Selective hydrolysis of plasmalogen phospholipids by
Ca2+-independent
PLA2 in hypoxic ventricular
myocytes
Jane
McHowat1,
Shi
Liu2, and
Michael H.
Creer1
1 Department of Pathology, St.
Louis University Medical School, St. Louis, Missouri 63104; and
2 Department of Biopharmaceutical
Sciences, University of Arkansas for Medical Sciences, Little Rock,
Arkansas 72205
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ABSTRACT |
Accelerated phospholipid catabolism occurs early after the onset
of myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipase
A2
(PLA2) activity in isolated
ventricular myocytes, resulting in increased lysophospholipid and
arachidonic acid production, contributing to arrhythmogenesis in
ischemic heart disease. The majority of ventricular myocyte arachidonic
acid was found in plasmalogen phospholipids. Hypoxia increased
membrane-associated,
Ca2+-independent,
plasmalogen-selective PLA2
activity, resulting in increased arachidonic acid release and
lysoplasmenylcholine production. Pretreatment with the specific
Ca2+-independent
PLA2 inhibitor bromoenol lactone
blocked hypoxia-induced increases in
PLA2 activity, arachidonic acid
release, and lysoplasmenylcholine production. Lysoplasmenylcholine
produced action potential derangements, including shortening of action
potential duration, and induced early and delayed afterdepolarizations
in normoxic myocytes. The electrophysiological alterations induced by
lysoplasmenylcholine would likely contribute to the initiation of
arrhythmogenesis in the ischemic heart.
lysoplasmenylcholine; lysophosphatidylcholine; membrane
phospholipids; bromoenol lactone; phospholipase
A2
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INTRODUCTION |
PHOSPHOLIPASE A2
(PLA2) has been isolated and
purified from the cytosolic fraction of myocardial tissue from several
species (13-15). These enzymes exhibit maximal activity in the
absence of Ca2+ and selectively
hydrolyze arachidonylated plasmalogen phospholipid substrates. However,
during ischemia, no significant change in myocardial cytosolic
PLA2 is observed, but an increase
in membrane-associated, Ca2+-independent,
plasmalogen-selective PLA2 occurs
(10, 12). Because of the cellular heterogeneity of whole myocardial
tissue, the specific source of the membrane-associated enzyme activity could not be established from these studies, and thus nothing is known
specifically regarding changes in
PLA2 activity in ischemic cardiac
myocytes.
The phospholipid composition of isolated cardiac myocytes is unique in
being comprised predominantly of plasmalogen molecular species (5).
Coupled with the observation that plasmalogens may be targeted for
hydrolysis during ischemia (8, 20), these findings suggest that
plasmalogen phospholipids may be the preferred substrates for
ischemia-activated phospholipases.
We thus asked the following questions:
1) Do isolated ventricular myocytes
exposed to hypoxia demonstrate an increase in
PLA2 activity?
2) Does the increase in
PLA2 activity result in increased production of lysophospholipids and free fatty acid?
3) Does the increase in
lysophospholipid content contribute to electrophysiological abnormalities in normal isolated ventricular myocytes?
We report that ventricular myocytes isolated from rabbit hearts and
exposed to brief intervals of hypoxia demonstrate an increase in
membrane-associated
Ca2+-independent
PLA2 activity that selectively
hydrolyzes plasmalogen phospholipids, resulting in increased
arachidonic acid release and lysoplasmenylcholine (LPlasC)
accumulation. LPlasC induces electrophysiological abnormalities in
ventricular myocytes that could lead to the production of arrhythmias
in the ischemic heart. This is the first study to show a direct link
between hypoxia-induced increases in
PLA2 activity, LPlasC
accumulation, and the production of electrophysiological abnormalities
in isolated ventricular myocytes.
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METHODS |
Isolation of ventricular myocytes.
Ventricular myocytes were isolated from adult female rabbit hearts as
described previously (22). Briefly, the heart was mounted on a
Langendorff perfusion apparatus and perfused at 37°C for 5 min with
a Tyrode solution containing (in mM) 118 NaCl, 4.8 KCl, 1.2 CaCl2, 1.2 MgCl2, 24 NaHCO3, 1.2 KH2PO4,
and 11 glucose; the Tyrode solution was saturated with 95%
O2-5%
CO2 to yield a pH of 7.4. This was
followed by 4-min perfusion with a
Ca2+-free Tyrode solution
containing EGTA (100 µM) and a final perfusion for 20 min with the Tyrode solution containing 100 µM
Ca2+ and 0.033% collagenase (type
II, Worthington Biochemical). The left and right ventricles were cut
into small pieces, placed in two Erlenmeyer flasks with 20 ml fresh
enzyme solution, and shaken at 37°C for 15 min, with 95%
O2-5%
CO2 blowing into each flask. Myocytes were washed with a HEPES buffer containing (in mM) 133.5 NaCl,
4.8 KCl, 1.2 MgCl2, 1.2 KH2PO4,
10 HEPES, and 10 glucose, plus 300 µM
CaCl2, pH adjusted to 7.4 with 10 N NaOH. Extracellular Ca2+
concentration was increased to 1.2 mM in three stages at intervals of
20 min. Elongated myocytes were separated from rounded nonviable cells
by repeated differential sedimentation.
Extraction, separation, and analysis of phospholipid classes.
Cellular phospholipids were extracted from isolated adult rabbit
ventricular myocytes (~20-40 mg total cellular protein suspended in 2 ml Ca-HEPES buffer) by the method of Bligh and Dyer (2) at
0-4°C. The chloroform layer was dried under
N2, and the lipid residue was
resuspended in 1 ml chloroform-methanol (1:1 vol/vol). Three 5-µl
aliquots were removed for measurement of total lipid phosphorus, and
200-µl aliquots were injected onto an Ultrasphere-Si (5 µm silica),
4.6 × 250-mm HPLC column (Beckmann Instruments, Fullerton, CA).
