
±9.8
±29.5
285.9c
±48.0
379.6B
±84.5
591.Oc
13.8A
±11.7
383.1B
32.2B
+29.3
75.3c
±55.6
220.4D
±63.5
±368.9
6.5A
±417.0
60.3A
+31.2
60.0A,C
Superscripts indicate results of statistical analysis (analysis of variance and Newman-Keuls test) (A # B t C + D).
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
lish the time course of intracellular electrolyte alterations in response to hypoxic and ischemic myocardial
injury.
We appreciate the excellent technical assistance of Carol
Greico, Linda Lopez, and Rebecca Lundswick, and the secretarial assistance of Deborah Granville.
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882
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Quantitative x-ray microanalysis of the elemental composition of individual myocytes in
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L M Buja, K P Burton, H K Hagler and J T Willerson
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Circulation. 1983;68:872-882
doi: 10.1161/01.CIR.68.4.872
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