Redistribution and abnormal activity of phospholipase A2 isoenzymes ...

Am J Physiol Cell Physiol 280: C573–C580, 2001.

Redistribution and abnormal activity of phospholipase A2 isoenzymes in postinfarct congestive heart failure JANE MCHOWAT,1 PARAMJIT S. TAPPIA,2 SONG-YAN LIU,2 RAETREAL McCRORY,1 AND VINCENZO PANAGIA2,† 1 Department of Pathology, St. Louis University Medical School, St. Louis, Missouri 63104; and 2Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Departments of Human Anatomy and Cell Science and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada R2H 2A6 Received 28 July 2000; accepted in final form 18 October 2000

McHowat, Jane, Paramjit S. Tappia, Song-Yan Liu, Raetreal McCrory, and Vincenzo Panagia. Redistribution and abnormal activity of phospholipase A2 isoenzymes in postinfarct congestive heart failure. Am J Physiol Cell Physiol 280: C573–C580, 2001.—Cardiac sarcolemmal (SL) cis-unsaturated fatty acid sensitive phospholipase D (cis-UFA PLD) is modulated by SL Ca2⫹-independent phospholipase A2 (iPLA2) activity via intramembrane release of cis-UFA. As PLD-derived phosphatidic acid influences intracellular Ca2⫹ concentration and contractile performance of the cardiomyocyte, changes in iPLA2 activity may contribute to abnormal function of the failing heart. We examined PLA2 immunoprotein expression and activity in the SL and cytosol from noninfarcted left ventricular (LV) tissue of rats in an overt stage of congestive heart failure (CHF). Hemodynamic assessment of CHF animals showed an increase of the LV end-diastolic pressure with loss of contractile function. In normal hearts, immunoblot analysis revealed the presence of cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) in the cytosol, with cPLA2 and iPLA2 in the SL. Intracellular PLA2 activity was predominantly Ca2⫹ independent, with minimal sPLA2 activity. CHF increased cPLA2 immunoprotein and PLA2 activity in the cytosol and decreased SL iPLA2 and cPLA2 immunoprotein and SL PLA2 activity. sPLA2 activity and abundance decreased in the cytosol and increased in SL in CHF. The results show that intrinsic to the pathophysiology of post-myocardial infarction CHF are abnormalities of SL PLA2 isoenzymes, suggesting that PLA2-mediated bioprocesses are altered in CHF.

(PLA2) isoenzymes represent a large family of distinct enzymes whose products are important for signal transduction processes, eicosanoid and platelet-activating factor formation, membrane remodeling, and lipid metabolism (1, 6, 31). At least three different PLA2s exist in mammalian cells, each of which demonstrates unique characteristics. Secretory PLA2 (sPLA2) have low molecular weights, require millimolar Ca2⫹ concentrations for activity, and are secreted into the extracellular space. The cytosolic

PLA2 (cPLA2) has a molecular mass of 85 kDa, requires increases in intracellular Ca2⫹ for phosphorylation of the enzyme and translocation to intracellular membranes but does not require Ca2⫹ for its catalytic activity. The Ca2⫹-independent PLA2 (iPLA2) has a molecular mass of 80 kDa and does not require Ca2⫹ for activity. In some cell types, several lines of evidence suggest that the different PLA2 isoenzymes are distinct in terms of signaling processes and that there is cross talk between them (6). We have reported that a form of phospholipase D (PLD), which is present in heart sarcolemma (SL), is stimulated by physiological concentrations of cis-unsaturated fatty acids (cis-UFA PLD) (18). This enzyme specifically hydrolyzes phosphatidylcholine to form phosphatidic acid (5, 12, 23). Various cis-unsaturated fatty acids showed different stimulatory potencies, with arachidonic acid being the most efficient agent (5, 18). The activity of SL Ca2⫹-independent PLA2 and subsequent intramembrane release of sn-2 unsaturated fatty acids modulates this PLD and the related formation of phosphatidic acid (18). Phosphatidic acid influences cardiac Ca2⫹ transport systems (9, 34) whose alterations are important determinants of the defective contractile performance during congestive heart failure (CHF) after a large myocardial infarction (MI) (8, 22). We hypothesized that abnormalities in protein abundance and activity, and cellular localization of myocardial PLA2 isoenzymes may occur at the overt stage of CHF after MI and result in PLA2 disordered function. This would raise the possibility of a secondary impairment in PLA2-mediated stimulation of SL cis-UFA PLD activity and related synthesis of phosphatidic acid. As a consequence, phosphatidic acid-induced increase in intracellular Ca2⫹ concentration and contractile performance of the heart could be affected during CHF by the impaired PLA2-PLD pathway. In this pathophysiological study, we examined the immunoprotein expression and activity of PLA2 isoenzymes in the SL and cytosolic compartments of the

