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Am J Physiol Heart Circ Physiol 293: H2148-H2154, 2007. First published July 27, 2007; doi:10.1152/ajpheart.00074.2007
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COX-2-dependent and potentially cardioprotective effects of negative inotropic substances released after ischemia

Katrin Birkenmeier,1,* Alexander Staudt,1,* Wolf-Hagen Schunck,2 Irka Janke,1 Corina Labitzke,1 Thomas Prange,1 Christiane Trimpert,1 Thomas Krieg,1 Martin Landsberger,1 Verena Stangl,3 and Stephan B. Felix1

1Innere Klinik B, Ernst-Moritz-Arndt-Universität, Greifswald, Germany; 2Max-Delbrück-Zentrum, Berlin, Germany; 3Medizinische Klinik mit Schwerpunkt Kardiologie und Angiologie, Charité Campus Mitte, Universitätsmedizin Berlin, Berlin, Germany

Submitted 18 January 2007 ; accepted in final form 18 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
During reperfusion, cardiodepressive factors are released from isolated rat hearts after ischemia. The present study analyzes the mechanisms by which these substances mediate their cardiodepressive effect. After 10 min of global stop-flow ischemia, rat hearts were reperfused and coronary effluent was collected over a period of 30 s. We tested the effect of this postischemic effluent on systolic cell shortening and Ca2+ metabolism by application of fluorescence microscopy of field-stimulated rat cardiomyocytes stained with fura-2 AM. Cells were preincubated with various inhibitors, e.g., the cyclooxygenase (COX) inhibitor indomethacin, the COX-2 inhibitors NS-398 and lumiracoxib, the COX-1 inhibitor SC-560, and the potassium (ATP) channel blocker glibenclamide. Lysates of cardiomyocytes and extracts from whole rat hearts were tested for expression of COX-2 with Western blot analysis. As a result, in contrast to nonischemic effluent (control), postischemic effluent induced a reduction of Ca2+ transient and systolic cell shortening in the rat cardiomyocytes (P < 0.001 vs. control). After preincubation of cells with indomethacin, NS-398, and lumiracoxib, the negative inotropic effect was attenuated. SC-560 did not influence the effect of postischemic effluent. The inducibly expressed COX-2 was detected in cardiomyocytes prepared for fluorescence microscopy. The effect of postischemic effluent was eliminated with applications of glibenclamide. Furthermore, postischemic effluent significantly reduced the intracellular diastolic and systolic Ca2+ increase (P < 0.01 vs. control). In conclusion, the cardiodepressive effect of postischemic effluent is COX-2 dependent and protective against Ca2+ overload in the cells.

cyclooxygenase; potassium adenosine 5'-triphosphate channels

EXPERIMENTAL EVIDENCE INDICATES that negative inotropic mediators are released from the postischemic myocardium during reperfusion (13, 14). We have previously shown that these substances reduce systolic cell shortening and Ca2+ transients in field-stimulated isolated rat cardiomyocytes. Reduction results from decreased maximal conductance and not from voltage- and time-dependent gating of the L-type Ca2+ channel (14). With the use of a double heart model, a marked decrease in left ventricular (LV) pressure (LVP) and in LV peak positive (LV dP/dtmax) and LV peak negative (LV dP/dtmin) coronary perfusion pressure was measured in heart II perfused with the postischemic effluent of heart I (13). The negative inotropic substances do not modulate tissue concentrations of cAMP and cGMP and do not affect the activities of cAMP-dependent protein kinase A and protein kinase C (14). Using a model of two sequentially perfused rat hearts, we demonstrated that these cardiodepressive mediators are not released from coronary endothelial cells (42). Until now, the precise mechanisms of these negative inotropic substances have remained unknown.

Ischemia and reperfusion are associated with an increase in arachidonic acid levels in cell membranes and induce profound alterations in myocardial eicosanoid generation (6, 37). Arachidonic acid is converted by cyclooxygenase (COX) to prostaglandin H2 (PGH2), which in turn is the precursor of further prostaglandins and thromboxanes. There is evidence that COX is involved in changes of myocardial metabolism after ischemia. A concentration of prostaglandins in cells increases under postischemic conditions (27). An inhibition of COX by indomethacin leads to a reduction in cardioprotection after ischemia (5). It has been speculated that a yet unknown cardioprotective factor is metabolized by COX. There are two different isoforms of COX, designated as COX-1 and COX-2. COX-1 is constitutively expressed, whereas COX-2 is inducible as an immediate early gene by various stress factors. Expression of COX-2 is intensified after cell damage (22, 33, 45) as well as after ischemia (1, 38).

