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Departments of 1Pathology, 2Cardiology, 3Clinical Chemistry, 4Physiology, and 5Gynecology, Vrije Universiteit Medical Center, and 6Institute for Cardiovascular Research, Vrije Universiteit, 1007 MB Amsterdam, The Netherlands; 7Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037; and 8Sanquin Reseach, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Department of Immunopathology, 1006 AN Amsterdam, The Netherlands
Submitted 8 October 2002 ; accepted in final form 3 June 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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myocardial infarction; inflammation
Type II secretory phospholipase A2 (sPLA2) hydrolyzes the sn-2-ester bond of phospholipids to produce free fatty acids and lysophospholipids (4), which is the rate-limiting step in the formation of several proinflammatory mediators like prostaglandins, leukotrienes, and platelet-activating factor (13). Hence, sPLA2 has been postulated to be a key factor in mediating tissue damage in human acute and chronic inflammatory diseases (13). Furthermore, plasma levels of the enzyme constitute a cardiovascular risk marker predicting coronary events in patients with coronary artery disease (6).
Recently, we have observed that the acute-phase proteins C-reactive protein (CRP) and sPLA2 are deposited in human infarcted myocardium. sPLA2 was found to occupy a larger area than CRP. Interestingly, the enzyme also bound to cardiomyocytes adjacent to the border zone of the infarcted area, where cells do not show morphological signs of cell damage (11). We therefore hypothesized that sPLA2 may enlarge the infarcted area (7). In the present study, we sought for evidence supporting this hypothesis. The cardiomyocytic cell line H9c2, as well as adult rabbit cardiomyocytes, were exposed to metabolic inhibition, mimicking ischemia, followed by reperfusion, while being exposed to sPLA2. In addition, the effect of sPLA2 on the binding of CRP was also analyzed. Our results indicate that sPLA2 can induce cell death in reversibly injured cardiomyocytes and tag these cells with CRP.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Adult rabbit cardiomyocytes. Male New Zealand White rabbits (2–2.5 kg) were tranquilized with midazolam (Dormicum; 1.5 mg/kg), anesthetized with fentanyl citrate (Hypnorm; 0.3 ml/kg), and anticoagulated with 1,000 units of heparin. Subsequently, the rabbits were euthanized with 100 mg of pentobarbital intravenously. The heart was then rapidly excised and attached to a Langendorff perfusion device via the aorta. Calcium-free buffer composed of (in mM) 118 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11.1 glucose was infused for 5 min under a constant pressure of 60 mmHg at 37°C. The hearts were then perfused with the same buffer containing collagenase (Worthington type II, 110 U/ml; Worthington Biochemical; Lakewood, NJ) and 4.7 µM CaCl2. After 30 min, the left ventricle was freed of other tissue, minced, and strained through a cell dissociation mesh (size 100, Sigma; St. Louis, MO). Sixty to eighty percent of the cells obtained were rod-shaped viable cardiomyocytes. The cells were suspended in medium containing 40 mM KCl, 20 mM KH2PO4, 50 mM L-glutamic acid, 20 mM taurine, 0.5 mM EGTA, 10 mM glucose, 10 mM HEPES, 77 mM KOH, and 4.9 mol/l MgCl2 and transferred into 15-ml conical tubes for experimental protocols. This study was approved by the Animal Ethics Committee of the Vrije Universiteit Medical Center Amsterdam and was performed according to the institutional guidelines.
Purification of sPLA2. sPLA2 was isolated from the supernatant of stimulated Hep G2 cells. Briefly, Hep G2 cells were cultured in 750-ml flasks containing Iscove's modified Dulbecco's medium (BioWhittaker), supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, until confluence. The cells were then incubated for several days with Iscove's modified Dulbecco's medium supplemented with a supernatant of endotoxin-treated monocytes until it contained a sufficient amount of sPLA2. sPLA2 was purified from the conditioned medium by affinity chromatography with the use of a monoclonal antibody coupled to Sepharose 4B beads. Low pH buffer (0.1 M glycin and 0.15 M NaCl, pH 2.5) was used to elute the enzyme from the column. Activity of the purified enzyme was measured with the use of a [14C]linoleoyl-phosphatidylethanolamine substrate. The actual concentration of sPLA2 used in the experiments was assessed with an ELISA as described previously (18).
