864
Volume Regulation and Plasma Membrane Injury in
Aerobic, Anaerobic, and Ischemic Myocardium
in Vitro
Effects of Osmotic Cell Swelling on Plasma Membrane Integrity
Charles Steenbergen, Mary L. Hill, and Robert B. Jennings
From the Department of Pathology, Duke University Medical Center, Durham, North Carolina
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SUMMARY. The relationship between cell swelling and plasma membrane disruption has been
evaluated in thin myocardial slices incubated in oxygenated or anoxic Krebs-Ringer phosphate
media. Electron microscopy and measurements of inulin-diffusible space were used to monitor
plasma membrane integrity. Inulin is excluded from the intracellular space of intact cells; therefore,
an increase in tissue inulin content is an excellent marker of loss of plasma membrane integrity.
Cell volume was increased during exposure of aerobic slices to hypotonic media, but the inulindiffusible space was not increased and electron micrographs showed no detectable plasma
membrane alterations. Likewise, during prolonged anoxic isotonic incubation, no evidence of
plasma membrane damage was observed. Incubation in anoxic hypotonic media for 60 minutes
resulted in a larger increase in cell volume than under aerobic conditions, but plasma membrane
integrity was maintained. Extended anoxic hypotonic incubation (300 minutes) produced no
further change in tissue water, but the inulin-diffusible space was increased and electron
micrographs revealed breaks in the plasma membranes primarily in association with large
subsarcolemmal blebs. Likewise, myocardial slices incubated in isotonic anoxic media for 240
minutes and hypotonic anoxic media for 60 minutes had an increased inulin-diffusible space and
the ultrastructural appearance was similar. This ultrastructural appearance is indistinguishable
from that observed in myocytes lethally injured by ischemia. Measurements of tissue osmolarity
during total ischemia showed that osmotically induced cell swelling could occur in ischemic
myocardium prior to the onset of plasma membrane disruption. Our results indicate that cell
swelling per se is incapable of rupturing plasma membranes; however, after prolonged periods of
energy deficiency, the plasma membrane or its cytoskeletal scaffold becomes injured, which
allows the membrane to rupture if the cell is swollen, as might occur during ischemia or
reperfusion. (Circ Res 57: 864-875, 1985)
Disruption of the plasma membrane, which is observed early during the irreversible phase of myocardial ischemic injury, may be the event which is
responsible for the transition from reversible to irreversible ischemic injury. Plasma membrane integrity has been evaluated by electron microscopy using
conventional thin sections (Jennings and Ganote,
1974; Ganote et al., 1975; Jennings et al., 1978;
Jennings and Hawkins 1980; Buja and Willerson,
1981), and the freeze-fracture technique (Ashraf and
Halverson, 1977), and by measurement of permeability of large molecular weight compounds such as
inulin (Jennings et al., 1978; Buja and Willerson,
1981; Reimer et al., 1981) or intracellular enzymes
(Ganote et al., 1975; Hearse et al., 1978). Electron
micrographs of irreversibly injured myocytes show
fragmented plasma membranes (Jennings et al.,
1978; Jennings and Hawkins, 1980; Buja and Willerson, 1981), and this lesion has been shown to be
associated with release of cytoplasmic enzymes
(Ganote et al., 1975) and increased permeability to
normally impermeant extracellular markers such as
inulin (Jennings et al., 1978; Reimer et al., 1981).
Numerous studies have demonstrated that myocardial infarction is associated with release of intracellular creatine kinase into the circulation; this release clearly indicates loss of plasma membrane
integrity in the lethally injured myocytes. However,
there is no consensus on the mechanism of membrane injury. Breaks in the plasma membrane can
be observed in electron micrographs of unreperfused
ischemic myocytes when the duration of ischemia is
sufficient to produce lethal injury, suggesting that
membrane damage occurs while the tissue is ischemic (Jennings et al., 1978; Jennings and Hawkins,
1980; Steenbergen and Jennings, 1984), and the
changes' become more pronounced during reperfusion. One possible mechanism of plasma membrane
disruption during permanent in vivo ischemia is
production of oxygen-free radicals, since there may
be enough collateral flow in the ischemic zone to
provide oxygen for free radical production. How-
Steenbergen et al. /Plasma Membrane Injury in Anoxic Myocardium
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ever, electron micrographs of myocardium subjected
to total ischemia in vitro at 37°C show the same
plasma membrane lesion, in a situation where no
collateral flow is possible, as is observed during in
vivo ischemia. Another hypothesis that could account for plasma membrane disruption during total
ischemia is activation of endogenous phosphofipases
resulting in degradation of membrane phospholipids
(Chien et al., 1979; Chien et al., 1981; Farber, 1982).
Measurements of phospholipid contents during in
vivo myocardial ischemia generally indicate that
phospholipid degradation is accelerated (Chien et
al., 1979, 1981; Shaikh and Downar, 1981; Corr et
al., 1982; Gross et al., 1982), but in an in vitro model
of total ischemia, it was found that there is no net
loss of tissue phospholipids, and lysophospholipids
accounted for less than 1% of tissue phospholipids
at a time when plasma membrane disruption is
widespread (Steenbergen and Jennings, 1984). Release of arachidonate from membrane phospholipids early during ischemia may alter membrane
permeability and may contribute to lethal injury
(Ghien et al., 1984), but no evidence has been produced to indicate that arachidonate production induces disintegration of the phospholipid backbone
of the membrane. Thus, we currently hypothesize
that plasma membrane disruption during ischemia
occurs on a basis other than degradation of the
phospholipids of the membrane.
The ultrastructural appearance of the disrupted
plasma membranes of irreversibly injured ischemic
myocytes suggests an alternative hypothesis to explain membrane disruption. Fragmentation of the
plasma membranes is most prominent in areas
where there are subsarcolemmal blebs which separate the membrane from the underlying myofibrils.