Interference with potassium (ATP) channels in cardiomyocytes is another mechanism by which arachidonic acid metabolites may modulate postischemic myocardial contractility (26). Ischemic conditions induce the opening of ATP channels in cardiomyocytes. There is evidence that potassium efflux improves cardiac function during reperfusion and that eicosapentaenoic acid is capable of opening the channels (2, 20, 26). The opening of potassium (ATP) channels has a negative inotropic effect on cardiac tissue (25, 28, 49).

Since alterations in arachidonic acid metabolism are intensively involved in the modulation of myocardial contractility after ischemia, in the present study we investigated in isolated rat cardiomyocytes whether the above-described negative inotropic substances are synthesized via arachidonic acid metabolism.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Collection of Postischemic Effluent From Isolated Rat Hearts

Rat hearts were perfused at a constant flow (10 ml/min) with Krebs-Henseleit buffer (KHB) in an open Langendorff system, as previously described (14).

The perfusion phase lasts 30 min; the control effluent (nonischemic effluent) was obtained during the final 30 s. Hearts were then subjected to 10 min of global stop-flow ischemia. Hearts were kept warm during this ischemic period. The isolated hearts were then subsequently reperfused. At the onset of reperfusion, postischemic effluent containing the negative inotropic mediators was collected over a period of 30 s (5 ml). The nonischemic and postischemic effluents were immediately used for in vitro experiments. Both nonischemic and postischemic effluents were diluted (1:4) with KHB (14).

Isolation of Rat Ventricular Myocytes

The ventricular myocytes were isolated in an open Langendorff system, as described elsewhere (14). Rat hearts were perfused with oxygenated Ca2+-free KHB (37°C, pH 7.4) containing (in mmol/l) 110 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 HEPES, and 11 glucose. After 3 min, hearts were perfused with KHB that contained 30 µmol/l Ca2+ and collagenase type II (355 U/ml). After 30 min of recirculation and collagenase digestion, hearts were minced and incubated for 15 min in the same solution. The cell suspension was filtered through a nylon sieve and then centrifuged at 43 relative centrifugal force for 1 min to separate cardiomyocytes from fibroblasts. The following washing phases increased Ca2+, in two steps, to 200 and 500 µmol/l.

Intact cells were collected using a Fuchs-Rosenthal chamber. Cells were considered intact if they exhibited the typical rod-shaped morphology with no blebs or granulation. Typically, about 2 x 106 cells were obtained per rat heart, 95% of them being intact. The cells were then plated on four-well-chamber glass slides (Nunc, Naperville, IL) that had been coated with 10 µg/ml of laminin. The chambers were incubated for 1 h at 4°C. Per chamber, 5,000 cells in 600 µl were filled in and subsequently incubated for 2 h at 4°C. The buffer was exchanged, and cells were stained with a solution containing 0.1% dimethyl sulfoxide (DMSO), 0.025% pluronic F-127, 0.2% BSA, and 5 µmol/l fura-2 AM. Cells were incubated for 10 min at room temperature on an orbital shaker with oscillation of 20 rpm. Afterward, the incubated cells were washed twice with 500 µl KHB.

Measurement of Ca2+ Transients and Systolic Cell Shortening

During the experiment, cardiomyocytes were superfused with experimental buffer at a flow rate of 2 ml/min and kept at room temperature to minimize the cell leakage of fluorescent probes. Postischemic and nonischemic effluents were likewise superfused in this manner, with stimulation by electrodes at a stimulation frequency of 1 Hz and with 5-ms pulse duration, and at a voltage of 12 V, with these conditions constant throughout the entire experiment (Myopacer, Ionoptix). A system consisting of a fluorescence microscope (Leica, DM-IRB) and a high-resolution camera (Myocam, Ionoptix) enabled analysis of both systolic cell shortening and Ca2+ transient per chamber. Cells were examined with a x20 immersion-oil objective. Ca2+-dependent fluorescence was measured by use of a laser with 6-mW excitation at 360 nm and 380 nm. Emitted light was detected at wavelengths over 515 nm. Changes in Ca2+ concentration were measured as changes in relative fluorescence of fura-2 AM. The difference between systolic and diastolic Ca2+ concentration was measured as the percent change in the ratio of fluorescence at excitation wavelengths of 360 nm to 380 nm. Changes in the Ca2+ transients after superfusion with postischemic or nonischemic effluents were measured as an increase or decrease of percent change in ratio.