Purification of CRP. CRP was purified from the ascitic fluid of patients with ovarian cancer with the use of phosphorylcholine affinity chromatography and gel filtration on a Biogel A 0.5 column (Bio-Rad; Richmond, CA) as described previously (17). The contaminating complexes of CRP and C4b, C4d, C3b, or C3d were removed by absorbing the preparations onto column Sepharose beads with appropriate monoclonal antibodies against C4 and C3 fragments. Purified CRP migrated as a single band with a molecular weight 25,000 on SDS-PAGE under reducing conditions and eluted as a single peak on mono-Q fast-performance liquid chromatography ion exchange chromatography (Pharmacia; Uppsala, Sweden). The final preparation was filtered through a 0.22-µm filter (Sartorius; Göttingen, Germany) and stored at a concentration of <1 mg/ml at 4°C (to minimize aggregation of CRP).
Metabolic inhibition protocol. Metabolic inhibition was induced by incubating the cells in "ischemic" buffer containing (in mM) 106 NaCl, 4.4 KCl, 1.0 MgCl2, 38 NaHCO3, 2.5 CaCl2, 20 2-deoxyglucose, and 1.0 NaCN, pH 6.6. The cells were then washed, and reperfusion was mimicked by incubating the cells in Dulbecco's modified Eagle's medium supplemented with 100 IE/ml sodium penicillin, 100 µg/ml streptomycin (Radiopharma-Fisiopharma), and 2 mM L-glutamine (GIBCO-BRL).
Assessment of cell viability. Annexin V (AV)-FITC and AV-phenylephrine (PE) (Bender MedSystems; Vienna, Austria) was used to assess flip-flop of the cell membrane. Propidium iodide (PI; Bender Medsystems, Vienna, Austria) or 7-aminoactinomycin D (7-AAD; Becton Dickinson, San Diego, CA) was used to assess cell membrane permeability and thus cell death. In brief, after being washed twice with phosphate-buffered saline (PBS, pH 7.4), the cardiomyocytes were suspended at a concentration of 106 cells/ml in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). The mixture was then incubated for 20 min in the dark at room temperature and analyzed with the use of flow cytometry (FACStar equipped with an argon ion laser 488 nm; Becton Dickinson). CaspaTag caspase-3 activity kit (DEVD, Intergen; Uden, The Netherlands) was used to determine active caspase-3 according to the manufacturer's instructions. Briefly, after metabolic inhibition and reperfusion, cells were labeled with FAM-DeVd-FMK, washed, and analyzed by flow cytometry (Becton Dickinson FACStar).
Cells staining negative for AV, PI/7-AAD, or caspase-3 were considered to be vital; cells staining positive for AV and negative for PI or 7-AAD were considered to have a flip-flopped membrane. AV-positive, PI/7-AAD-negative cells that had no active caspase-3 were considered to represent reversibly injured cells, whereas those having active caspase-3 were considered to be early apoptotic cells. Cells positive for AV as well as for PI/7-AAD were considered to be late apoptotic or necrotic cells.
Binding of sPLA2 or CRP to cells. Binding of sPLA2 to cells was evaluated with the use of a two-step staining procedure with monoclonal antibody 4A1 (subclass IgG1) against human sPLA2 (kindly provided by Dr. F. B. Taylor, Jr., Oklahoma Medical Research Foundation, Oklahoma City, OK). Binding of CRP to cells was evaluated with the use of a two-step staining procedure with monoclonal 5G4 (subclass IgG1) against CRP. Subsequently, goat anti-mouse immunoglobulin antibodies coupled to R-phycoerythrin (G-anti-mouse PE; DAKO; Glostrup, Denmark) were added. Cells were evaluated with the use of an immunofluorescence microscope (Leica DMBR) or by flow cytometry.