Phospholipids were separated into different classes based on
differences in polar headgroup composition using gradient elution with
a mobile phase comprised of hexane-isopropanol-water (7). Figure
1 shows a typical separation of
phospholipid classes derived from adult rabbit ventricular myocytes
using this gradient elution system. Phospholipid classes were
quantified in the isolated fractions by measurement of lipid phosphorus
by microphosphate assay (3). The fatty acid composition of the isolated
glycerophospholipid classes was determined by gas chromatographic (GC)
analysis of the fatty acid methyl ester (FAME) and dimethylacetal (DMA)
derivatives produced after acid-catalyzed methanolysis (11). Figure
2 shows a typical GC tracing of the
volatile FAME and DMA derivatives produced after methanolysis of
diradyl choline phospholipids derived from rabbit ventricular myocytes.
Identification of individual FAME species was established by comparison
of their GC retention times with commercial standards (Alltech,
Deerfield, IL). Individual DMA species were identified by comparison of
their GC retention times with the DMA derivatives produced after
acid-catalyzed methanolysis of LPlasC derived from bovine heart choline
glycerophospholipids (5). The alkylacyl glycerophospholipid content of
phosphatidylcholine and phosphatidylethanolamine was determined by
quantification of lipid phosphorus in the lysophospholipid fraction
remaining after sequential, exhaustive base- and acid-catalyzed
hydrolysis of the diradylphospholipids (11).

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Fig. 1.
A representative HPLC chromatogram (ultraviolet detector response at
203 nm vs. elution time) for class separation of phospholipids derived
from isolated rabbit ventricular myocytes. Samples were injected onto
an Ultrasphere Si 5-µm HPLC column and eluted with a gradient elution
mobile phase of hexane-isopropanol-water at a flow rate of 1.5 ml/min.
In order of elution, phospholipids are separated into
diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE),
phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine
(PC), and sphingomyelin (sphing). Choline lysophospholipids
[lysoplasmenylcholine (LPlasC) and lysophosphatidylcholine
(LPC)] elute after elution of all other cellular phospholipids.
SF refers to solvent front.
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Fig. 2.
Gas chromotographic (GC) elution profiles of volatile fatty acid methyl
ester (FAME) and dimethylacetal (DMA) derivatives produced after
acid-catalyzed methanolysis of choline glycerophospholipids derived
from isolated rabbit ventricular myocytes. Chart speed varies
throughout GC separation; thus relationship between peak widths and
component mass also varies throughout separation. Calculation of
relative percent is based on integrated peak areas and use of relative
response factors for each FAME and DMA component as described
previously (5). FID, flame ionization detector.
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Separation and quantification of individual choline and
ethanolamine glycerophospholipid molecular species.
Individual choline and ethanolamine glycerophospholipid molecular
species were isolated by reverse-phase HPLC using an Ultrasphere ODS (5 µm, C18) column, 4.6 × 250 mm (Beckmann Instruments). Individual molecular species were
separated using a gradient elution system with a mobile phase comprised
of acetonitrile-methanol-water with 20 mM choline chloride (23). The
molecular identity of individual molecular species was established by
GC characterization of the FAME and DMA derivatives produced after
acid-catalyzed methanolysis of the phospholipid species recovered in
column effluents and by comparison of absolute retention time, relative
retention time, and order of elution of individual species with
previously injected phospholipids of known composition. Quantification
of individual phospholipid molecular species was achieved by
determination of lipid phosphorus in reverse-phase HPLC column
effluents by the method of Itaya and Ui (19).
Induction of hypoxia.
A glucose-free 1.2 mM Ca-HEPES buffer, pH 7.4, was degassed under
vacuum for 1 h and then bubbled with 100% prepurified
N2 for at least 2 h to attain a
PO2 of <15 mmHg. Myocytes suspended
in 1.2 mM Ca-HEPES (2 × 106
cells/sample) in a glass vial were washed with glucose-free 1.2 mM
Ca-HEPES and then subjected to 10 min of hypoxia. Room air was
exchanged with 100% N2 delivered
into the glass vial for 1 min. Hypoxic glucose-free 1.2 mM Ca-HEPES
buffer (2 ml) was then transferred to the myocyte pellet via a
spring-loaded glass syringe. The 100%
N2 atmosphere was maintained above
the hypoxic solution and cells for the entire hypoxic interval.
Measurement of PLA2 activity.