These blebs, which contain abnormal accumulations
of intracellular fluid, are absent in reversibly injured
myocytes, but appear early during the irreversible
phase of ischemic injury (Jennings and Hawkins,
1980), and are an objective sign of cell swelling. The
association between ultrastructural signs of plasma
membrane disruption and the appearance of subsarcolemmal blebs suggest that cell swelling may
contribute to membrane fragmentation. An attempt
to quantify cell volume changes during permanent
ischemia using ultrastructure was inconclusive because myocyte diameters were strongly influenced
by the osmolarity of the fixative (Tranum-Jensen et
al., 1981). Comparison between ischemic and nonischemic cells, each fixed with solutions that had
approximately the same osmolalities as the tissue in
situ, shows that the ischemic myocytes were only
slightly larger than the nonischemic myocytes. Another approach, based on measurements of tissue
osmolaliry, predicts an increase in intracellular volume of 13% during ischemia (Friedrich et al., 1981).
In the present study, the effect of cell swelling on
plasma membrane integrity was investigated by ultrastructural analysis and measurements of inulin
865
permeability to determine whether the plasma membrane was intact. The data are consistent with the
hypothesis that energy deficiency of sufficient duration causes an ultrastructurally inapparent injury
to the plasma membrane or its supports which
makes the membrane susceptible to osmotic swelling. Although this injury, per se, does not rupture
the plasma membrane, disruption can occur if cells
are osmotically swollen, as might occur during ischemia and/or reperfusion.
Methods
Hearts from adult mongrel dogs of either sex, anesthetized with intravenous sodium pentobarbital, were excised
rapidly and immersed in 750 ml of ice-cold isotonic KG.
Sections of the wall of the left ventricle containing the
papillary muscles were used to prepare thin slices for
incubation, and in some experiments, portions of the
septum were subjected to total ischemia by placing the
tissue in sealed containers immersed in a 37°C water bath
(Reimer et al., 1981). For the experiments in which tissue
osmolality measurements were made, a sample of septum
was frozen in liquid nitrogen at the same time as other
portions of the septum were placed in the water bath.
Samples were removed from the water bath after 60, 75,
90, 120, and 150 minutes of total ischemia and were
frozen in liquid nitrogen. In some experiments, a sample
also was removed after 180 minutes of total ischemia to
assess volume regulation of incubated thin tissue slices.
Tissue Osmolality Measurements
Tissue osmolality was measured with a Wescor model
5100C vapor pressure osmometer, according to the procedures of Tornheim (1980) and Knepper (1982). The
instrument was balanced and then calibrated with commercially prepared 100, 290, and 1000 mOsm standard
solutions. To calibrate, 8 pi of the standard solution were
pipetted onto a filter papeT disc, inserted into the chamber,
and the chamber was sealed. A 50-second delay before
initiation of the instrument cycle gave reproducible results
with the 290 mOsm standard; moreover, increasing the
duration of the delay had no effect on the osmolaliry
reading. However, a greater delay was necessary to obtain
reproducible readings with the 100 mOsm standard. Since
the tissue samples had osmolality values of over 300
mOsm, a 50-second delay was used in most experiments,
but a 3-minute delay was used in one experiment, without
any discernible difference in the results. The same delay
was used for standards and tissue. The initial calibrations
were performed with each of the three standard solutions,
followed by 5-10 readings with the 290 mOsm standard
to ensure reproducibility. After each tissue sample, the
instrument was rechecked with the 290 mOsm standard.
Values were accepted if the 290 mOsm standard read 290
± 2mOsm. If the thin tissue slice curled up during warming in the sample chamber, it sometimes came into contact
with the thermocouple. This usually interfered with stabilization of the instrument as it approached the end of
the operation cycle and resulted in erroneous readings.
This event always was followed by failure of the 290
mOsm standard to give a reproducible reading. In this
case, the thermocouple was replaced with a clean one
while the dirty one was cleaned. The new thermocouple
was allowed to come into thermal equilibrium with the
866
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instrument, was balanced, and then was calibrated as
described above, before being used for tissue samples.
The tissue was kept frozen in liquid nitrogen before
preparation for osmolality determination. The sections of
control and permanently ischemic septum were allowed
to warm up slightly, and cylindrical full-thickness cores,
approximately 6 mm in diameter, were cut with a cork
borer which had been precooled in liquid nitrogen. The
core of tissue was subsequently removed from the liquid
nitrogen and allowed to warm up slightly. The endocardial
connective tissue as well as 2-3 mm of subendocardial
tissue was removed with a precooled razor blade, and
then disc-shaped slices were cut, approximately 1 mm
thick. In each experiment, three to five slices were used
for osmolality measurements at each duration of total
ischemia. These measurements were averaged to obtain a
single osmolality value for each duration of ischemia for
each experiment. The tissue slices were dried to determine
the tissue dry weight. Using a water content of 3.5 ml/g
dry weight, we calculated tissue volume to be certain that
none of the slices had volumes less than 10 m m . A
minimum volume of 10 mm has been shown to be
necessary for reproducible, accurate tissue osmolality
measurements (Tomheim, 1980). The dry weights of the
slices ranged from 4.0-10.7 mg, which corresponded to
water contents of 14-37 fil.
Slice Incubation Protocols
Thin slices of myocardium were prepared for incubation
as described elsewhere (Grochowski et al., 1976; Ganote
et al., 1976; Jennings et al., 1978; Reimer et al., 1981). The
slices were cut free-hand. They were approximately 0.5
mm thick and weighed between 30 and 100 mg (wet
weight). Slices were placed in 25-ml Erlenmeyer flasks
filled with 15 ml of media, six slices per flask. The flasks
were prewarmed in a shaker bath at 37°C and preequilibrated with N2 or O2. The flasks were shaken 180 times
per minute in a 37°C water bath, and were continuously
equilibrated with humidified N2 or O2. The basic medium
is a Krebs-Ringer phosphate (KRP) solution containing 30
mM mannitol, 1% bovine serum albumin, and trace
amounts of [14C]hydroxymethyl inulin (Reimer et al.,
1981). When an anoxic medium was to be used, it was
prepared fresh from boiled water and was saturated continuously with N2 gas before use (Jennings et al., 1983).
For some experiments, the osmolality of the basic KRP
solution was lowered by the addition of deionized water,
which caused a proportional reduction in all media constituents. Additional [C]hydroxymethyl inulin was added
to the incubation flasks if the media C activity was
reduced by more than 50%.
At the end of the period of incubation, the slices were
removed from the flasks, dipped briefly in 0.25 M sucrose
to remove excess KRP and inulin, blotted on Whatman
number 1 filter paper, and weighed quickly on a Cahn
model DTL micTobalance. In some experiments, one slice
was immediately placed in ice-cold 3.6% perchloric acid
(PCA) for extraction of tissue metabolites. In most experiments, a small piece from the center of one of the remaining slices was processed for electron microscopy.