Inhibition of calcium mobilization and of cytosolic PLA2. Intracellular calcium mobilization was inhibited by incubation cells in the reperfusion medium containing 50 µM BAPTA-AM (Calbiochem; Nottingham, UK). Extracellular calcium was inhibited by the addition of 20 mM EGTA to the reperfusion medium (Sigma) (19). Cytosolic PLA2 (cPLA2) was inhibited by incubating cells in reperfusion medium supplemented with 20 µM methyl-arachidonyl-fluorophosphonate (MAFP; Cayman; Ann Arbor, MI) (1, 8).
Assessment of proliferation capacity of H9c2 cells. The proliferative capacity of H9c2 cells, a measure for injury, was assessed by staining with sulforhodamine B (SRB) using a method that was adapted from that for viable cells of adherent cell lines (14). Briefly, cells were cultured in 96-well plates for 1 day and incubated in ischemic buffer. After 45 min, the supernatant was replaced by standard culture medium, and cells were incubated during 3 additional days in the presence or absence of sPLA2. On day 4, the cells were fixed and stained with SRB. After SRB was dissolved in a Tris base, the optical density, which reflects the number of viable cells in a well, was measured at 540 nm with a standard ELISA plate reader. Injury to the cells was quantified as the percentage of cells staining with the dye after 96 h compared with untreated cells.
Statistics. Data were analyzed with the use of SPSS for Windows version 9.0 (SPSS; Chicago, IL). Means ± SD were calculated to express data according to distribution. Student's t-test was used to calculate the significance of the observed differences in case of normal distribution; the Wilcoxon-Mann-Whitney test was used otherwise. Statistical significance was defined as a value of P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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25% of the AV single-positive population (Fig. 2)] appeared to be caspase-3 negative and thus presumably represented reversibly injured, flip-flopped cells.
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Effects of sPLA2 on metabolic inhibition of H9c2 cells. In a previous study, we (11) found that circulating levels of sPLA2 increase after acute myocardial infarction (AMI). To mimic the clinical situation of AMI, sPLA2 was added in the reperfusion phase, at a concentration (800 ng/ml) that may occur after AMI (11). When added to control cells (nonischemic), sPLA2 bound to up to 20% of the total cell population (Fig. 3). These sPLA2-binding cells all stained positively with PI and thus represented nonviable cells in the population. H9c2 cells incubated during a 2-h "reperfusion" period in the presence of sPLA2 after exposure to a 45-min "ischemia" period bound the enzyme significantly more than control cells (50.0 ± 5.6% positive cells vs. 19.15 ± 5.0%, P < 0.01; see Fig. 3). When cells exposed to ischemia and reperfusion in the absence or presence of sPLA2 were compared regarding viability and membrane flip-flop, it appeared that the fraction of 7-AAD-positive cells was increased by sPLA2 in control cells (14.1 ± 1.6%) versus 25.7 ± 1.4% (P < 0.01) in the ischemic cells (Fig. 4). This was accompanied by a decrease of the single AV-positive population in control cells (8.7% ± 1.1%) versus 23.9% ± 1.9% (P < 0.01) in the ischemic cells (Fig. 4). Notably, triple staining analysis (sPLA2, 7-AAD, and AV) revealed that sPLA2 bound to the AV- and 7-AAD-positive population as well as to single AV-positive cells. These results pointed to a cytotoxic effect of sPLA2 on the cells exposed to metabolic inhibition. This putative cytotoxic effect of sPLA2 was confirmed by the SRB cytotoxicity assay, which revealed that subsequent to 45 min of metabolic inhibition, sPLA2 (800 ng/ml) produced a significant decrease in cell viability after 3 days from 56.6 ± 14.8% to 33.2 ± 9.9% viable cells (P < 0.01) (Fig. 5).