† Deceased Nov 24 2000. Address for reprint requests and other correspondence: J. McHowat, Dept. of Pathology, St. Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

myocardial infarction; signal transduction; phospholipase D

PHOSPHOLIPASE A2

http://www.ajpcell.org

0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society

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PLA2 ISOENZYMES IN CONGESTIVE HEART FAILURE

noninfarcted left ventricular tissue of rats at 8 wk after occlusion of the left anterior descending coronary artery when the animals were in the overt stage of CHF (10, 16, 30). MATERIALS AND METHODS

Experimental model. All experimental protocols for animal studies were approved by the Animal Care Committee of the University of Manitoba, following the guidelines established by the Canadian Council on Animal Care. MI was produced in male Sprague-Dawley rats (175–200 g) by surgical occlusion of the left anterior descending coronary artery as described previously (16, 30). Animals were anesthetized with 5% isoflurane, and the heart was exteriorized through an incision in the intercostal space. The left anterior descending coronary artery was ligated 2–3 mm from its origin using a 6–0 silk suture, the heart was repositioned, and the chest was closed. The mortality of the experimental animals operated on in this manner was ⬃40% within the first 48 h after surgery. Age-matched, sham-operated animals were used as controls and were treated similarly, except the suture around the artery was not tied. The animals were allowed to recover and maintained on food and water ad libitum for a period of 8 wk. At that time, the cardiac function of some randomly selected animals was assessed, and then those animals were killed for further biochemical analysis. As in previous studies (16, 19, 30), animals (n ⫽ 17) with large transmural infarcts [40% of the left ventricular (LV) free wall] and sham-operated animals (n ⫽ 17) were used. The above rat infarct model has been characterized in past studies (10). On the basis of retrograde pulmonary edema and in vivo LV mechanical function, we determine the animals to be in an overt stage of CHF at 8 wk after the occurrence of a large (⬎40% of the LV free wall) transmural infarct (10). This is considered a good experimental model of CHF that mimics the clinical conditions occurring in humans after a large transmural MI (15). LV function. The LV function of five randomly selected animals from each group (control and CHF) was assessed (30). Rats were anesthetized using an injection of ketaminexylazine (100:10 mg/kg ip). After intubation of the trachea to maintain adequate ventilation, the right carotid artery was exposed and a micromanometer-tipped catheter (2–0; Millar SPR-249) was inserted and advanced into the left ventricle. The catheter was secured with a silk ligature around the artery, and, after a 15-min stabilization of the heart function, LV pressures and maximum rates of isovolumic pressure development (⫹dP/dtmax) and decay (-dP/dtmax) were recorded. Hemodynamic data were computed instantaneously and displayed on a computer data-acquisition workstation (Biopac, Harvard Apparatus). After the hemodynamic assessment, each animal was killed by decapitation. The heart was rapidly excised, immersed in ice-cold 0.6 M sucrose and 10 mM imidazole (pH 7.0), and washed to remove any contaminating blood. The atrial, macrovascular, and connective tissues were removed, and the right ventricle was separated. The infarcted area was excised 2 mm distal to the scar to include the border region (26). The noninfarcted LV tissue (including intraventricular septum) of each assessed animal was frozen in liquid nitrogen and kept at ⫺80°C. When the hemodynamic experiments were concluded, the frozen LV tissues were thawed at 0–4°C, pooled, and homogenized to obtain one SL preparation and one cytosolic preparation (see Preparation of cardiac cytosolic and SL fractions) from pooled control LV tissues as well as from pooled failing LV tissues. These SL and cytosolic preparations from hemodynamically assessed animals were used for the biochemical experiments