Subsequently, the five slices were weighed and then
placed in an oven at 105°C overnight to determine the
dry weight and the total tissue water in each slice. Two of
the slices were used for electrolyte determination, and
three were used to measure the inulin-diffusible space
(IDS).
Circulation Research/Vol. 57, No. 6, December 1985
Electrolytes
The dried tissue slices were extracted in 5 ml of 0.75 N
HNO3 and tissue Na+, K+, Mg^, and Ca++ contents were
measured by atomic absorption spectrophotometry, using
a previously published procedure (Jennings et al., 1970,
1978).
Inulin-Diffusible Space
Dried slices were rehydrated with two drops of deionized water. After several hours were allowed for the tissue
to become rehydrated, 1 ml of Soluene 350 (Packard) was
added to each vial to solubilize the tissue. The extracellular
14
C activity was determined by addition of 0.1 ml of the
media from each incubation flask to scintillation vials. The
media vials were prepared in duplicate and were treated
the same as the tissue vials, starting with the addition of
soluene. Once the tissue samples were fully dissolved, 15
ml of acidified Instagel (Packard) were added to each vial.
The Instagel was acidified 9:1 with 0.5 N HC1. The vials
were counted in a Packard model 3255 liquid scintillation
counter. The IDS was calculated as described elsewhere
(Grochowski et al., 1976; Ganote et al., 1976).
Electron Microscopy
The tissue was minced under 4% glutaraldehyde in
0.1 M sodium cacodylate buffer at pH 7.4 (osmolality
approximately 650 mOsm). The tissue was fixed for at
least 4 hours, postosmicated for 1 hour in 1% osmium
tetroxide, dehydrated in a graded ethanol series, rinsed in
propylene-oxide, and embedded in Epon 812. Two grids
consisting of three to five thin sections were prepared
from each specimen. Thin sections were stained with
uranyl acetate and lead citrate and examined with a JEOL
JEM 100B electron microscope. The central portions of the
thin sections were examined thoroughly, and low- and
high-magnification photographs were taken of plasma
membranes to evaluate membrane integrity. The criteria
for ultrastructural evaluation of plasma membranes are
given elsewhere (Jennings and Hawkins, 1980).
Metabolites
For determination of tissue metabolite contents, a blotted, weighed tissue slice was placed in ice-cold 3.6% PCA,
homogenized with a Tri-R homogenizer, centrifuged, and
neutralized to pH 5 with K2CO3 and KOH. The extracts
were centrifuged to remove precipitated KCIO4, and ATP,
creatine phosphate, and lactate were assayed enzymatically as described previously (Jennings et al., 1981).
Statistics
All results are expressed as the mean ± SEM. For determination of statistical significance between experimental
groups, paired f-tests are used where the experiments were
designed to compare aerobic and anoxic slices from the
same heart under otherwise identical conditions. One-way
analysis of variance was used to determine whether there
are significant differences among three or more groups. If
the one-way analysis of variance indicates differences
among the groups (P < 0.01), then unpaired f-tests are
used to establish which groups are significantly different
from control and from each other.
Steenbergen et al. /Plasma Membrane Injury in Anoxic Myocardium
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Minutes of In Vitro Ischemia
Results
Tissue Osmolality Changes during Total
Ischemia in Vitro
To estimate the extent of cell swelling which
occurs during myocardial ischemia, tissue osmolality
was measured in an in vitro model of total ischemia
where tissue water content cannot change during
the ischemic interval. If it is assumed that there is
little movement of osmotically active particles across
the plasma membrane during ischemia, then water
will move from the extracellular space to the sarcoplasm to maintain osmotic equilibrium as anaerobic
metabolism continues to generate small molecular
weight molecules intracellularly. The time-course of
tissue osmolality changes during permanent total in
vitro ischemia is shown in Figure 1. The control
samples had an osmolality of 324 ± 3 mOsm (mean
± SEM). This is undoubtedly higher than the osmolality of normal myocardium in vivo because, during
excision and cooling of the heart before sampling,
much of the creatine phosphate was hydrolyzed to
creatine and inorganic phosphate, and some lactate
was produced. This would increase tissue osmolality
by at least 15-20 mOsm. Therefore, it is likely that
the normal myocardial osmolality in vivo is in the
range of 305 mOsm. The data in Figure 1 indicate
that tissue osmolality increases to a maximum value
of approximately 420 mOsm after 75-90 minutes of
total ischemia in vitro, and that the first evidence of
plasma membrane disruption, such as ultrastructural
discontinuities and increased IDS during subsequent
incubation, is coincident with the maximum increase
in osmolality. Assuming that there is no net movement of osmotically active molecules or ions across
867
FIGURE 1. The relationship between tissue osmolality and plasma membrane damage during
total ischemia in vitro is illustrated as a function of duration of ischemia. Tissue osmolality
was measured in permanently ischemic myocardial slices, as described in the text. Plasma
membrane damage was assessed from the
change in inulin-diffusible space. Electron micrographs of myocardium subjected to total ischemia in vitro show intact plasma membranes
at 60 minutes and progressively more numerous
breaks in plasma membranes as the duration of
ischemia exceeds 75 minutes (Jennings el al.,
1980; Steenbergen and Jennings, 1984). When
slices are prepared from the ischemic tissue and
incubated for 60 minutes in Krebs-Ringer phosphate containing inulin, the inulin-diffusible
space flDS) increases in parallel with the increase in size and frequency of the breaks in the
plasmalemma, indicating that the increase in
IDS is due to plasma membrane disruption
which exists prior to incubation. The IDS data
were reported previously (Reimer et al., 1981;
Steenbergen and Jennings, 1984). Data are presented as mean ± SEM.
the plasma membrane during the initial 75-90 minutes of total ischemia in vitro, that the number of
osmotic particles in the extracellular space remains
constant, and that osmotic equilibrium is maintained
by net water movement, then the magnitude of the
water shift can be determined from the initial osmolality (OSMi), the final osmolality (OSMf), and
the initial extracellular volume (ECVj). The final
extracellular volume is calculated as: (ECV|)(OSMi)/
(OSMF). If ECV, is 23% of tissue water in normal
myocardium (Polimeni and Al-Sadir, 1975) and total
tissue water is 3.65 ml/g dry weight (Jennings et al.,
1984), then the net water movement will be 0.23
ml/g dry weight. This would correspond to an 8%
increase in intracellular volume (ICV)/ from 2.81 to
3.04 ml/g dry weight.