Ventricular myocytes were exposed to normoxic or hypoxic conditions
(<15 mmHg PO2) and then placed on
ice and sonicated for 10 s. After initial sonication, 2 mM
dithiothreitol (DTT) and 10% glycerol were added to the cell
suspension. The suspension was sonicated on ice a further three times
for 10 s, and the sonicate was centrifuged at 14,000 g for 10 min. The resultant
supernatant fraction was centrifuged at 100,000 g for 60 min to separate the membrane
fraction (pellet) from the cytosolic fraction (supernatant). The
membranes were resuspended in buffer containing (in mM) 250 sucrose, 10 KCl, 10 imidazole, 5 EDTA, and 2 DTT with 10% glycerol, pH 7.8. PLA2 activity in subcellular
fractions was assessed by incubating enzyme (8 µg membrane protein or
200 µg cytosolic protein) with 100 µM plasmenylcholine,
phosphatidylcholine, or alkylacyl glycerophosphorylcholine radiolabeled
with oleate (18:1) or arachidonate (20:4) at the
sn-2 position and containing a
saturated 16-carbon aliphatic moiety at the
sn-1 position (16:0). Synthesis of
radiolabeled substrates has been described previously (22). Incubations
were performed in assay buffer containing 10 mM Tris and 10% glycerol, pH 7.0, with either 4 mM EGTA or 10 mM
Ca2+ at 37°C for 5 min in a
total volume of 200 µl. Reactions were terminated by the addition of
100 µl butanol, vortexed, and centrifuged at 2,000 g for 5 min. Released radiolabeled
fatty acid was isolated by application of 25 µl of the butanol phase
to channeled silica gel G plates, development in petroleum
ether-diethyl ether-acetic acid (70:30:1), and subsequent
quantification by liquid scintillation spectrometry. These reaction
conditions resulted in linear reaction velocities with respect to both
time and enzyme concentration for each substrate examined. The 100 µM
substrate concentration was selected to ensure that maximal reaction
velocities were obtained and to ensure negligible isotope dilution
effects by endogenous substrate. At the concentration of substrate and
amount of protein used in the assay, endogenous phospholipids were
<10% of exogenous substrate. Protein content of each sample was
determined by the Lowry method using freeze-dried BSA (Bio-Rad
laboratories) as the protein standard as described previously (21).
Measurement of choline lysophospholipid content in isolated
myocytes.
Choline lysophospholipids were quantified using a modification of a
radiometric assay method described previously from our laboratory (6).
Lipids were extracted from the myocytes by the method of Bligh and Dyer
(2), followed by the separation of the lysophospholipids from other
phospholipids by HPLC using a silica column (5-µm Ultrasphere Si)
with a mobile phase consisting of hexane, isopropyl alcohol, and water
(465:465:70 vol/vol/vol). This HPLC system provides complete separation
of LPlasC and lysophosphatidylcholine (LPC), enabling subsequent
quantification of each lysophospholipid subclass (Fig.
3). The purified LPlasC and LPC fractions
as well as known amounts of LPlasC and LPC standards were then
acetylated with
[3H]acetic anhydride
using 0.33 M dimethylaminopyridine as a catalyst. The acetylated
lysophospholipids were then separated by thin-layer chromatography and
scraped, and radioactivity was quantified by liquid scintillation
spectrometry. Standard curves were constructed for LPlasC and LPC
standards, and corresponding choline lysophospholipid levels were
derived for all samples and normalized according to the protein content
of the myocytes measured as described by Markwell et al. (21) with the
use of lyophilized BSA (Bio-Rad Laboratories, Richmond, CA) as the
protein standard.
[14C]LPC was added as
an internal standard to all samples and standards to correct for any
loss that may occur during acetylation.

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Fig. 3.
Chromatogram of HPLC separation of LPlasC and LPC from PC. Samples were
injected onto an Ultrasphere Si 5-µm HPLC column and eluted with a
mobile phase of hexane-isopropanol-water (465:465:70) at a flow rate of
1.5 ml/min. Absorbance was measured at 203 nm, and LPlasC and LPC
fractions were collected separately for subsequent quantitation.
Definitions are as in Fig. 1.
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Measurement of total arachidonic acid release.
Arachidonic acid release was determined by measuring
[3H]arachidonic acid
released into the surrounding medium from myocyte suspensions
prelabeled with
[3H]arachidonic acid.
Briefly, myocyte suspensions (106
myocytes in 10 ml culture medium) were incubated at 37°C with 3 µCi [3H]arachidonic
acid for 18 h. This incubation resulted in >70% incorporation of radioactivity into the myocytes. Eighty-five percent
of incorporated radioactivity was recovered from phosphatidylcholine or
phosphatidylethanolamine phospholipids. After incubation, myocyte suspensions were washed three times with Tyrode solution containing 3.6% BSA to remove unincorporated
[3H]arachidonic acid.
Myocytes were incubated at 37°C for 15 min before being subjected
to experimental conditions. At the end of the hypoxic interval or a
corresponding period in HEPES buffer for controls, myocyte suspensions
were centrifuged, and the supernatant was removed. Myocyte pellets were
dissolved in 10% BSA, and radioactivity in both supernatant and pellet
was quantified by liquid scintillation spectrometry.
Electrophysiological measurements.
Ventricular myocytes were placed on the heated stage of an inverted
microscope (Nikon Diaphot) and perfused with a control Tyrode solution.
Cells were patch clamped using perforated-patch techniques (17) with a
patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City,
CA). Briefly, patch electrodes were fabricated from borosilicate glass
(7052, Garner Glass, Claremont, CA) and filled with a pipette solution
consisting of (in mM) 110 potassium aspartate, 25 KCl, 2 MgCl2, 5 Na2ATP, 5.6 glucose, 5 HEPES, and
5 Tris base (pH adjusted to 7.2 with KOH). Filled pipette electrodes
had a tip resistance of 2-5 M
. The tip of pipette electrodes
was filled with a small amount of nystatin-free pipette solution and
then back-filled with the pipette solution containing nystatin (250 µg/ml). After the gigaseal formation, a repetitive 5-mV
hyperpolarizing pulse was applied to monitor decline of the series
resistance, which was typically smaller than 20 M
in 10-15 min.
In the current-clamp mode, action potentials (AP) of myocytes were
elicited with a 2-ms depolarizing pulse. The recorded AP was filtered
at 2 kHz through a four-pole low-pass Bessel filter and sampled at 10 kHz with a PC/AT computer using PClamp 6.0 software (Axon Instruments)
through Axon TL-1 labmaster DMA acquisition system. All experiments
were conducted at 37°C.
Biochemical measurements.