shown in Figs. 1–5. The related results were consistent with those of the corresponding SL and cytosolic preparations from nonassessed animals. Therefore, the results from hemodynamically assessed and nonassessed hearts were pooled and subjected to statistical analysis. Preparation of cardiac cytosolic and SL fractions. After killing the sham-operated and experimental animals by decapitation, we rapidly excised the hearts and immersed them in ice-cold buffer containing 0.6 M sucrose and 10 mM imidazole (pH 7.0) as indicated above. The noninfarcted LV tissue (including intraventricular septum) from three to five hearts was pooled, homogenized (small aliquots of each homogenate were frozen in liquid nitrogen and kept at ⫺80°C for marker enzymes studies), and centrifuged at 12,000 g for 30 min at 4°C. Part of the supernatant was centrifuged at 110,000 g for 60 min at 4°C, and aliquots of the resulting supernatant were frozen in liquid nitrogen, kept at ⫺80°C, and used as the cytosolic fraction. The rest of the 12,000 g supernatant was diluted with 300 mM KCl buffer to solubilize myofibrillar proteins (7) and further processed for the preparation of SL according to the method of Pitts (27) used previously (18, 30). The final SL pellet was resuspended in 250 mM sucrose and 10 mM histidine and divided into aliquots, which were frozen in liquid N2 and stored at ⫺80°C until assayed. All of the above steps were performed at 0–4°C. Marker enzymes (30) were assessed (n ⫽ 3 separate SL preparations from control and failing hearts). The activity of K⫹-p-nitrophenol phosphatase, an SL marker, was decreased in 8-wk post-MI left ventricle compared with sham controls (3.4 ⫾ 0.2 vs. 5.8 ⫾ 0.4 ␮mol 䡠 mg SL protein⫺1 䡠 h⫺1, respectively). This enzyme was similarly decreased in the homogenate from post-MI LV. Its relative specific activity (specific activity in SL/specific activity in the homogenate) was 14.7 ⫾ 0.9 and 14.1 ⫾ 0.5 in sham and MI groups, respectively, indicating an equal degree of enrichment of the SL membrane in both groups. The relative specific activity of rotenone-insensitive NADPH-cytochrome c reductase (sarcoplasmic reticular marker) was 0.36 ⫾ 0.05 and 0.38 ⫾ 0.06, while that of cytochrome c-oxidase (mitochondrial marker) was 0.42 ⫾ 0.07 and 0.47 ⫾ 0.05 in sham and MI hearts, respectively. These results indicate that both SL fractions under study were relatively pure with only minimal but equal contamination from other subcellular organelles. Protein concentrations were determined by the Lowry method as described elsewhere (30). Immunoblot analysis of PLA2 isoforms. Equivalent amounts (20 ␮g) of SL or cytosolic protein were mixed with an equal volume of SDS sample buffer and heated at 95°C for 5 min before being loaded onto a 10–15% polyacrylamide gel. Protein was separated by SDS-PAGE at 150 V for 50 min and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Richmond, CA) at 200 V for 1 h. Nonspecific sites were blocked by incubating the membranes with Tris buffer solution containing 0.05% (vol/vol) Tween 20 (TBST) and 5% (wt/vol) nonfat milk 1 h at room temperature. The blocked PVDF membrane was incubated with primary antibodies to cytosolic PLA2 (cPLA2, 1:1,000 dilution, Santa Cruz), Ca2⫹-independent PLA2 (iPLA2, 1:2,000 dilution, Cayman Chemical), or secretory PLA2 (sPLA2, 1:1,000 dilution, Upstate Biotechnology) for 1 h at room temperature. Unbound antibodies were removed with three washes with TBST solution, and membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution). After six washes with TBST, regions of antibody binding were detected with enhanced chemiluminescence (Amersham, Arlington Heights, IL) and exposure to film (Hyperfilm, Amersham). Band intensities of the immunoblot

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were quantified by using a charge-coupled device camera imaging densitometer (Bio-Rad GS670). Phospholipase A2 activity. Intracellular PLA2 activity (cPLA2 and iPLA2) was assessed by incubating enzyme (8 ␮g SL protein or 100 ␮g cytosolic protein) with 100 ␮M plasmenylcholine or phosphatidylcholine substrates radiolabeled with oleic acid (16:0, [3H]18:1) or arachidonic acid (16:0, [3H]20:4) at the sn-2 position. The substrate was introduced into the incubation mixture by injection in ethanol (5 ␮l). Incubations were performed in assay buffer containing 10 mM Tris, 10% glycerol, pH 7.0, with either 4 mM EGTA or 1 mM Ca2⫹ at 37°C for 5 min in a total volume of 200 ␮l. Reactions were terminated by the addition of 100 ␮l butanol, and released radiolabeled oleic acid ([3H]18:1) was isolated by application of 25 ␮l of the butanol phase to channeled Silica Gel G TLC plates (Analtech, Newark, DE) development in petroleum ether-diethyl ether-acetic acid (70:30:1 vol/vol), 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-␮M substrate concentration was selected to ensure negligible isotope dilution effects by endogenous substrate. To define PLA2 specific activity, total PLA2 activity was normalized according to protein content. sPLA2 activity was assessed by incubating SL or cytosolic proteins with 100 ␮M (16:0, [3H]18:1) phosphatidylcholine substrate and 1.2 mM Ca2⫹ in the presence of 10 ␮M bromoenol lactone (BEL) and 20 ␮M methyl arachidonyl fluorophosphonate (MAFP). Because, at these concentrations, BEL inhibits specifically iPLA2, and MAFP inhibits both cytosolic iPLA2 and cPLA2 but not sPLA2 (1), the residual PLA2 activity measured represents sPLA2. Statistical analysis. All values are expressed as means ⫾ SE. The differences between two groups were evaluated by Student’s t-test. A probability of 95% or more was considered significant. RESULTS