Effects of Cell Swelling on Aerobic Myocardium
The structural integrity of the plasma membranes
was evaluated in two ways. Portions of the incubated slices were examined by electron microscopy
to determine whether plasma membranes were intact. Only myocytes in the center of the slices were
evaluated. In addition, inulin, a 5000 molecular
weight polysaccharide which is excluded from intact
myocytes, was used to monitor plasma membrane
integrity (Grochowski et al., 1976; Jennings et al.,
1978; Reimer et al., 1981). Inulin diffuses from the
media into the interstitial space during incubation
of thin myocardial slices and rapidly approaches
equilibrium within 30 minutes (Grochowski et al.,
1976). The IDS varies slightly under different experimental conditions even when electron micrographs show intact plasma membranes, but it increases substantially when disruption of the plasma
868
Circulation Research/Vo/. 57, No. 6, December 1985
membrane is apparent. This is consistent with the
ultrastructural observation that the interstitial space
is relatively constant in size, regardless of the incubation conditions.
To determine whether, and under what circumstances, cell swelling can disrupt plasma membranes, we incubated thin slices of control myocardium in continuously oxygenated hypotonic media
for 60 minutes at 37°C. The hypotonic media were
prepared by diluting the standard KRP phosphate
media with deionized water. The results, plotted in
Figure 2, show that TTW and intracellular volume
(ICV) increase when the extracellular osmolality is
reduced, but that there is no increase in the IDS;
moreover, ultrastructural evaluation shows no evidence of plasma membrane disruption. The ICV,
calculated as the difference between the TTW and
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Total Tissue Water
Introcellular Volume
Inulin Diffusible Space
Predicted Intracellular Volume
8-
6--
E
o
4--
2--
(7)
300
(4)
(4)
(6)
(3)
240
180
120
Media Osmolality
(mOsm)
(
7)
60
FIGURE 2. The relationship between intracellular volume and media
osmolality after 60 minutes of aerobic incubation is illustrated. Total
tissue water (TTW and inulin-diffusible space (IDS) were measured
as described in the text. Since electron micrographs showed intact
plasma membranes under the conditions ustd for these experiments,
the IDS can be used to estimate slice extracellular volume. Intracellular volume (ICV) was calculated as TTW-1DS The predicted ICV
was determined assuming that there is no net movement of osmotically active molecules or ions into or out of myocytes during hypotonic
incubation, that osmotic equilibrium is maintained solely by water
influx, and that the number of intracellular osmotically active particles remains constant. The predicted ICF was calculated from the ICV
under isotonic conditions QCVJ and the media osmolality (OSMJ,
using the equation: predicted ICV = (7CViX300,)/(OSMJ. Data are
presented as mean ± SEAL
the IDS, increased to nearly 4 ml/g dry weight at an
extracellular osmolality of 60 mOsm. Since the maximum expansion of ICV during total ischemia can
never exceed the TTW, and since the intracellular
volume of the tissue incubated in 60 mOsm media
(3.92 ml/g dry weight) was greater than the total
water content observed in totally ischemic tissue
(3.58 ml/g dry weight) when membrane disruption
has occurred, cell swelling per se cannot be the cause
of plasma membrane disruption during myocardial
ischemia. These data also demonstrate that the ICV
calculated as TTW—IDS, is much less than the predicted ICV, which was determined by assuming that
osmotic equilibration during exposure to hypotonic
media would be accomplished solely by water influx, without any efflux of osmotically active particles.
Effects of Cell Swelling on Anaerobic
Myocardium
The data described above indicate that the plasma
membranes of aerobic myocytes withstand an increase in cellular volume of over 50%, and that
volume-regulating mechanisms prevent explosive
cell swelling during severely hypotonic conditions.
In the next series of experiments, we explored the
possibility that the plasma membranes of ischemic
or anoxic myocytes might be more susceptible to cell
swelling than those of aerobic myocytes. Thin slices
of control myocardium were incubated under aerobic and anoxic conditions for 1-4 hours; the medium
then was diluted 1:1 with deionized water, which
had been prewarmed and preequilibrated with O2
or N2, to lower the osmolarity to 150 mOsm. As
shown in Figure 3, the IDS is unchanged by 1 hour
of anoxic incubation followed by 1 hour of hypotonic anoxic incubation (1 + 1). The IDS values are
somewhat higher than in Figure 2 because inulin
equilibration is only about 90% completed in 1 hour,
and therefore the IDS slightly underestimates the
extracellular space in Figure 2, whereas, in Figure 3,
the IDS slightly overestimates the extracellular space
because the shces were prelabeled during the isotonic incubation with inulin at twice the final media
inulin concentration. No evidence of plasma membrane damage developed after 2 hours of anoxic
incubation followed by 1 hour of hypotonic (150
mOsm) incubation (2 + 1), but the 4 + 1 anoxic
slices showed markedly increased IDS values, compared with aerobic slices subjected to similar conditions (P < 0.01). Thus, 4 hours of anoxia produced
a change in the tissue which allowed the inulin
space to increase when the myocytes were subjected
to hypotonic conditions.
Direct measurement of the TTW showed that
swelling occurred during the hypotonic period of
incubation. However, hypotonic incubation consistently induced higher water levels in the anoxic than
in the oxygenated slices. In the oxygenated slices,
TTW values were 4.00 ± 0.11 (1 + 1), 3.76 + 0.03
(2 + 1), and 3.73 ± 0.03 (4 + 1), whereas, in the
Steenbergen et al. /Plasma Membrane Injury in Anoxic Myocardium
869
-1.8
1.6
FIGURE 3. The effect of osmotic cell swelling on
plasma membrane integrity, as assessed by
changes in inulin-diffusible space, is shown as
a function of the duration of incubation in
oxygenated or anoxic medium. Slices of control
myocardium were incubated for 1-4 hours in
isotonic medium followed by 1 hour in 750
mOsm media prepared by diluting the isotonic
medium 1:1 with deionized water. The deionized water was prewarmed and preequilibrated
with either 100% O2 or 100% N2 to avoid any
change in media temperature or oxygen content.