Lactate dehydrogenase was used as a marker of cell death/lysis and was
determined as described previously (25). Long-chain acylcarnitine
measurements in isolated myocytes were made using a method described
recently by our laboratory (22). ATP content of isolated myocytes was
quantified by HPLC separation on a Hi-Pore RP-318 reverse-phase HPLC
column eluted with ammonium phosphate (0.1 M, pH 5.5) as described
previously by our laboratory (31).
Statistics.
Statistical comparison of values was performed by the Student's
t-test or ANOVA with the Fisher
multiple-comparison test as appropriate. All results are expressed as
means ± SE. Statistical significance was considered to be
P < 0.05.
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RESULTS |
Phospholipid composition of rabbit ventricular myocytes.
Total phospholipid phosphorus in isolated rabbit ventricular myocytes
was found to be 134 ± 4 nmol/mg protein. The major phospholipid classes were found to be choline (49 ± 1.5%) and ethanolamine (40.7 ± 3.4%) glycerophospholipids (see Table
1). Small amounts of phosphatidylinositol,
phosphatidylserine, and cardiolipin were also present (Table 1). Rabbit
ventricular myocyte phospholipids contained 54 ± 4% of
plasmalogens. Plasmalogen phospholipids comprised 53% of choline
glycerophospholipids and 65% of ethanolamine glycerophospholipids, with a small amount detected in phosphatidylserine (Table 1). Alkylacyl
glycerophospholipids comprised 1% of choline and ethanolamine glycerophospholipids (Table 1).
The mass of arachidonylated species in plasmalogen and diacyl
phospholipids in the choline and ethanolamine classes is shown in Fig.
4. In ethanolamine glycerophospholipids,
the amount of arachidonylated species was 18.0 ± 1.5 nmol/mg
protein in plasmalogen and 18.5 ± 1.2 nmol/mg protein in diacyl
phospholipids (Fig. 4A). Arachidonylated species accounted for 11.1 ± 1.0 nmol/mg protein in
plasmalogens and 6.8 ± 0.6 nmol/mg protein in diacyl phospholipids in choline glycerophospholipids (Fig.
4A). Comparison of arachidonylated species to total species in each subclass demonstrated that 70% of
plasmalogens and 60% of diacyl species in ethanolamine
glycerophospholipids contained arachidonic acid at the
sn-2 position (Fig.
4B), whereas 58% of
plasmenylcholine and 13% of phosphatidylcholine species were
arachidonylated (Fig. 4B).

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Fig. 4.
Isolated ventricular myocyte content of arachidonylated plasmalogen and
diacyl phospholipid species in ethanolamine and choline
glycerophospholipid species (A).
Amount of arachidonylated species expressed as a percentage of total of
each subclass is also shown (B).
Values shown represent means ± SE for independent results from 8 separate animals.
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Hypoxia induces a time-dependent, reversible increase in
Ca2+-independent,
plasmalogen-selective PLA2.
Isolated rabbit ventricular myocytes were subjected to hypoxia for
increasing time intervals, and
PLA2 activity was measured in
isolated cytosolic and membrane fractions (Fig.
5). After only 5 min of hypoxia,
PLA2 activity measured in the
membrane fraction using (16:0,
[3H]18:1)
plasmenylcholine in the absence of
Ca2+ (4 mM EGTA) was increased
1.5-fold (Fig. 5). No corresponding increase in the membrane fraction
was observed using (16:0,
[3H]18:1)
phosphatidylcholine, and no significant changes in
PLA2 activity were observed in the
cytosol (data not shown). The increase in membrane-associated
PLA2 using (16:0,
[3H]18:1)
plasmenylcholine was further increased to twofold for up to 20 min
(Fig. 5). A 10-min hypoxic interval followed by 30-min reoxygenation
resulted in return of PLA2
activity to basal levels (Fig. 5). No change in membrane-associated
PLA2 activity using (16:0,
[3H]18:1)
phosphatidylcholine was observed at any period of hypoxia or hypoxia
plus reoxygenation.

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Fig. 5.
Changes in membrane-associated phospholipase
A2
(PLA2) activity in response to
increasing intervals of hypoxia and its reversal by 30-min reperfusion
after 10-min hypoxia. Activity measurements were made using (16:0,
[3H]18:1)
plasmenylcholine in absence of
Ca2+ (4 mM EGTA). Values shown
represent means ± SE for independent results from 6 separate
animals. * P < 0.05 and
** P < 0.01 compared with
control values.
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We did not observe any decrease in cytosolic
PLA2 activity corresponding to the
increase in membrane-associated
PLA2 activity measured using
plasmalogen substrate. This suggests that induction of hypoxia may
cause activation of an integral membrane
PLA2 rather than translocation of
PLA2 activity from cytosol to
membrane. This mechanism has been proposed in earlier studies in whole
rabbit myocardium subjected to ischemia by Gross and co-workers
(12). Additionally, the enzyme we have found in isolated ventricular myocytes bears many of the same features as that characterized in the
whole rabbit myocardium in that it cannot be removed from the membrane
by exposure to EGTA, repeated sonication, or submicellar concentrations
of detergent (12) and that removal of oxygen results in an increase in
PLA2 activity demonstrable only
with the use of plasmalogen substrates.
After 10 min of hypoxia, ATP levels fell from 36.8 ± 0.4 nmol/mg
protein (n = 4) to 27.1 ± 0.5 nmol/mg protein (n = 6, P < 0.01) and returned to normal
after 30-min reoxygenation. Long-chain acylcarnitine levels increased
from 40.7 ± 5.1 to 196.5 ± 25.4 pmol/mg protein
(n = 6, P < 0.01) after 10-min hypoxia and
had returned to control levels after 30-min reoxygenation. These
myocytes showed no significant decrease in viability during the hypoxic interval when examined under light microscopy. In addition, lactate dehydrogenase release in myocytes subjected to 10-min hypoxia was not
greater than that in normoxic myocytes (13.2 ± 2.8% for normoxic
cells vs. 14.7 ± 3.0% for hypoxic cells). As a result of these
experiments, we decided to choose a hypoxic interval of 10 min for
further studies. This allowed enough time to induce significant
biochemical changes but was short enough to minimize cell death and to
be reversible upon reoxygenation.