The coronary occlusion resulted in the presence of reproducible transmural infarcts in the LV free wall. As in past studies (16, 30), the noninfarcted heart muscle of the experimental animals underwent significant hypertrophy during the 8-wk post-MI period, as indicated by an increase of the noninfarcted LV (septum and noninfarcted LV free wall remote from infarct) weight and by the augmented ratio of noninfarcted LV weight to body weight compared with control values (Table 1). Consistent with previous findings (16), marked interstitial fibrosis of the LV free wall was observed in horizontal sections of the heart under light microscopy (not shown). A significant increase in the wet-to-dry weight ratio of the lungs showed the presence of pulmonary edema in CHF animals. Augmented LV end-diastolic pressure with concomitant loss of contractile function (⫾dP/dtmax) was also detected in the CHF group (Table 1). These results are consistent with previous observations in this model indicating that the animals, at 8 wk after the occurrence of a large MI, were in a stage of overt CHF (10, 16, 19, 21, 30). Measurement of intracellular PLA2 activity (cPLA2 and/or iPLA2) in the presence (1 mM) and absence (4 mM EGTA) of Ca2⫹ and with plasmenylcholine and

Table 1. General characteristics and LV function of post-MI CHF animals Parameter

Body wt, g Noninfarcted LV wt, g Noninfarcted LV/body wt, mg/g Lung wet wt/dry wt ratio Scar wt, g LVSP, mmHg LVEDP, mmHg ⫹dP/dtmax, mmHg/s ⫺dP/dtmax, mmHg/s

Control

CHF

509 ⫾ 14 0.89 ⫾ 0.04 1.75 ⫾ 0.05 4.07 ⫾ 0.36 ND 138 ⫾ 10 3.2 ⫾ 0.7 5,481 ⫾ 389 5,233 ⫾ 300

483 ⫾ 19 1.13 ⫾ 0.06* 2.33 ⫾ 0.12* 5.47 ⫾ 0.51* 0.33 ⫾ 0.05 120 ⫾ 13 13.4 ⫾ 2.0* 3,826 ⫾ 296* 3,328 ⫾ 329*

Values are means ⫾ SE of 5 experiments with 5 different control or congestive heart failure (CHF) animals except for scar weight values (n ⫽ 4). Assessment of the left ventricular (LV) function and further processing of the hearts are indicated in MATERIALS AND METHODS. The noninfarcted LV weight of the experimental animals (at the 8th wk after induction of myocardial infarct) refers to the weight of LV free wall (plus septum) after removal of scar tissue. The ratio of wet to dry weight for lungs was obtained as indicated elsewhere (14). Control, noninfarcted, sham-operated age-matched animals; CHF, experimental animals with large transmural infarct of the LV free wall; ND, not detectable; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; ⫹dP/dtmax, maximum rate of isovolumic pressure development; ⫺dP/dtmax, maximum rate of isovolumic pressure decay; MI, myocardial infarction. * P ⬍ 0.05 vs. control.