Incubation time is given as hours of isotonic
incubation plus hours of hypotonic incubation.
Only the 4+ 1 anoxic slices had a significantly
(P < 0.01) elevated IDS, compared with the
corresponding aerobic slices. Data are presented
• = Anoxic
o = Aerobic
I 1.4
« 10
1+)
2+1
3+1
Hours of Incubation Isotonic + Hypotonic
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anoxic slices, TTW values were 4.35 ± 0.13 (1 + 1),
4.16 ± 0.13 (2 + 1), 4.09 ± 0.12 (3 + 1), and 4.30 ±
0.07 (4 + 1). Nevertheless, the extent of swelling
was not the primary determinant of plasma membrane disruption, since membrane disruption was
not observed in the slices with the highest TTW, i.e.,
slices given 1 hour of isotonic and 1 hour of hypotonic anoxic incubation.
Previous studies 0ennings et al., 1983) have
shown that slice adenosine triphosphate (ATP) and
creatine phosphate (CP) contents are well maintained during prolonged aerobic, isotonic incubation
except for an early loss of adenine nucleotides from
the myocytes on the cut edges. On the other hand,
anoxic, isotonic incubation reduces ATP and CP to
40% and 12% of that in aerobic slices after 1 hour
and to nearly undetectable levels after 3 hours.
Incubation in hypotonic media further enhances the
loss of ATP and CP, under both aerobic and anoxic
conditions. The ATP/g dry weight of the aerobic
slices in Figure 3 was 80 ± 9% of that in control
isotonic slices from the same experiments and the
CP/g dry weight was 46 ± 6% of control, with no
significant difference between the 1 + 1,2 + 1, and
4 + 1 aerobic groups. The magnitude of the drop in
high energy phosphate compounds is proportional
to the reduction in media osmolarity. Virtually no
ATP or CP remains after 1 hour of aerobic incubation when media osmolarity is decreased to 60
mOsm. The anoxic slices in Figure 3 had virtually
no ATP or CP after any duration of isotonic plus
hypotonic incubation.
Continuous incubation in media of different osmolarities was used to study the effect of media
osmolarity on myocardial volume regulation under
aerobic and anoxic conditions. Unincubated control
myocardium as well as anoxic slices incubated for 1
hour in 300 mOsm media had essentially the same
TTW, 3.7 ml/g dry weight, while oxygenated slices
had 3.4 ml/g dry weight when incubated for 1 hour
4+]
as mean ± SEW.
in isotonic media (P < 0.01, aerobic vs. anoxic incubation). As shown in Figure 4, when slices were
placed in hypotonic media, tissue water increased
markedly; however, the water content was significantly higher after 1 hour of incubation under anoxic
than aerobic conditions when incubated in 150
mOsm media (P < 0.01). Myocardial slices incubated
in oxygenated hypotonic media lost approximately
15% of tissue water between 1 and 5 hours of
incubation. Thus, oxygenated slices are able to
dampen the volume increase during osmotic swelling, but volume regulation in anoxic slices exposed
to an osmotic gradient is relatively ineffective. Only
a 3% decrease in tissue water occurred between 1
and 5 hours of incubation in the hypotonic anoxic
media.
The effects of continuous exposure to hypotonic
aerobic or anoxic media on plasma membrane integrity was examined by electron microscopy (see next
section) and by measurement of IDS. After 1 hour
of incubation, the IDS values (Fig. 4) are indistinguishable. The IDS increased slightly under all conditions during the subsequent 4 hours of incubation
as inulin equilibration was completed. At 5 hours,
the IDS values for the anoxic isotonic slices and the
oxygenated hypotonic (150 mOsm) slices are not
significantly different from the oxygenated isotonic
values. However, after 5 hours of incubation, there
is a significant increase (P < 0.01) in IDS under
anoxic hypotonic (150 mOsm) or aerobic severely
hypotonic (60 mOsm) conditions, as compared with
any of the other three experimental conditions. The
experimental conditions that produce elevated inulin spaces are the conditions that cause the most
pronounced destruction of high energy phosphate
reserves, with ATP plus creatine phosphate levels
reduced to 5% of control or less after 1 hour of
incubation, i.e., at a time when plasma membranes
are intact. This contrasts with the oxygenated hypotonic (150 mOsm) slices which maintain high
Circulation Research/VoJ. 57, No. 6, December 1985
870
TABLE 1
5.0-•
Effect of Media Osmolarity and Oxygen Content on Tissue
Water and Inulin Permeability of Incubated Myocardial
Slices after 180 Minutes of Total Ischemia in Vitro
Experimental conditions
a>
Q 4.0
E
180 Min total ischemia in
vitro + 60 min in oxygenated isotonic media
o
3.0--
_L
a>
o
a
Q.
CO
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• — • O2lsofonic (300 mOsm)
• — • N2 lsotonic (300 m0sm)
o—0O2Hypotonic ( 150 mOsm)
o — a N2 Hypotonic (150mOsm)
- • A—&O2Hypofonic ( 60 mOsm)
1.0-0.5-•
60
180
300
Duration of Incubation(min)
FIGURE 4. The effect of media osmolality on myocardial slice TTW
and IDS under aerobic and anoxic conditions is illustrated. The
number of experiments for each incubation condition is shown in
parentheses in the upper panel Data are presented as mean ± SEW.
No significant difference in the 1-hour IDS measurements could be
demonstrated by one-way analysis of variance (P > 0.01), but there
are significant differences in the IDS measurements at 5 hours and in
the TTW data at 1 and 5 hours. The 1-hour TTW data are significantly
different (P < 0.01) except for the aerobic 60 mOsm value, which is
not significantly different from the anoxic 150 mOsm value or the
aerobic 150 mOsm value. At 5 hours, the only TTW values that are
not significantly different are the two aerobic hypotonic values. The
5-hour IDS values for the 60 mOsm slices and for the anoxic 150
mOsm slices are not significantly different from each other, but each
is significantly different (P < 0.01) from the other three 5-hour values.
There is no significant difference between the aerobic isotonic IDS
value and the aerobic 150 mOsm value at 5 hours.
energy phosphate reserves, albeit at a reduced level
(37 ± 11% of control after 5 hours).