For the experiments, cells were used that demonstrated rectangular morphology and that contracted without interruption. A Ca2+ transient was required to show constant values over a period of 2 min. In the experiments, cardiomyocytes were used to show at least 15% contractility and 25% change in ratio at baseline. Testing took place in preceding experiments to determine whether baseline values for contractility and Ca2+ transients were affected by preincubation with the various inhibitors used in this study.

We carried out detection under basal conditions, after which we superfused 2 ml of substances (e.g., postischemic and nonischemic effluent in 1:4 dilution). We next performed acute measurements immediately, which were repeated after 120 s (2 min) and a further 170 s (5 min). Measurements took place over 10 s.

In a further series of experiments, cells were preincubated with the nonselective COX inhibitor indomethacin (5 µmol/l), the selective COX-2 inhibitors NS-398 (0.25 µmol/l) and lumiracoxib (0.1 µmol/l), the selective COX-1 inhibitor SC-560 (0.25 µmol/l), and the nonselective potassium (ATP) channel inhibitor glibenclamide (1 µmol/l). These inhibitors did not affect the baseline of Ca2+ transients or systolic cell shortening in the concentrations applied.

Detection of COX-2 and COX-1 in Adult Cardiomyocytes

Adult rat cells were plated into laminated six-well plates. After 2 h, the cells were stained with fura-2 AM. For cell lysis, radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz) was used, with additions of 10 µl of protease inhibitor, PMSF, and sodium orthovanadate per 1 ml RIPA buffer. Protein quantification took place with a BCA protein assay kit (Pierce). RAW 264.7 lysate (mouse leukemia monocyte macrophage cell line; Santa Cruz) was used as a positive control for COX-2. Samples were mixed 1:1 with 2x sample buffer (Santa Cruz), which were then incubated at 95°C for 3–5 min and run on 10% SDS polyacrylamide gel. Total protein (40 µg) per lane were taken for SDS-PAGE. The proteins were transferred onto membranes (Hybond ECL, Amersham) and subjected to immunoblot analysis. Membranes were then blocked for 1 h at room temperature in blocking solution (5 g milk powder diluted in 1 ml 1x TBS-0.1% Tween). Membranes were subsequently incubated overnight with goat anti-COX-2 (0.5 µg/ml, Cayman Chemicals) or mouse anti-COX-1 (Cayman Chemicals), diluted in 5% BSA-0.1% Tween 20 in 1x TBS. Membranes were washed and incubated for 90 min at room temperature with secondary antibody bovine anti-goat IgG or bovine anti-mouse IgG, both coupled to horseradish peroxidase at a dilution of 1:5,000 in washing buffer. Proteins were revealed by chemiluminescence with the use of the chemiluminescence kit Super Signal West Pico (Pierce).

Detection of COX-2 in Whole Adult Rat Hearts

Hearts of adult rats were isolated, and protein was extracted. Therefore, hearts were minced and frozen at –80°C. After 2 days, heart pieces were minced again in liquid nitrogen to obtain very small pieces for production of homogenates and to avoid thawing. Homogenates were produced in lysis buffer (RIPA buffer, Santa Cruz). After centrifugation at 14,000 rpm and 4°C for 15 min, protein extracts were tested for COX-2 expression with Western blot analysis.

In accordance with the collection of postischemic and nonischemic effluent, protein extraction and analysis of COX-2 expression were performed in hearts perfused for 30 min only (control) or perfused for 30 min and subjected to 10 min of global stop-flow ischemia and reperfused for 30 s. COX-2 expression was additionally analyzed after a reperfusion period of 3 and 5 h after ischemia. Control hearts (nonischemic hearts), accordingly, were perfused for a total period of 3.5 and 5.5 h before analyzing for COX-2 expression. Analysis of the protein extracts took place under the same experimental conditions as used for analysis of expression of isolated adult cardiomyocytes.

Materials

Various buffers were applied for our experiments. Buffers were sterile filtered and stored at 4°C. Isolated rat cardiomyocytes were resuspended in buffer containing (in mmol/l) 117 NaCl, 2.8 KCl, 0.6 MgCl2, 1.2 KH2PO4, 1.2 CaCl2, 10.0 HEPES, and 20.0 glucose at pH 7.3. In the Langendorff system, fixed hearts were perfused with buffer containing (in mmol/l) 110 NaCl, 2.6 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25.0 HEPES, and 11.0 glucose at pH 7.4, before isolation of cells. For production of postischemic effluent, the isolated rat heart was fixed in the Langendorff system and perfused with KHB containing (in mmol/l) 116.4 NaCl, 4.02 KCl, 1.16 MgSO4, 1.19 KH2PO4, 1.26 CaCl2, 7.0 HEPES, 9.9 glucose, and 24.9 NaHCO3 at pH 7.3–7.5, to permit heartbeat. Substances were purchased from Merck (Darmstadt, Germany), except for MgCl2, which was supplied by Sigma-Aldrich (Deisenhofen, Germany).