phosphatidylcholine substrates demonstrated that most of the activity was Ca2⫹ independent and selective for arachidonylated phospholipid substrates (Table 2). PLA2 activity was approximately equal for plasmenylcholine and phosphatidylcholine substrates. The presence of Ca2⫹ significantly decreased PLA2 activity measured using plasmenylcholine but had no effect on PLA2 activity measured using phosphatidylcholine. Specific activity in the SL was at least one order of magnitude greater than that measured in the cytosol (Table 2). Intracellular PLA2 activity was measured in the cytosolic and SL fractions from control and failing rat hearts in the presence and absence of Ca2⫹ with both (16:0, [3H]18:1) plasmenylcholine and (16:0, [3H]18:1) phosphatidylcholine substrates. PLA2 activity was found to be ⬃10-fold greater in SL than cytosol under all assay conditions (Figs. 1 and 2). Most PLA2 activity in both subcellular fractions was measured in the absence of Ca2⫹ (Figs. 1 and 2) and demonstrated a preference for arachidonylated phospholipid substrates (data not shown) but little preference for either plasmenylcholine or phosphatidylcholine substrates (Figs. 1 and 2). In the cytosol from failing rat hearts, PLA2 activity measured with both substrates in the presence and absence of Ca2⫹ was found to be significantly increased over that measured in the cytosol of normal rat hearts (Fig. 1). Conversely, SL PLA2 activity was significantly decreased in failing hearts under all conditions studied (Fig. 2). In a separate series of experiments, we measured sPLA2 activity using (16:0, [3H]18:1) phosphatidylcholine in the presence of 1.2 mM Ca2⫹. sPLA2 activity in the cytosolic fraction was not significantly altered by the presence of heart failure (Fig. 3). However, sPLA2

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Table 2. Phospholipase A2 activity in sarcolemmal and cytosolic subcellular fractions from control rat hearts Cell Fraction

Substrate

Sarcolemma

Plasmenylcholine 16:0, [3H]18:1 16:0, [3H]20:4 Phosphatidylcholine 16:0, [3H]18:1 16:0, [3H]20:4 Plasmenylcholine 16:0, [3H]18:1 16:0, [3H]20:4 Phosphatidylcholine 16:0, [3H]18:1 16:0, [3H]20:4

Cytosol

EGTA

Ca2⫹

8.84 ⫹ 0.80 16.03 ⫹ 1.12

6.94 ⫹ 0.45* 13.97 ⫹ 1.21*

6.44 ⫹ 0.50 12.48 ⫹ 1.17

7.53 ⫹ 0.54 13.21 ⫹ 1.42

0.45 ⫹ 0.06 1.27 ⫹ 0.11

0.25 ⫹ 0.01* 1.05 ⫹ 0.32

0.25 ⫹ 0.03 0.93 ⫹ 0.16

0.25 ⫹ 0.03 1.12 ⫹ 0.22

Values represent means ⫾ SE in nmol 䡠 mg protein⫺1 䡠 min⫺1 for separate measurements from 6 different animals. Activity defined by using plasmenylcholine or phosphatidylcholine substrates in the absence (4 mM EGTA) or presence (1 mM Ca2⫹) of calcium. Substrate composition is represented as a:b, c:d where a:b and c:d represent the chain length: number of double bonds for the aliphatic groups at the sn-1 and sn-2 positions, respectively, of the corresponding phospholipid substrate molecule. * P ⬍ 0.05 compared to corresponding value obtained in the presence of EGTA.

activity in the SL was significantly increased in failing hearts compared with normal heart (Fig. 3). Measurement of PLA2 activity in subcellular fractions using synthetic phospholipid substrates does not determine which specific PLA2 isoenzyme is responsible for the hydrolysis. Although alteration of PLA2 assay conditions with respect to phospholipid substrate utilized or the presence of Ca2⫹ may indicate the

Fig. 1. Phospholipase A2 (PLA2) activity in the cytosolic fraction (cPLA2) from control and failing rat hearts. PLA2 activity was measured (each assay in duplicate) using 100 ␮M (16:0, [3H]18:1) plasmenylcholine (PlsCho) or (16:0, [3H]18:1) phosphatidylcholine (PtdCho) in the absence (4 mM EGTA) or presence (1 mM) of Ca2⫹. Data represent means ⫾ SE for 4 values obtained from 4 separate cytosolic preparations. Each cytosolic preparation was isolated from the left ventricular (LV) tissue as indicated in MATERIALS AND METHODS. CHF, congestive heart failure. Other details are as in Table 1. *P ⬍ 0.05, **P ⬍ 0.01 when compared with corresponding control values.

Fig. 2. Sarcolemmal (SL) PLA2 activity in control and failing hearts. PLA2 activity was measured (each assay in duplicate) using 100 ␮M (16:0, [3H]18:1) PlsCho or (16:0, [3H]18:1) PtdCho in the absence (4 mM EGTA) or presence (1 mM) of Ca2⫹. Data represent means ⫾ SE for 4 values obtained from 4 separate SL preparations. Each SL preparation was isolated from the LV tissue as indicated in MATERIALS AND METHODS. **P ⬍ 0.01 when compared with corresponding control values.

activity of a particular PLA2 isoenzyme, further studies are necessary to elucidate fully the presence of PLA2 isoenzymes in subcellular fractions. To determine whether changes in PLA2 activities in the subcellular fractions from failing hearts were due to changes in expression of previously characterized PLA2 isoen-

Fig. 3. Secretory PLA2 (sPLA2) activity measured in the cytosolic and SL subcellular fractions from control and failing hearts. PLA2 activity was measured (each assay in duplicate) using 100 ␮M (16:0, [3H]18:1) PtdCho in the presence of 1.2 mM Ca2⫹. Data represent means ⫾ SE for 4 values obtained from 4 separate cytosolic or SL preparations which were isolated as indicated in MATERIALS AND METHODS. **P ⬍ 0.01 when compared with corresponding control values.


