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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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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Abstract
Introduction
Methods
Results
Discussion
References

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.

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.

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 MOmega . 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 MOmega 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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Abstract
Introduction
Methods
Results
Discussion
References

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).

                              
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Table 1.   Phospholipid composition of isolated adult rabbit ventricular myocytes

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.

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.

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).

                              
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Table 2.   Basal (control) and hypoxia-stimulated PLA2 activity in isolated rabbit cardiac myocytes

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.

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 (bullet ) 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.

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 (bullet ). Effect of pretreatment with BEL (10 µM, 10 min) on hypoxia-induced [3H]arachidonic acid release is also shown (square ). triangle , 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.

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.

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.

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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