For isolation of adult rat cardiomyocytes and for production of postischemic effluent, female rats were used (albino Wistar rats; Schönwalde; Berlin, Germany). Animals were 49 to 56 days old and had a weight of 180 to 200 g. Rats were anesthetized with thiopental sodium from Byk Gulden (Konstanz, Germany).

For staining intracellular Ca2+, fura-2 AM was employed from Sigma-Aldrich. Laminin was obtained from Harbor Bio-Products (Norwood, MA). For cell isolation, collagenase type II (Cell Systems) was applied.

For inhibitor experiments, indomethacin, SC-560, NS-398, and glibenclamide were obtained from Sigma-Aldrich. Lumiracoxib was obtained from Novartis Pharma (Nuremberg, Germany). Inhibitors were dissolved in DMSO, provided by Merck.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, Revised 1996), and the protocol was approved by the District Veterinary Office.

Statistical Analysis

The results are expressed as means ± SE for n calculations. The analysis entailed comparisons between and within the groups. Univariate post hoc analyses were performed in the form of Mann-Whitney U-tests and Wilcoxon tests after overall testing. Accounts for multiple comparisons were carried out using the sequentially rejective Bonferroni-Holm procedure.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
To characterize the effects of postischemic and nonischemic effluent (dilution with 1:4 KHB), we performed measurements of Ca2+ transients (difference between systolic and diastolic Ca2+ concentrations) and systolic cell shortening of isolated cardiomyocytes. Nonischemic effluent was used as a negative control and demonstrated no effect (Fig. 1A). Reduction of Ca2+ transients and systolic cell shortening was clearly evident during superfusion of the isolated cardiomyocytes with postischemic coronary effluent: the negative inotropic effect is shown by a 15 ± 1% reduction of Ca2+ transients and a 25 ± 2% reduction of systolic cell shortening during steady state (P < 0.001 vs. control).


Figure 1
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  		Fig. 1. A–D: effects of postischemic and nonischemic coronary effluent on Ca2+ transients and systolic cell shortening of the isolated rat cardiomyocytes in the presence and absence of indomethacin (Indo; A), the selective cyclooxygenase (COX)-1 inhibitor SC-560 (B), the selective COX-2 inhibitor NS-398 (C), and the selective COX-2 inhibitor lumiracoxib (Lum; D). Data are presented as means ± SE for 6 different myocytes for each group. +++P < 0.001 vs. nonischemic coronary effluent.

 
Metabolism of the Negative Inotropic Substances

Potential role of COX. The assumption that postischemic substances derive from arachidonic acid metabolism prompted us to speculate that the factors are metabolized in the cell by COX to their active form. We accordingly tested the effect of indomethacin, a nonselective inhibitor of COX (8, 24).

Preincubation of the cells with 5 µmol/l of indomethacin completely abolished the negative inotropic effect of postischemic effluent (Fig. 1A).

To further elucidate which COX isoform is involved, we tested SC-560 [a selective inhibitor of COX-1 (16, 41)], as well as NS-398 and lumiracoxib [two selective inhibitors of COX-2 (11, 18, 34, 44, 46)]. The results are shown in Fig. 1, B–D. Preincubation of the cells with SC-560 (0.25 µmol/l) failed to modulate the negative inotropic effect of postischemic effluent. In contrast, preincubation with NS-398 (0.25 µmol/l) and lumiracoxib (0.1 µmol/l) totally prevented the effect of postischemic effluent on Ca2+ transients and contractility.

Potential Cardioprotective Effect of the Negative Inotropic Substances

Potential role of potassium (ATP) channels. We tested the effect of the nonselective potassium (ATP) channel-blocking agent glibenclamide (25), which equally blocks sarcolemmal and mitochondrial potassium (ATP) channels. As shown in Fig. 2, glibenclamide completely blunted the negative inotropic effect of postischemic effluent (Fig. 2).