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⬃110 kDa corresponding to cPLA2, and no significant band at ⬃14 kDa that would indicate the presence of sPLA2 (Fig. 5A). In the SL from failing hearts, immunoblot analysis revealed a decrease in the protein amount of both cPLA2 and iPLA2 isoforms and the presence of sPLA2 (Fig. 5). Thus the decrease in SL PLA2 activity observed in failing hearts is likely due to a decrease in SL iPLA2 and may, to a lesser extent, be due to an accompanying decrease in cPLA2. The increase in SL sPLA2 immunoprotein in the failing heart is accompanied by a marked increase in sPLA2 activity. Thus CHF is associated with a marked redistribution of PLA2 isoenzymes resulting in a redistribution of cytosolic and SL PLA2 activity in the failing heart. DISCUSSION

The rat infarct model employed in this and other studies in our laboratory (16, 21, 30) results in a form of CHF that resembles that occurring in humans after a large transmural MI (15). In the present pathophysiological study, we examined the immunoprotein expression and activity of SL and cytosolic PLA2 isoenzymes at the overt stage of CHF after MI. Our findings show for the first time that intrinsic to the pathophysFig. 4. Immunoblots of cytosolic PLA2 isoforms in CHF. A: representative Western blots showing the presence of cPLA2 and sPLA2 in sham-operated control (C1) and CHF (CHF1 and CHF2 are from two different preparations) animals. Independent PLA2 (iPLA2) was not detectable. B: quantified data of PLA2 isoforms protein concentration. The bands corresponding to cPLA2 and sPLA2 were subjected to densitometric analysis, as described in MATERIALS AND METHODS. Data are means ⫾ SE from 3 separate cytosolic preparations obtained as indicated in MATERIALS AND METHODS. *P ⬍ 0.05 compared with controls.

zymes, we used antibodies to known cPLA2 and iPLA2 enzymes and to sPLA2 for immunoblot analysis. Immunoblot analysis of the cytosolic fraction from control hearts demonstrated bands at 110 and 14 kDa, indicating the presence of cPLA2 and sPLA2 but not of iPLA2 (Fig. 4A). In the cytosolic fraction from failing hearts, the density of bands corresponding to cPLA2 was increased and the bands corresponding to sPLA2 were decreased (Fig. 4). Thus, although PLA2 activity in the cytosol was measured in the absence of Ca2⫹ and not significantly enhanced by the addition of Ca2⫹, immunoblot analysis indicates that any PLA2 activity in this fraction would be due to the presence of cPLA2 and not iPLA2. In response to an increase in intracellular Ca2⫹, cPLA2 is translocated from the cytosol to intracellular membranes, primarily the nucleus and endoplasmic reticulum, and associates with the membrane phospholipids through its Ca2⫹-dependent lipid binding domain. However, cPLA2 does not require Ca2⫹ for catalytic activity and can demonstrate PLA2 activity in the absence of Ca2⫹ in in vitro assay systems (20) and may contribute to the cytosolic PLA2 activity measured in the absence of Ca2⫹ in this study. Immunoblot analysis of the SL fraction from control hearts demonstrated the presence of a dense band at ⬃80 kDa corresponding to iPLA2, a weaker band at

Fig. 5. Immunoblots of SL PLA2 isoforms in CHF. A: representative Western blots showing the PLA2 isoforms present in control (C1, C2, C3) and CHF (CHF1, CHF2, CHF3) animals. B: quantified data of PLA2 isoform protein concentration. The bands corresponding to each PLA2 isoform were subjected to densitometric analysis. Data are means ⫾ SE from 3 separate SL preparations obtained as indicated in MATERIALS AND METHODS. *P ⬍ 0.05 compared with controls.