Myocytic membrane-associated and cytosolic
PLA2 activity was measured using
phospholipid substrates radiolabeled at the
sn-2 position with oleic acid (16:0,
[3H]18:1) or
arachidonic acid (16:0,
[3H]20:4) in the
presence or absence of Ca2+ under
control conditions and after 10-min hypoxia (Table
2). The majority of
PLA2 activity in isolated
ventricular myocytes was membrane associated,
Ca2+ independent, and selective
for sn-2 arachidonylated substrates (Table 2).
After 10-min hypoxia, an increase in
PLA2 activity was detected in the
membrane fraction using plasmenylcholine substrate labeled at the
sn-2 position with oleate or
archidonate and in the absence or presence of
Ca2+ (Table 2). Ten-minute hypoxia
had no effect on membrane-associated PLA2 activity using
phosphatidylcholine or alkylacyl glycerophosphorylcholine substrates.
No change in cytosolic PLA2
activity was observed during hypoxia under any conditions tested (Table
2).
Because we determined that hypoxia specifically activated
PLA2 that was present in the
membrane fraction, was maximal in the presence of EGTA, and was
selective for plasmalogen substrates, we subsequently refer to this
enzyme as membrane-associated,
Ca2+-independent,
plasmalogen-selective PLA2.
Cardiac myocyte PLA2 activity is
inhibited by the presence of ATP.
PLA2 purified from the cytosolic
fraction of whole rabbit myocardium has been shown to be augmented by
ATP (14). We included 10 mM ATP in our
PLA2 assay buffer and determined
PLA2 activity measurements both
with and without ATP. PLA2
activity measurements in the membrane fraction measured with and
without 10 mM ATP are presented in Fig. 6.
With the use of (16:0,
[3H]18:1)
plasmenylcholine, the presence of ATP significantly inhibited membrane-associated PLA2 activity
measured in normoxic and hypoxic myocytes (Fig. 6). A decrease in
membrane-associated PLA2 activity measured using (16:0,
[3H]18:1)
phosphatidylcholine was also observed in the presence of ATP.

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Fig. 6.
Effect of presence of 10 mM ATP in assay buffer (open bars) on
membrane-associated PLA2 activity
using (16:0, [3H]18:1)
plasmenylcholine substrate in presence of 4 mM EGTA. Solid bars,
control. Values represent means ± SE for independent results from 4 separate animals. * P < 0.05, ** P < 0.01 compared with
corresponding values in absence of ATP.
+ P < 0.05 when comparing hypoxia with corresponding control value.
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Thus, although our enzyme isolated from ventricular myocytes possesses
many of the characteristics of that isolated from the whole rabbit
myocardium, there are some differences between the two that may suggest
that other PLA2 isoforms may be
present in whole myocardium (from cells such as endothelial or smooth
muscle cells) that contribute to the increase in
PLA2 activity in response to
global ischemia. In addition, these studies highlight a
possible mechanism for PLA2
activation during hypoxia or ischemia in the cardiac myocyte in
that a decrease in ATP levels, such as that which occurs early after
the onset of oxygen depletion, may in itself activate
PLA2 activity.
Hypoxia-induced increase in PLA2 is
blocked by pretreatment with bromoenol lactone.
Bromoenol lactone (BEL) is a potent, irreversible, mechanism-based
inhibitor of myocardial
Ca2+-independent
PLA2 that is more than 1,000-fold
specific for inhibition of
Ca2+-independent
PLA2(s) in comparison with
multiple Ca2+-dependent
PLA2(s) (16). Myocytes were
incubated with 1-10 µM BEL for 10 min before induction of
hypoxia. Basal membrane-associated PLA2 activity was reduced
significantly by BEL concentrations greater than 2 µM using both
plasmenylcholine (Fig. 7) and
phosphatidylcholine substrates (data not shown). The hypoxia-induced
increase in membrane-associated PLA2 activity measured using
(16:0, [3H]18:1)
plasmenylcholine was completely abolished by BEL concentrations greater
than 2 µM (Fig. 7). Cytosolic
PLA2 activity under control or
hypoxic conditions was not significantly altered by BEL.

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Fig. 7.
Effect of pretreatment of isolated rabbit ventricular myocytes with
increasing concentrations of bromoenol lactone (BEL) (10 min) on
membrane-associated PLA2 in
control conditions (X) and after 10 min of hypoxia ( ) using (16:0,
[3H]18:1)
plasmenylcholine substrate in presence of 4 mM EGTA. Values shown
represent means ± SE for independent results from 5 animals.
* P < 0.05, ** P < 0.01 compared with
corresponding control values.
+ P < 0.05 compared with corresponding control myocytes with no BEL
pretreatment.
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Mepacrine and dibucaine act through interactions with the lipid-water
interface physically preventing
PLA2 from hydrolyzing the
phospholipid substrate (27). Under the strictest terms, it is
inaccurate to consider these agents as direct enzyme inhibitors, and
conclusions drawn from their use in physiological/pathophysiological experiments should be considered carefully (27). However, these agents
have been used historically as "classic" inhibitors of Ca2+-dependent
PLA2(s). Pretreatment of myocytes
with 10 µM mepacrine or 50 µM dibucaine for 30 min had no
significant effect on basal membrane-associated
PLA2 activity or the increase in
PLA2 activity in response to
hypoxia using plasmenylcholine substrate (data not shown). Taken
together, these results demonstrate that a short interval of hypoxia
results in the selective activation of a
Ca2+-independent
PLA2 enzyme, since the increase is
not blocked by classic
Ca2+-dependent
PLA2 inhibitors such as mepacrine
or dibucaine but is completely inhibited by pretreatment with BEL, a
specific Ca2+-independent
PLA2 inhibitor.