Cell Swelling in Ischemic Myocardium
Previous studies from this laboratory have demonstrated that tissue slices prepared from left ventricular myocardium after 100 or more minutes of
total ischemia in vitro develop a markedly elevated
inulin diffusible space during an hour of incubation;
ultrastructural analysis suggests that the plasma
membrane disruption is produced prior to incubation (Jennings and Hawkins, 1980; Steenbergen and
Jennings, 1984). However, it is possible that mem-
Total tissue
water
Inulin-diffusible
space
ml/g dry wt
2.44 ± 0.06
4.28 ± 0.05
(8)'
180 Min total ischemia in
vitro + 60 min in anoxic
isotonic media
4.43 ±0.10
180 Min total ischemia in
vitro + 60 min in anoxic
hypertonic media (KRP +
100 HIM polyethylene glycol)
2.55 ± 0.02
2.67 ± 0.07
(6)
1.51 ±0.08
(3)
After 180 minutes of total ischemia in vitro at 37°C, portions
of the left ventricular myocardium were used to prepare thin
tissue slices which were incubated for 60 minutes at 37°C as
indicated. The hypertonic medium was prepared by adding 100
mM polyethylene glycol to the standard Krebs-Ringer phosphate,
giving a final osmolality of approximately 400 mOsm. The data
are presented as mean ± SEM. Tissue water before incubation is
3.7 ml/g dry weight.
* Numbers in parentheses = number of experiments.
brane disruption is produced when the hypertonic
ischemic myocardial slices are placed in isotonic
incubation media, or it could be that disruption
occurs as a result of exposure of the ischemic tissue
to oxygen. To explore these possibilities, we prepared slices from myocardium which was totally
ischemic for 180 minutes, and the slices were incubated in either oxygenated isotonic media, anoxic
isotonic media, or anoxic media supplemented with
100 mM polyethylene glycol (PEG) to increase media
osmolality to the measured tissue osmolality in prolonged total ischemia (see Fig. 1). As shown in Table
1, the ischemic slices swell when incubated for 1
hour in isotonic media. The IDS is elevated markedly
and to a nearly equal extent, regardless of whether
the slices are oxygenated or maintained anoxic. The
ischemic slices incubated in anoxic media containing
100 mM PEG do not swell, and at the end of 1 hour
of incubation, TTW is decreased by approximately
30%, compared with the water content prior to
incubation (3.7 ml/g dry weight). However, prevention of swelling does not prevent the rise in IDS.
IDS is approximately 60% of TTW regardless of the
incubation conditions. Thus, the data are consistent
with the ultrastructural evidence that ischemia-induced plasma membrane disruption is present before incubation or reoxygenation, and does not require swelling during incubation to become manifest.
Steenbergen et al./Plasma Membrane Injury in Anoxic Myocardium
871
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Effects of Cell Swelling on infrastructure
Ultrastructural evaluation of the incubated slices
shows normal cellular morphology after 300 minutes of aerobic, isotonic incubation (Fig. 5). The
plasmalemma exhibits its control scalloped appearance with attachments between the membrane and
the underlying myofibrils at the Z-lines. Mitochondria are compact and show densely packed cristae.
After 60 minutes of aerobic incubation in 150 mOsm
media (Fig. 6A), cell swelling is manifest by increased separation of myofibrils; however, the mitochondria generally remain dense and exhibit
prominent matrix granules. An occasional swollen
mitochondrion is evident. The nuclear chromatin
pattern is indistinguishable from control. The glycocalyx and plasmalemma are opposed closely to
the underlying myofibrils and appear intact. After
300 minutes of aerobic hypotonic incubation (Fig.
6B), cell swelling is less marked than at 60 minutes.
Mitochondrial cristae remain tightly packed and matrix granules are abundant. Endothelial cells show
numerous pinocytotic vacuoles. The glycocalyx and
plasmalemma remain unchanged from control.
As reported previously (Jennings et al., 1983),
amorphous matrix densities (AMD) appear in mitochondria after 300 minutes of anoxic isotonic incubation, but no ultrastructural evidence of sarcolemmal damage is detectable (Fig. 7). After 60 minutes
FIGURE 6. The effect of aerobic hypotonic incubation on myocardial
ultrastructure. Panel A shows myocytes after 60 minutes of incubation
in 150 mOsm oxygenated media and panel B shows the effect of 300
minutes in 150 mOsm oxygenated media. Nuclear chromatin (N) is
evenly distributed. Mitochondria generally are dense and have numerous prominent matrix granules (MC), although an occassional
swollen mitochondrion (SM) is present. Capillary endothelium (CAP)
is unremarkable. The inset shows the intact plasma membrane (P) at
higher magnification. Panel A: 10,950x; panel B: 2O,95OX; inset.
50,00Ox.
FIGURE 5. Representative electron micrographs of several myocytes
incubated for 300 minutes m isotonic, oxygenated Krebs-Ringer phosphate media at 37°C Nuclear chromatin (N) is evenly distributed,
the plasmalemma (P) and glycocalyx (G) are intact. Panel A: 10,950X;
panel B- 43,750X.
of anaerobic incubation in 150 mOsm media (Fig.
8), most of the mitochondria are severely swollen
and some of the cristae are disrupted. A similar
ultrastructural appearance is produced by 60 minutes of aerobic incubation in 60 mOsm media. However, despite the marked degree of cell swelling, the
plasmalemma remains opposed to the underlying
myofibrils. The plasma membrane retains its scalloped appearance, and definite breaks in the plasmalemma are not identified. The glycocalyx likewise
remains intact. More prolonged anaerobic hypotonic
incubation produces more severe ultrastructural
changes. AMD are prominent, and there are linear
matrix densities as well. There is increased sarcoplasmic space beneath the sarcolemma, and the
sarcolemma often is separated from the underlying
myofibrils. In some cells, the attachments between
872
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the sarcolemma and the underlying myofibrils are
broken over a length of membrane equal to 10 or
more sarcomeres. The result is a large subsarcolemmal bleb; the plasma membrane overlying the bleb
generally is markedly disrupted although the glycocalyx remains intact. Short segments of sarcolemma often retain a normal ulrrastructural appearance with the plasma membrane immediately beneath the glycocalyx, but generally the glycocalyx is
devoid of underlying membrane or is associated
with plasmalemmal vesicles. The subsarcolemmal
blebs often contain membrane vesicles derived from
swollen mitochondria or the sarcoplasmic reticulum.