Figure 2
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  		Fig. 2. Effects of postischemic and nonischemic coronary effluent on Ca2+ transients and systolic cell shortening of the isolated rat cardiomyocytes in the presence and absence of glibenclamide (Glib). Data are presented as means ± SE for 6 different myocytes. +++P < 0.001 vs. nonischemic coronary effluent.

 
To study the putative protective effect of postischemic effluent on cardiomyocytes under the condition of increased extracellular Ca2+ load, we increased extracellular Ca2+ concentration from 1.2 to 2.4 mmol/l. When compared with the nonischemic effluent, postischemic effluent significantly attenuated intracellular systolic and diastolic Ca2+ increase, thereby suggesting that postischemic effluent protects cardiomyocytes from Ca2+ overload in the cells (P < 0.01, Fig. 3).


Figure 3
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  		Fig. 3. Effects of postischemic and nonischemic coronary effluent on Ca2+ transients and systolic cell shortening of the isolated rat cardiomyocytes after increasing the extracellular Ca2+ concentration from 1.2 to 2.4 mmol/l. Data are presented as means ± SE for 6 different myocytes. ++P < 0.01 vs. nonischemic coronary effluent.

 
COX-2 Expression in Isolated Adult Cardiomyocytes

COX-2 is inducibly expressed in cells (1, 33). We therefore performed Western blot analysis to investigate whether COX-2 is expressed in isolated cardiomyocytes laminated and stained with fura-2 AM. As shown in Fig. 4, COX-2 is expressed in the cardiomyocytes used in our experiments. Cells that were not laminated and stained with fura-2 AM demonstrate less expression of COX-2 in Western blot analysis than do cells that were laminated with or without staining with fura-2 AM.


Figure 4
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  		Fig. 4. Western blot analysis of protein expression of COX-2 in cardiomyocyte lysates laminated and stained with fura-2 AM (A, lane 2, and B, lane 5), without being laminated or stained (B, lane 2), only stained with fura-2 AM (B, lane 4), or only laminated (B, lane 3). RAW 264.7 lysate (mouse leukemia monocyte macrophage cell line) was used as a positive control (A and B, lane 1). The presence of constitutively expressed COX-1 in cardiomyocyte lysate is shown in C. Shown is 1 representative blot for the various lysates.

 
COX-2 Expression in Isolated Whole Rat Hearts

To investigate whether COX-2 expression is induced by 10 min of global stop-flow ischemia, we additionally performed Western blot analysis of protein extracts from whole rat hearts before and after 10 min of global ischemia. Rat hearts basally express COX-2 (Fig. 5A), and the expression is not upregulated after 10 min of global stop-flow ischemia either after 30 s or 3 h of reperfusion (Fig. 5, A and B). After a reperfusion period of 5 h, COX-2 is upregulated in postischemic and nonischemic hearts (Fig. 5C).


Figure 5
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  		Fig. 5. Western blot analysis of protein expression of COX-2 in whole rat hearts perfused for 30 min only (control; A, lane 1) or perfused for 30 min and subjected to 10 min of global stop-flow ischemia and reperfused for 30 s (A, lane 2). COX-2 expression after 3 and 5 h of reperfusion with and without preceding 10 min of global ischemia is shown in B and C. Shown is 1 representative blot for the various protein extracts.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study provides further insights into the mechanisms by which previously described negative inotropic substance(s) released after myocardial ischemia during reperfusion mediate their negative inotropic effects. Our results suggest that these postischemic substance(s) are most likely a product of the arachidonic acid metabolism.

Several studies have shown that eicosanoids, in particular prostaglandins, are generated during myocardial ischemia. Mobert and Becker (27) detected an increase in prostaglandin concentration in cells of guinea pig hearts after ischemia. Synthesis of prostaglandins by COX-2 requires the release of arachidonic acid or epoxyeicosatrienoic acids (EETs) from phospholipids. The main mediator of postischemic effluent released from myocardial tissue is possibly metabolized to its active form by COX-2.

Role of COX-2 in the Negative Inotropic Effect of Postischemic Effluent

Our data indicate a complex signal transduction process initiated by the main mediator of postischemic effluent. COX-2 evidently plays a role in metabolism of postischemic effluent and its effects on cardiomyocytes; the negative inotropic effect of postischemic coronary effluent on Ca2+ transients and systolic cell shortening was completely prevented by preincubation of the isolated cardiomyocytes with the nonselective cyclooxygenase inhibitor indomethacin and the COX-2 inhibitors NS-398 and lumiracoxib. These findings suggest that the main mediator of postischemic effluent is metabolized by COX-2 to its active form, which leads to a reduction of Ca2+ transients and systolic cell shortening. The effects of the postischemic effluent may be attributed to the effects of oxygen free radicals or different proteins such as cytokines. Previous studies, however, have demonstrated that pretreatment of the postischemic effluent with the free radical scavengers superoxide dismutase and catalase, or various proteases as well as heating, does not modulate the effect of the postischemic effluent (13).