Exposure of ventricular myocytes to 10-min hypoxia results in an
increase in arachidonic acid release and LPlasC production.
Because isolated ventricular myocytes exposed to hypoxia demonstrate an
increase in membrane-associated
PLA2 activity that is selective
for plasmalogen phospholipids and because we have shown that the
majority of arachidonic acid in phospholipids is esterified to
plasmalogens, we examined the effect of hypoxia on arachidonic acid
release and the production of LPlasC.
Ventricular myocytes prelabeled with
[3H]arachidonic acid
and exposed to increasing intervals of hypoxia demonstrated a
significant increase in
[3H]arachidonic acid
release over control after 5 min that was sustained over a 30-min
hypoxic interval (Fig. 8).
Pretreatment of the myocytes with 10 µM BEL for 10 min before
induction of hypoxia abrogated the hypoxia-induced increase in
arachidonic acid release (Fig. 8). Thus inhibition of
Ca2+-independent
PLA2 by BEL resulted in inhibition
of arachidonic acid production during hypoxia.

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Fig. 8.
Effect of increasing intervals of hypoxia on
[3H]arachidonic acid
release from isolated ventricular myocytes ( ). Effect of
pretreatment with BEL (10 µM, 10 min) on hypoxia-induced
[3H]arachidonic acid
release is also shown ( ). , Control. Arachidonic acid release
from untreated ventricular myocytes is indicated by dotted line. Values
shown represent means ± SE for independent results from 4 separate
animals. ** P < 0.01 when
compared with control myocytes.
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|
We have demonstrated previously (22) that hypoxia alone failed to
increase total choline lysophospholipid mass in the absence of thrombin
stimulation. In this study, we measured LPlasC and LPC mass together.
We have now modified our assay method to enable us to measure LPlasC
and LPC separately after isolation by HPLC (Fig. 3). Ventricular
myocytes exposed to 10-min hypoxia demonstrated a significant increase
in LPlasC that was inhibited completely by pretreatment with 10 µM
BEL for 10 min (Fig. 9). Accompanying the
hypoxia-induced increase in LPlasC we found a decrease in LPC content
that would explain why we were unable to detect an increase in total
choline lysophospholipid mass in our previous study (22). Thus
ventricular myocytes exposed to short intervals of hypoxia demonstrate
an increase in membrane-associated,
Ca2+-independent
PLA2 activity that hydrolyzes
plasmalogen phospholipids selectively. Activation of this
PLA2 enzyme results in increased arachidonic acid release and increased LPlasC production, both of which
may be inhibited by the
Ca2+-independent
PLA2 inhibitor BEL.

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Fig. 9.
Total choline lysophospholipid (hatched bars), LPlasC (solid bars), and
LPC (open bars) content in control, hypoxic (10 min), and
BEL-pretreated (10 µM, 10 min) hypoxic ventricular myocytes. Values
shown represent means ± SE for independent results from 5 separate
animals. Definitions are as in Fig. 1.
* P < 0.05, ** P < 0.01 when compared with
corresponding control values.
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|
Effect of LPlasC on the AP of ventricular myocytes.
Because we have demonstrated an increase in LPlasC in response to
hypoxia and because the effect of LPlasC on the electrophysiological properties of ventricular myocytes remain unknown, we determined the
effect of adding LPlasC to the perfusate on the AP of isolated ventricular myocytes perfused in vitro. Figure
10 illustrates a representative recording
of the AP of a ventricular myocyte before, during, and after exposure
to 1 µM
1-O-hexadecyl-1'-enyl-2-LPlasC [(16:0) LPlasC]. Exposure of the cardiac myocyte to 1 µM
LPlasC caused a rapid decrease in the amplitude of plateau phase within 1 min with no change in the resting membrane potential (RMP)
(trace 1, Fig.
10A). Within 1.5 min, in addition to
reduction of the plateau potential, LPlasC shortened the AP duration
and depolarized the membrane potential that involved a phase 4 diastolic depolarization (trace 2,
Fig. 10A). After 2 min of perfusion,
an early afterdepolarization developed (trace
3, Fig. 10A).
LPlasC-induced spontaneous beating was arrested by a spontaneous
hyperpolarization (Fig. 10B).
Thereafter, the elicited AP maintained a low plateau potential, short
duration, and depolarization of membrane potential after 3-min exposure to LPlasC. Upon removal of LPlasC, the AP recovered within 5 min (Fig.
10C). Thus LPlasC is capable of
reversibly eliciting rapid and profound changes in the AP properties of
isolated ventricular myocytes.

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Fig. 10.
Effect of LPlasC on action potential of rabbit ventricular myocytes.
A: 1 min after exposure to 1 µM
LPlasC, plateau potential and action potential duration (APD) gradually
decreased (trace 1 vs.
trace C, control). At 1.5 min in
presence of LPlasC, plateau potential and APD further decreased along
with development of a diastolic depolarization (trace
2). Two minutes after exposure, an early
afterdepolarization was developed on a reduced APD
(trace 3).
B: after afterdepolariztion,
spontaneous activities were observed and followed by a gradual
hyperpolarization and resumption of stimulated action potential.
C: during removal of LPlasC, action
potential was recovered.