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iology of post-MI CHF is abnormal compartmentalization of PLA2 isoenzymes with concomitant alterations in subcellular PLA2 activities. Immunoblot analysis revealed the presence of multiple PLA2 isoenzymes in both control and failing hearts. In the normal heart cytosol, both cPLA2 and sPLA2 isoenzymes were present, but no iPLA2 was detected by immunoblot. In the SL, both cPLA2 and iPLA2 were detected with no evidence of sPLA2. In failing hearts, the SL protein content of intracellular PLA2 isoenzymes (iPLA2 and cPLA2) was decreased, whereas sPLA2 was now detectable. Conversely, in the cytosolic fraction, sPLA2 was decreased and cPLA2 was increased. Thus there is specific compartmentalization of PLA2 isoenzymes in the rat myocardium and evidence of PLA2 isoenzyme translocation in heart failure. Accompanying the decrease in SL cPLA2 and iPLA2 immunoprotein expression in the failing heart, SL PLA2 activity, measured using both plasmenylcholine and phosphatidylcholine substrates in the presence or absence of Ca2⫹, was decreased. The majority of the SL PLA2 activity was Ca2⫹ independent, suggesting that iPLA2 mediated the majority of substrate hydrolysis. The assumption that the majority of SL PLA2 activity is due to the presence of iPLA2 is supported by immunoblot analysis that demonstrates dense bands at ⬃80 kDa using an iPLA2 antibody to the cytosolic form of iPLA2 described previously in P388D1 macrophages and Chinese hamster ovary (CHO) cells. Thus membrane-associated iPLA2 in the rat heart has sufficient homology with the previously cloned iPLA2 for the antibody to recognize it. However, the iPLA2 in the rat heart possesses several characteristics that distinguish it from previously described iPLA2 isoforms, for example, subcellular localization and its preference for arachidonylated phospholipids. Additionally, iPLA2 in the rat heart effectively hydrolyzes plasmalogen phospholipids; however, there are no data regarding the hydrolysis of plasmalogen phospholipids in previously characterized iPLA2 from P388D1 or CHO cells. At this time, it is not known if the iPLA2 isoforms in the rat heart and CHO or P388D1 represent alternative splicing products or posttranslational modifications of the same gene product with different localizations and intracellular function in different cell types. The difference in PLA2 activity we detect using phosphatidylcholine substrates in the presence vs. the absence of Ca2⫹ may indicate hydrolytic activity by cPLA2. This difference was not significant, but was consistent, in control hearts, whereas it was not evident in failing hearts, suggesting that any cPLA2 activity had been severely depressed during heart failure. Because PLA2 is subject to strict regulation, it is conceivable that its regulatory mechanisms could determine the subcellular distribution of the PLA2 isoenzymes. For example, sequence analysis has revealed the presence of a pleckstrin homology domain in cPLA2 (24). This domain binds to membrane phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) with high affinity, which results in an intense increase in substrate

hydrolysis (24). Our recent finding of a low SL PtdIns 4,5-P2 mass in CHF (30) may account, at least partially, for the decreased SL cPLA2. Both SL and cytosolic sPLA2 activities were found to be minimal in control hearts. CHF did not significantly alter cytosolic sPLA2 activity, although immunoprotein expression was decreased. Alternatively, SL sPLA2 activity and immunoprotein expression were significantly increased in failing hearts. Little is known about the regulation of sPLA2 activity; however, we do know that it requires millimolar Ca2⫹ for catalytic activity and is modulated by accessory proteins (1, 11, 31). In this regard, a phospholipase-activating protein (PLAP) has been isolated and cloned (3, 4) and shown to stimulate sPLA2 activity selectively (4). Also of note is that tumor necrosis factor (TNF)-␣ treatment of endothelial cells augmented the expression of both PLAP and sPLA2 (2). Because localized production of TNF-␣ by failing myocardial cells has been reported (13), this may provide the stimulus for the increase in SL sPLA2 abundance and activity observed in the present study. Antibodies against sPLA2 provide substantial protection against ischemia-reperfusion damage in the rat heart (28), which suggests that the observed increase in SL sPLA2 activity and abundance in CHF may contribute, in part, to cardiac dysfunction. Moreover, we have previously demonstrated that TNF-␣ induces a decrease in membrane-associated iPLA2 activity in normal rat ventricular cardiomyocytes (17). The above actions of TNF-␣ on PLA2 isoenzymes favor the view that TNF-␣ may mediate the redistribution of PLA2 isoenzymes and the changes in PLA2 activity during CHF. The relevance of the observed redistribution and defective activity of PLA2 isoforms in the failing heart may arise from the action of membrane PLA2 in originating intrinsic signaling molecules (31). In particular, increasing evidence suggests that all three PLA2 isoenzymes may participate in some manner in arachidonic acid mobilization, dependent on the cell type and agonist used. In support of this, we demonstrated that in isolated cardiomyocytes, arachidonic acid release due to activation of either cPLA2 or iPLA2 is dependent on the agonist used (17). Recently, the ␤2-adrenoceptor (␤2-AR)-mediated signaling and positive inotropic response in cardiomyocytes were related to the activation of cPLA2 and intramembrane mobilization of arachidonic acid (25, 29). The positive inotropic effect induced by ␤2-AR is diminished in CHF in humans and in animal models of heart failure (33). Therefore, we cannot exclude that the observed decrease of SL cPLA2 immunoprotein abundance and activity, with subsequent impairment of the ␤2-AR-cPLA2-arachidonic acid pathway, may be a factor in the depressed cardiac response to ␤2-AR stimulation in CHF. Of further interest could be our recent evidence that activation of SL Ca2⫹-independent PLA2 activity and subsequent intramembrane mobilization of sn-2 unsaturated fatty acids modulate the cis-UFA PLD isoenzyme (18), which is a major source of signaling phosphatidic acid in heart SL (32). Phosphatidic


