INTRODUCTION

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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.

	METHODS

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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).

View larger version (18K): [in this window] [in a new window]   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.

View larger version (26K): [in this window] [in a new window]   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.

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.

View larger version (20K): [in this window] [in a new window]   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.

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.

	RESULTS

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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).

View larger version (20K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   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.

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.

View larger version (16K): [in this window] [in a new window]   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.

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.

View larger version (14K): [in this window] [in a new window]   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.

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.

View larger version (15K): [in this window] [in a new window]   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.

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.

View larger version (13K): [in this window] [in a new window]   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.

View larger version (24K): [in this window] [in a new window]   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.

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.

View larger version (12K): [in this window] [in a new window]   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.

	DISCUSSION

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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.