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Requirement of calcium or cPLA2 for effects of sPLA2 on the H9c2 cells. sPLA2 activity is calcium dependent (10, 19). Therefore, we studied the influence of extracellular calcium on the binding of sPLA2 to ischemically challenged H9c2 cells by adding EGTA (20 mM) during the incubation with sPLA2. This resulted in a significant, but not complete, reduction of the binding of sPLA2 to the cells (Fig. 6). EGTA had no significant influence on the amount of PI-positive cells subsequent to metabolic inhibition and reperfusion with sPLA2 (not shown). We then studied the role of intracellular calcium by preincubation of the cells with 50 µM BAPTA-AM for 30 min. BAPTA-AM also significantly diminished binding of sPLA2 to ischemically challenged cells, to a similar extent as EGTA did (Fig. 6). Analysis of the effects of BAPTA-AM on cell viability revealed that this intracellular calcium chelator protected the cells against the ischemia-reperfusion challenge (P < 0.01, data not shown). This effect was more or less proportional to its inhibitory effect on sPLA2 binding. Thus the effect of BAPTA-AM on sPLA2 binding likely was due to its effect on cell viability. When BAPTA-AM and EGTA were combined, no additional inhibition of sPLA2 binding was found (not shown).
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The susceptibility of cellular membranes for sPLA2 may be influenced by the activity of cPLA2 (16). Preincubation of the cells for 30 min with the cPLA2 inhibitor MAFP reduced the binding of sPLA2 to ischemically challenged cells, which the decrease was similar to that found with BAPTA-AM or EGTA (not shown). Preincubation with MAFP had no effect on the increase of cell death subsequent to metabolic inhibition and sPLA2 reperfusion.
Effect of sPLA2 on adult rabbit cardiomyocytes subjected to metabolic inhibition. To extrapolate the effects of sPLA2 on H9c2 cells to adult cardiomyocytes, we also studied the effects of sPLA2 on adult cardiomyocytes isolated from male New Zealand White rabbits. After isolation, three morphologically distinct cell populations were observed, as has been shown by others as well: a majority of rectangular cells (AV negative/PI negative) and a minority of square (AV positive/PI negative) and round cells (most cells being AV positive/PI positive) (Fig. 7A). In pilot experiments, it was established that, compared with H9c2 cells, a shorter metabolic inhibition period (20 min), a shorter reperfusion period (45 min), and a lower concentration of sPLA2 (100 ng/ml compared with 800 ng/ml) yielded an optimal number of single AV-positive cells (not shown). Remarkably, after metabolic inhibition, a small though not significant increase (2–9%) in the rectangular cells that bound sPLA2 was observed (Fig. 7B). Also, the percentage of round cells binding sPLA2 slightly increased from 61% to 70% after metabolic inhibition. Metabolic inhibition, however, had the most pronounced effect on sPLA2 binding to the square cells, resulting in a 26.7 ± 15.0% increase (P < 0.02) of single AV-positive cells binding sPLA2.
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Effect of CRP in metabolically inhibited H9c2 cells. On the basis of immunohistochemical observations in humans with AMI, we postulated that sPLA2 could induce binding sites for CRP on cardiomyocytes (11). Therefore, we also studied CRP in this respect in our in vitro system. Because of a lack of species specificity of the anti-CRP monoclonal antibody 5G4 on cardiomyocytes derived from rabbits, we had to limit these experiments to H9c2 cells. Two hours of incubation with CRP (300 µg/ml) after ischemia, in the absence of sPLA2, resulted in a significant increase of CRP binding compared with the control situation (Fig. 8). This enhanced binding was limited to PI/AV positive, thus late apoptotic/necrotic cells (not shown).
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Binding of CRP irrespective of sPLA2 theoretically may result from the ability of a cPLA2 to form binding sites for CRP. Therefore, we performed these experiments by preincubating the cells for 30 min with the cPLA2 inhibitor MAFP. However, no effect of this inhibitor, used at concentrations from 10–100 µM, on the binding of CRP to the cells was found (not shown).