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In a series of experiments, we found that exposure to LPlasC caused a
transient increase in the AP duration (APD) within 1 min by 30% at
levels of 0 mV (APD0) and 90%
complete repolarization (APD90),
respectively (Table 3). Thereafter, LPlasC
gradually decreased APD and slightly depolarized the RMP before the
development of afterdepolarizations. After LPlasC-induced
afterdepolarization, some cells remained depolarized at approximately
40 mV, whereas others displayed a very short action potential
duration before they became unexcitable. Recovery of the action
potential upon removal of LPlasC also varied from cell to cell. Some
cells remained at approximately
40 mV, whereas others fully
recovered as shown in Fig. 10C. The
transient increase in APD may result from an increase in L-type
Ca2+ channel current, with the
later shortened APD resulting from an activation of
K+ channels. Mechanisms underlying
alterations of membrane ion currents induced by LPlasC are currently
under investigation.
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Table 3.
Effects of 1 µM (16:0) lysoplasmenylcholine on action
potential duration and resting membrane potential of isolated rabbit
ventricular myocytes
|
|
 |
DISCUSSION |
This is the first study to demonstrate a reversible increase in
PLA2 activity in response to short
intervals of hypoxia and reperfusion in isolated rabbit ventricular
myocytes which results in increased arachidonic acid release and LPlasC
production. An increase in LPlasC in the ventricular myocyte sarcolemma
contributes to the development of electrophysiological abnormalities
that could contribute to arrhythmogenesis in the ischemic heart.
The majority of PLA2 activity was
found to be membrane associated,
Ca2+ independent, and selective
for sn-1 ether-linked phospholipid substrates. Cardiac myocyte PLA2
activity also displayed a distinct preference for
sn-2 arachidonylated phospholipid
substrates. The differential responses to
Ca2+ and different substrate
selectivity profiles of cytosolic and membrane-associated
PLA2 suggest that the
PLA2 activities in these fractions
may be mediated by different PLA2
isoforms. Exposure of isolated rabbit ventricular myocytes to short
intervals of hypoxia leads to activation of membrane-associated,
Ca2+-independent,
plasmalogen-selective PLA2. The
increase in membrane-associated PLA2 activity occurs without
substantial decrements in cytosolic PLA2 activity, suggesting that
there is little, if any, translocation of activity from cytosol to
membrane in response to hypoxia. The activation of membrane-associated
PLA2 during hypoxia may result from the activation of a novel, latent
PLA2 activity in the membrane fraction; however, we cannot rule out the possibility that these results could represent hypoxia-induced changes in activity and substrate selectivity of basal membrane-associated
PLA2 activity.
Pretreatment of ventricular myocytes with 10 µM BEL resulted in
complete inhibition of hypoxia-induced increases in membrane-associated PLA2 activity, arachidonic acid
release, and LPlasC production. BEL is a selective inhibitor for
Ca2+-independent
PLA2 but has been shown recently
to inhibit phosphatidic acid phosphohydrolase (PAP) at similar
concentrations to those used for
PLA2 inhibition (1). Because
arachidonic acid release may also result from sequential phospholipase
D, PAP, and diacylglycerol lipase action, studies using BEL may be
difficult to interpret if arachidonic acid release is the only measured
end point of PLA2 activity.
However, in this study, we have observed inhibition of
PLA2 activity at BEL
concentrations lower than those reported for PAP inhibition (1) and
have demonstrated that 10 µM BEL not only abolishes hypoxia-induced
arachidonic acid release but also
PLA2 activity and LPlasC
production. Because we have measured both
PLA2 enzyme activity and the
metabolic products of PLA2
hydrolysis in BEL-pretreated cells in control and hypoxic conditions,
we are confident that the PAP pathway in response to hypoxia is
unlikely to contribute significantly to the increased arachidonic acid release. The goal of the BEL inhibition studies was to demonstrate that
BEL pretreatment blocked subsequent responses to hypoxia based on
1)
PLA2 activity measurements,
2) arachidonic acid release, and
3) LPlasC production. Thus it was
important to preincubate ventricular myocytes with BEL before the onset
of hypoxia to establish the role of BEL-inhibitable
Ca2+-independent
PLA2 in the observed changes in
these PLA2 activity and
phospholipid metabolic end points. Addition of 1 µM BEL to the
isolated subcellular membrane fractions immediately before substrate
addition inhibited completely basal and hypoxia-induced increases in
Ca2+-independent
PLA2 activity measured using
(16:0, [3H]18:1)
plasmenylcholine substrate. Thus BEL inhibits
Ca2+-independent
PLA2 when added to the cells
before induction of hypoxia and also inhibits the membrane-associated
PLA2 activity when added to the
isolated membrane fraction immediately before substrate addition.
Because BEL blocks LPlasC production and arachidonic acid release
during hypoxia but does not inhibit
Ca2+-dependent secretory
PLA2
(sPLA2) or cytosolic
PLA2
(cPLA2) (1, 16), our results
demonstrate that activation of
sPLA2 or
cPLA2 during hypoxia does not
occur in isolated cardiac myocytes and is not required as a
prerequisite for the hypoxia-induced selective increase in
membrane-associated,
Ca2+-independent,
plasmalogen-selective PLA2.