The interstitial space is not expanded appreciably
under these incubation conditions. A similar ultrastructural appearance is obtained if myocardial slices
are incubated in isotonic anoxic media for 240 minutes followed by 60 minutes in 150 mOsm anoxic
media (Fig. 9). Overall myocardial ultrastructure is
distorted less under these conditions than during
300 minutes of anaerobic hypotonic incubation; the
mitochondrial AMD are smaller and less numerous
and mitochondrial swelling is less marked. Nevertheless, large subsarcolemmal blebs and plasma
membrane disruption are apparent in most myocytes. The ulrrastructural appearance of the fragmented plasma membranes in Figure 9 is indistinguishable from that observed during lethal ischemic
Circulation Reseaich/Vo/. 57, No. 6, December 1985
FIGURE 8. The effect of 60 minutes of hypotonic (150 mOsm) anoxic
incubation on myocardial ultrastructure. Mitochondria (M) are severely swollen, but there is no evidence of plasma membrane disruption. Panel A: 10.950X; panel B: 43J5OX.
injury in vivo or in vitro (see Jennings et al., 1978,
1983; Reimer et al., 1981).
Discussion
FIGURE 7. The ultrastructural appearance of myocytes after 300
minutes of anoxic isotonic incubation. Tissue glycogen is depleted
and small amorphous matrix densities (AMD) are present in most
mitochondria, but the sarcolemma appears to be intact. Panel A:
10,950-X; panel B: 3Z8OOX.
The data presented in this paper demonstrate that
cell swelling and high energy phosphate depletion
in combination, but not individually, result in myocardial plasma membrane disruption, morphologically identical to that observed during lethal ischemic injury. Thus, plasma membrane fragmentation appears to be a multifactorial process. There is
an initial, ultrastructurally inapparent injury to the
membrane or its cytoskeletal scaffold which is associated with prolonged energy deficiency. This initial injury makes the membrane susceptible to stress
which, in this model, is in the form of cell swelling.
However, this may not be the only mechanism of
rupturing weakened plasma membranes. It has been
suggested that contracture and/or contraction band
formation also rupture plasma membranes (Ganote,
1983). The significance of the present data is that it
establishes the presence of a form of plasma membrane injury which develops during the period of
energy deficiency and weakens the membrane so
that it ruptures when stressed. When the same stress
is applied to myocytes which have not experienced
prolonged energy deficiency, it does not alter plasma
membrane integrity. Thus, it is not the stress per se
but the initial ultrastructurally inapparent injury to
the membrane or the cytoskeleton which is the basis
of myocardial plasma membrane disruption during
prolonged oxygen and substrate deprivation.
Steenbergen et al. /Plasma Membrane Injury in Anoxic Myocardium
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FIGURE 9. The ultrastructural appearance of myocytes after 240
minutes of incubation in isoiomc anoxic media followed by 60 minutes
in hypotonic (150 mOsm) anoxic media. Small mitochondrial amorphous matrix densities (AMD) are evident. There are breaks in the
plasmalemma (indicated by arrows) associated with a large subsarcolemmal bleb (SB), but the glycocalyx (G) appears to be intact. Panel
A- 1Q950X; panel B- 31800*.
Previous work on plasma membrane damage during in vivo and in vitro ischemia provides important
background data. Plasma membrane disruption occurs at or shortly after the transition from reversible
to irreversible ischemic injury during permanent
severe in vivo myocardial ischemia 0ennings et al.,
1978; Jennings and Hawkins, 1980; Jennings and
Reimer, 1981). Furthermore, after shorter intervals
of ischemia, where breaks in the plasma membranes
are not detectable at the end of the ischemic episode,
reperfusion or reoxygenation does not result in
plasma membrane fragmentation (Jennings and
Hawkins, 1980; Jennings et al., 1983), even though
considerable cell swelling occurs at the onset of
reperfusion due to the high osmolality of the ischemic myocytes (Tranum-Jensen et al., 1981). The
mechanism of plasma membrane disruption in
zones of severe ischemia in vivo is probably similar
to that demonstrated to be present in total ischemia,
since the zone of severe ischemia in the dog heart
often receives virtually no collateral flow. However,
a small amount of collateral flow, if present, could
provide a limited supply of oxygen, water, and
electrolytes, and thereby could create the possibility
of oxygen-mediated free radical formation, calcium
influx, and water diffusion into the severely ischemic area. It seems unlikely that these changes
873
occur to a significant extent during the time that
membrane damage develops in vivo, but they cannot be ruled out. Furthermore, the in vivo ischemic
myocardium is affected by systemic neural and hormonal influences. On the other hand, total ischemia
in vitro is not subject to these complications. High
energy phosphate and adenine nucleotide depletion,
and ultrastructural changes including plasma membrane disruption, develop more slowly during total
ischemia in vitro than during severe ischemia in
vivo, but the same pattern of alterations is observed
(Jennings et al., 1981). Thus, plasma membrane
disruption can occur without any influx of new
exogenous calcium, water, or oxygen into the ischemic tissue. Therefore, it is clear that there must
be a mechanism of plasma membrane damage
which does not require involvement of extrinsic
factors.
Previous work on lethal injury during substratefree high-flow anoxic perfusion of isolated hearts
provides an important contrast to the preceding
observations on ischemic injury. Plasma membrane
damage during 45 minutes of substrate-free anoxic
perfusion is minimal, but reoxygenation results in
sudden massive enzyme release and ultrastructural
plasma membrane disintegration similar to that observed during ischemia (Ganote et al., 1975; Hearse
et al., 1978). Thus, plasma membrane injury during
ischemia occurs spontaneously while the tissue is
ischemic, but membrane injury is not apparent during anoxic perfusion, suggesting that there are factors which are present in ischemia and absent in
high-flow anoxia which have a major impact on
membrane integrity. Furthermore, the observations
on anoxic injury have promoted the concept that
membrane damage under energy-deficient conditions is due to reintroduction of molecular oxygen
(Hearse et al., 1978).