Effect of sPLA2 on CRP binding in metabolic inhibited H9c2 cells. We then studied whether sPLA2 could facilitate binding of CRP to ischemically challenged H9c2 cells. For this, we preincubated H9c2 cells with sPLA2 (800 ng/ml) for 1 h subsequent to ischemia, whereafter CRP (300 µg/ml) was incubated for 1 h. In a former study, we found that the presence of sPLA2 preceded CRP in serum in patients with AMI (6). As shown in Fig. 8, sPLA2 did not potentiate binding of CRP to nonischemic cells. However, sPLA2 significantly increased the binding of CRP to ischemically challenged H9c2 cells, mainly PI/AV-positive cells. Single AV-positive cells (5% of the cells) were among the cells that bound CRP on which sPLA2 also potentiated binding of CRP.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Binding of sPLA2 to the H9c2 cells was strongly dependent on intra- and extracellular calcium. Chelation of extracellular calcium abrogated the binding of sPLA2 to the ischemically challenged cells. The effect of intracellular calcium on sPLA2 binding presumably was due to a cell protective effect (12, 15). However, it cannot be excluded that the extracellular release of calcium by dead cells, and the subsequent stimulated sPLA2 binding potential was also inhibited by preventing lethal cell damage via inhibition of a rise in intracellular calcium with the use of BAPTA-AM. Theoretically, an alternative explanation for the decline in sPLA2 binding to the challenged H9c2 cells by BAPTA-AM could be that inhibition of actin depolymerization and a subsequent inhibition of the membrane flip-flop may have reduced the formation of binding sites for sPLA2. Nevertheless, a calcium-dependent binding of human sPLA2 to ischemically challenged cardiomyocytes is to the best of our knowledge a new observation. Furthermore, we found that the binding of sPLA2 to the ischemic H9c2 cells was also dependent on cPLA2 activity. cPLA2-dependent binding of sPLA2 to cells has been established before. However, this was not done with human sPLA2 as we have used in the present study (16). Notably, the inhibition of sPLA2 binding to cardiomyocytes subsequent to inhibition of the increase of intracellular calcium thus could also result from inhibition of calcium-dependent intracellular cPLA2.
In vivo, a small amount (5–10%) of cardiomyocytes in the area adjacent to the border zone of the infarction area in patients with AMI (11) bind sPLA2, despite normal morphology. In analogy, also in the in vitro system, we identified a minority of the cardiomyocytes that stained positive for AV, but not for PI, and lacked caspase-3 activity. These cells, presumably reversibly damaged from the ischemic challenge, bound sPLA2. The question then arose as to whether sPLA2 itself could induce a transformation from single flip-flopped cells to late apoptotic/necrotic cells. Indeed, as shown in Fig. 4, sPLA2 decreased the amount of single flip-flopped cells while at the same time increasing the amount of late apoptotic/necrotic cells. This significant decrease in cell viability by sPLA2 was also demonstrated in the cytotoxicity assay (Fig. 5). We also found that the acute phase protein CRP bound to late apoptotic and/or necrotic, nonviable cells, independent of sPLA2 and/or cPLA2 (Fig. 8). However, we also found that sPLA2 significantly increased the binding of CRP not only to late apoptotic and/or necrotic cells but also to single AV-positive cells.
These results thus suggest a devastating effect of sPLA2 itself on cardiomyocytes during ischemia, not only via a direct cytotoxic effect but also via facilitating an inflammatory response. This is in contrast with a recent study of de Windt et al. (2), who found in isolated hearts of sPLA2-deficient mice no effect of sPLA2 on the necrotic infarcted area compared with wild-type mice. However, these authors perfused the heart with Krebs-Henseleit buffer, which may not yield similar results as these with blood.
In conclusion, our data show that sPLA2 can bind to ischemically challenged cardiomyocytes and is capable of transforming flip-flopped, caspase-3-negative cells into a late apoptotic/necrotic stage. We suggest that inhibition of sPLA2, for example, with specific inhibitors, as have recently been developed (9), could salvage at that moment nonlethally ischemic jeopardized cardiomyocytes and thus could result in inhibition of the infarction area of patients with AMI.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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