acid influences SL Ca2⫹ transport systems and increases intracellular free Ca2⫹ and cardiac force of contraction (34). Various cis-unsaturated fatty acids showed different stimulatory potencies. Arachidonic acid, which is released in isolated cardiomyocytes by the agonist-induced activation of cPLA2 or iPLA2 (17), is the most efficient agent (5). Therefore, the decrease of SL cPLA2 and iPLA2 isoforms observed in this study may reduce the stimulatory potential of SL PLA2 on cis-UFA PLD. Indeed, our most recent experiments with melittin, which stimulates PLA2 (18), show that melittin-evoked activation of cis-UFA PLD via PLA2 is significantly depressed in SL membranes from post-MI CHF animals [1,630 ⫾ 81 and 868 ⫾ 70 in control and CHF, respectively; values (n ⫽ 3) were obtained as indicated in (18) and are expressed as a percentage of the corresponding basal values; P ⬍ 0.05 vs. control]. This would impact negatively on SL cis-UFA PLD activity and related synthesis of phosphatidic acid, thus contributing to the defective Ca2⫹ movements and contractile performance of the failing heart in post-MI CHF (8, 22). In summary, congestive heart failure secondary to myocardial infarction is associated with atypical compartmentalization of PLA2 isoenzymes and concomitant changes in activities. This suggests that the bioprocesses mediated by PLA2 are altered in CHF. In particular, PLA2-dependent stimulation of SL cis-UFA PLD and related synthesis of phosphatidic acid may be affected, with possible consequences for Ca2⫹ homeostasis and contractile performance of the failing hearts. That said, a question to be considered is whether PLA2-related abnormalities precede CHF and contribute to the pathogenesis of post-MI CHF. Further investigations are needed to address this issue. This study was supported by the American Heart Association, Missouri Affiliate and National Heart, Lung, and Blood Institute Grant HL-54907 (J. McHowat), and by the Canadian Institutes of Health Research (V. Panagia). Presented in part at the XXI Annual Meeting of the American Section of the International Society for Heart Research, San Diego, California, June 9–12, 1999. REFERENCES 1. Balsinde J, Balboa MA, Insel PA, and Dennis EA. Regulation and inhibition of phospholipase A2. Annu Rev Pharmacol Toxicol 39: 175–189, 1999. 2. Clark MA, Chen MJ, and Crooke ST. Tumour necrosis factor induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells. Biochem J 250: 125–132, 1988. 3. Clark MA, Conway TM, Shorr RGL, and Crooke ST. Identification and isolation of a mammalian protein which is antigenically and functionally related to the phospholipase A2 stimulatory peptide melittin. J Biol Chem 262: 4402–4406, 1987. 4. Clark MA, Ozgur LE, Conway TM, Dispoto J, Crooke ST, and Bomalaski JS. Cloning of a phospholipase A2-activating protein. Proc Natl Acad Sci USA 88: 5418–5422, 1991. 5. Dai J, Williams SA, Ziegelhoffer A, and Panagia V. Structure-activity relationship of the effect of cis-unsaturated fatty acids on heart sarcolemmal phospholipase D activity. Prostaglandins Leukot Essent Fatty Acids 52: 167–171, 1995. 6. Dennis EA. Diversity of group types, regulation, and function of phospholipase A2. J Biol Chem 269: 13057–13060, 1994.

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