The present data suggest a unifying explanation
for these previous observations. During ischemia,
cellular metabolism results in increased tissue osmolality (Fig. 1, and Tranum-Jensen et al., 1981;
Friedrich et al., 1981). Although aerobic myocytes
can limit the extent of osmotically induced volume
expansion by releasing potassium, a volume-regulatory response observed in a variety of types of
cells (see Cala, 1980), anaerobic myocytes do not
regulate volume as effectively. Furthermore, release
of potassium by ischemic myocytes is restricted, due
to accumulation of potassium in the interstitium (Hill
and Gettes, 1980; Friedrich et al., 1981; Weiss and
Shine, 1981; Kleber, 1983). Because the interstitial
volume is so much less than the cytosolic volume
(Polimeni and Al-Sadir, 1975), the increase in interstitial potassium concentration is much greater than
the decrease in cytoplasmic potassium concentration. The measurements of tissue osmolality during
total ischemia in vitro indicate that cell swelling due
to osmotic forces would be maximal at the time
when plasma membrane disruption begins, providing a potential mechanism for rupturing plasma
874
membranes. Other factors, such as inhibition of
plasma membrane ion pumps, may also contribute
to cell swelling late in the reversible phase of ischemia. Generalized cell swelling may not be the only
or even the primary mechanism which promotes
plasma membrane disintegration during ischemia,
but it is a factor which is present in ischemia and
absent in high-flow anoxia and which could account
for the presence of ultrastructurally detectable
plasma membrane lesions in lethally injured ischemic myocardium but not in lethally injured anoxic myocardium.
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Mechanism of Alterations in Membrane
Structure
In addition to providing information on the importance of volume regulation and cell swelling in
plasma membrane injury during ischemia, the ultrastructural appearance of the disrupted plasma membranes suggest a possible mechanism for the ultrastructurally inapparent initial membrane injury. In
control myocardium, electron microscopy reveals
attachments between the plasma membrane and the
underlying myofibrils at each Z-line, producing a
scalloped appearance in contracted myocytes (Sommer and Johnson, 1979; Jennings and Hawkins,
1980; Chiesi et al., 1981). The morphology of irreversibly injured ischemic myocytes (Jennings et al.,
1978, 1983; Jennings and Hawkins, 1980; Steenbergen and Jennings, 1984) or anoxic myocytes which
are osmotically swollen after 240 minutes of oxygen
deprivation, in contrast, is characterized by breaks
in plasma membranes which occur prominently
where there are subsarcolemmal blebs separating
the plasmalemma from the myofibrils. Subsarcolemmal blebs are large collections of intracellular fluid
extending over many Z-lines. They are not observed
in reversibly injured ischemic myocardium, even
though increased tissue osmolality and cell swelling
occur during this phase of ischemia, but they are
observed early in the irreversible phase, coincident
with fragmentation of plasma membranes. Thus the
presence of subsarcolemmal blebs is objective evidence of loss of Z-line attachments to the plasmalemma, and the appearance of subsarcolemmal blebs
in close temporal proximity to the development of
plasma membrane disruption suggests that the Zline connections are important for maintenance of
plasma membrane integrity during cell swelling.
Both microfilaments and intermediate filaments
interact with cell membranes, and several different
types of cytoskeletal-membrane attachments have
been described in a variety of types of cells (Geiger
et al., 1981, 1984; Price and Sanger, 1983). In adult
cardiac muscle, intermediate filaments have been
observed at Z-lines (Tokuyasu et al., 1983) and
linking Z-lines to adjacent subsarcolemmal densities
(Chiesi et al., 1981; Price and Sanger, 1983; Forbes
and Sperelakis, 1983), and vinculin has been identified as one probable constituent of the attachment
complex between the intermediate filaments and
Circulation Research/Vol. 57, No. 6, December 1985
integral proteins of the plasma membrane (Pardo et
al., 1983). Although it is unclear how ischemia might
damage cytoskeletal interactions with the plasma
membrane, one possibility is activation of calciumactivated proteases which have been localized to Zbands (Reddy et al., 1975; Dayton and Schollmeyer,
1981), and in close proximity to the plasma membrane (Dayton and Schollmeyer, 1981). However,
during total ischemia, the rise in cystolic free calcium
cannot exceed 10~4 M and probably is much lower
(Jennings et al., 1985). Thus, the mechanism responsible for loss of Z-line attachments to the plasma
membrane is uncertain.
The progressive increase in the inulin diffusible
space as the duration of total ischemia prior to
incubation is increased from 75-150 minutes suggests that the development of plasma membrane
disruption is not synchronous, despite the uniformity of the flow and oxygen deprivation. This is also
reflected in the electron micrographs, where subsarcolemmal blebs and plasma membrane discontinuities become increasingly more frequent as the duration of ischemia is increased. However, it is virtually impossible to quantify the proportion of lethally injured myocytes as a function of duration of
ischemia by the present ultrastructural methods. If
ultrastructurally detectable breaks in plasma membranes are irreparable, as the correlation between
irreversible injury and plasma membrane disruption
suggests, then a myocyte will be lethally injured
once it has one membrane lesion. However, the
probability of finding that lesion by transmission
electron microscopy is small unless the myocyte has
more than one lesion. Furthermore, once the plasma
membrane is broken in one area of the cell, this
could permit efflux of intracellular water, electrolytes, and protein which would reduce swelling
elsewhere in the cell. Thus, the stimulus for formation of subsarcolemmal blebs would be diminished,
and this might reduce the rate of development of
subsequent lesions in the same cell. This could account for the observation that even after prolonged
total ischemia, substantial lengths of plasma membrane appear to be intact.
This study was supported by National Institutes of Health Grant
HL 23138, Clinical Investigator Award K08-HL01337, and North
Carolina Heart Association Crant-in-Aid 1982-1985 - A-47.
Address for reprints: Dr. Charles Steenbergen, Department of
Pathology, Duke University Medical Center, Durham, North Carolina
27710.
Received June 4, 1985, accepted for publication September 12,
7985
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INDEX TERMS: Myocardial ischemia • Plasma membrane disruption • Myocardial osmolality • Volume regulation • Cell
swelling
Volume regulation and plasma membrane injury in aerobic, anaerobic, and ischemic
myocardium in vitro. Effects of osmotic cell swelling on plasma membrane integrity.
C Steenbergen, M L Hill and R B Jennings
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Circ Res. 1985;57:864-875
doi: 10.1161/01.RES.57.6.864
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