Purified rabbit myocardial cytosolic
Ca2+-independent
PLA2 has been reported previously
to be activated and stabilized by ATP (14). In contrast to these
findings, we have demonstrated that both cytosolic and
membrane-associated PLA2 in
cardiac myocytes is inhibited by ATP, GTP, and their nonhydrolyzable
analogs (data not shown). The fact that
PLA2 activity measured in the
absence of ATP is greater than in the presence of ATP provides an
interesting mechanism whereby PLA2
may be activated under hypoxic conditions. On the basis of previous
estimates of the volume of the cytosolic space (0.4 ml/g wet tissue wt)
(26), the concentration of cytosolic ATP in isolated normoxic
ventricular myocytes is ~13 mM. Changes in ATP concentration in the
millimolar range have been shown to affect the properties of
membrane-associated functions, such as the function of ATP-dependent
potassium channels, even though the channel is regulated by micromolar
concentrations of ATP (28). Thus membrane-associated ATP-dependent
functions within the membrane can occur if ATP levels fall at all below
normal levels. It has been suggested that the myocyte membrane senses a
different pool of ATP than the rest of the cytoplasm, and thus a small
reduction in ATP concentrations can have a significant effect on
myocyte function (28). Thus a fall in ATP concentration within the
hypoxic cardiac myocyte may play a role in activating
membrane-associated PLA2 and may
exacerbate the electrophysiological perturbations resulting from
PLA2-induced accumulation of
LPlasC during hypoxia.
Our findings that hypoxia induces a membrane-associated,
Ca2+-independent
PLA2 that selectively hydrolyzes
plasmenylcholine substrate agree with those published previously
measuring PLA2 activity isolated
from whole myocardium (10, 12). Gross and co-workers (12) demonstrated
activation of a membrane-associated, Ca2+-independent
PLA2 that was selective for
plasmenylcholine substrate after global ischemia in rabbit
hearts. Activation of PLA2 was time dependent and reversible upon reperfusion (10). In contrast to
these findings, Vesterqvist et al. (30) could not demonstrate an
increase in PLA2 during global
ischemia when activity was measured using endogenous
phospholipids as substrate but did observe an increase in LPlasC
content in ischemic myocardium. However, it should be noted that in the
former studies an increase in PLA2 activity was measured in the membrane fraction only. Because the latter
authors measured PLA2 activity in
the whole myocardium without isolating subcellular fractions, it is
possible that they could be measuring several
PLA2 isoforms that may be
influenced differently by ischemia.
Detailed analysis of the phospholipid composition of isolated rabbit
ventricular myocytes revealed that the majority of arachidonic acid was
found at the sn-2 position of
phospholipids with a vinyl ether linkage at the
sn-1 position, i.e., most arachidonic
acid in isolated ventricular myocytes is found in plasmalogen species. Plasmalogens have been shown to be present in highest concentrations in
the surface membranes of cells in which intrinsic electrical activity
plays an important physiological role and to represent a highly
metabolically active pool of phospholipids (9, 18, 29). In addition,
accelerated plasmalogen catabolism has been demonstrated during
ischemia (see Ref. 9 for review). Activation of a
membrane-associated, plasmalogen-selective
PLA2 in isolated cardiac myocytes
in response to hypoxic conditions will lead to accumulation of
arachidonic acid and LPlasC within the sarcolemma, which results in
rapid dramatic alterations in the electrophysiological properties of
the cell (for review, see Refs. 4, 24).
In this study, we have demonstrated for the first time the production
of alterations in the action potential properties of isolated cardiac
myocytes in the presence of LPlasC. The LPlasC-induced shortening of
APD and reduction of RMP were similar to those induced by LPC or
palmitoylcarnitine (4, 24). However, the concentration of LPlasC
required to elicit alterations in action potentials was much lower than
other amphiphilic compounds. In addition to a greater potency, within 1 min LPlasC produced a transient increase in APD accompanied by positive
inotropism (data not shown), which probably resulted from an increase
in L-type Ca2+ currents. The
decrease in APD after 1-min exposure to LPlasC may result from an
activation of K+ channels
and/or a consecutive reduction of
Ca2+ current, similar to that
induced by LPC and palmitoylcarnitine (4, 24). The shortened APD
results in a decrease in the duration of the refractory period, which
leads to an early afterdepolarization as shown in Fig.
7A (trace
3). The potential arrhythmogenic effects of LPlasC
may also result from an increase in inward currents as demonstrated
with palmitoylcarnitine in rabbit ventricular myocytes (32). Considered
collectively, the results of our electrophysiological studies suggest a
specific interaction of LPlasC with ion channel proteins in the
membrane in addition to its ability to elicit nonselective
perturbations in the biophysical properties of the phospholipid
bilayer. The fact that LPlasC can induce action potential alterations
at lower concentrations than LPC is important, since we demonstrate
that LPlasC content increases threefold during hypoxia, whereas there
is a decrease in LPC. Thus, although total choline lysophospholipid
content does not change during hypoxia, there is an increase in LPlasC
relative to LPC that would be expected to contribute to the development
of electrophysiological abnormalities.
In summary, this is the first study to demonstrate the reversible
activation of a membrane-associated,
Ca2+-independent,
plasmalogen-selective PLA2 in
isolated rabbit ventricular myocytes in response to hypoxic conditions.
Accompanying the increase in PLA2
activity is an increase in arachidonic acid release and selective
LPlasC production. The accumulation of these metabolites within the
ventricular myocyte sarcolemma is capable of inducing profound
alterations in electrophysiological properties that may contribute
directly to arrhythmogenesis in the ischemic heart.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the technical assistance of Jan Jones,
Meei Liu, and Rae Treal McCrory.
 |
FOOTNOTES |
Research from the authors' laboratory was supported in part by the
Veterans Administration Research Career Development Award Program (to
M. H. Creer), the Veterans Administration Merit Review Grant Program
(to M. H. Creer), and the American Heart Association, Arkansas
Affiliate (to J. McHowat, S. Liu, and M. H. Creer).
Address for reprint requests: J. McHowat, Dept. of Pathology, St. Louis
Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104.
Received 15 December 1997; accepted in final form 20 February
1998.
 |
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