COX-2 metabolizes arachidonic acid to PGH2, which is the precursor of further prostaglandins and thromboxanes. COX-2 is not constitutively expressed in cardiomyocytes but is rather induced by various stress factors, e.g., cytokines, mechanical stress factors, or phenomena, that occur during ischemia-reperfusion (1, 21, 31–33). COX-2 is basally expressed in the cardiomyocytes used in our experiments. The expression is assumably upregulated due to the preparation process and during lamination of cells for fluorescence microscopy (Fig. 4).

Several studies demonstrate that COX-2 is upregulated in preconditioned myocardium and that this phenomenon plays an essential role in ischemic preconditioning. COX-2 is accordingly discussed as an important factor of heart protection in the late phase of ischemic preconditioning (4, 23, 40, 48). COX-2 selective inhibitors interfere with protective effects in the late phase of ischemic preconditioning (40). In the isolated rat hearts used in our experiments, we were not able to detect upregulated COX-2 expression in Western blot analysis after 10 min of global ischemia. Detection of upregulated COX-2 expression in Western blot analysis after induction by various stress factors (e.g., after ischemia) is normally possible at the earliest after 3 h (3, 10). We therefore perfused rat hearts for 3 h after subjection to 10 min of global stop-flow ischemia in the open Langendorff system and conducted Western blot analysis of the protein extracts. We did not detect upregulated COX-2 expression in these hearts. A longer reperfusion period of the rat hearts leads to intensified COX-2 in the ischemic and control heart, most likely due to myocardial stress (e.g., myocardial edema formation) caused by longer perfusion periods of the isolated hearts (Fig. 5C).

Cardioprotective Effect of Postischemic Effluent

To further elucidate the pathophysiological role of these negative inotropic substance(s), we increased extracellular Ca2+ concentration to simulate Ca2+ overload. Cardiac ischemia-reperfusion injury is associated with Ca2+ overload in the cardiomyocytes (15, 19, 43, 47). Ca2+ overload is limited by ischemic preconditioning (43, 47). As shown in Fig. 3, postischemic effluent significantly reduced intracellular diastolic and systolic Ca2+ increase, whereas incubation of cells with the nonischemic effluent (control) had no influence on intracellular diastolic and systolic Ca2+ concentration. Accordingly, from a teleological point of view, we speculate that one important function of the negative inotropic substance(s) is to protect cardiomyocytes against Ca2+ overload after ischemia during reperfusion.

The negative inotropic effect of postischemic effluent is antagonized by glibenclamide, which is an antidiabetic sulfonylurease and an inhibitor of sarcolemmal and mitochondrial potassium (ATP) channels. This finding suggests that potassium (ATP) channels play a role in the negative inotropic effect of postischemic effluent. The opening of potassium (ATP) channels occurs during the repolarization phase of the action potential. The efflux of potassium ions returns the cells to the resting potential. In cardiac tissue, the opening of potassium (ATP) channels by various potassium channel openers has a negative inotropic effect on the cells (25, 28, 49). Glibenclamide specifically antagonizes the negative inotropic effects of potassium channel openers in cardiac tissue (36). Under ischemic conditions, potassium (ATP) channels are opened, which in turn elicits a decrease in action potential duration (12). Various studies show that the potassium (ATP) channel is influenced by substances released from COX metabolism. PGE2 has an activating effect on potassium (ATP) channels through E-prostanoid (EP) receptor type 2 (EP2) in cultured interstitial cells of Cajal from murine small intestine (7). Aimond et al. (2) describe a group of potassium (ATP) channels in the hearts of mice and rats that are regulated by intracellular acidose, free fatty acids, and arachidonic acids. Other studies provide evidence that potassium (ATP) channels are opened by EETs (26).

The opening of potassium (ATP) channels during ischemia likewise has a cardioprotective effect and is associated with ischemic preconditioning (9). The protective effects persist into the reperfusion phase following prolonged ischemia (17). Substances released from arachidonic acid metabolism show significant cardioprotective effects in several studies (29, 35). In a recent canine study, 11,12-EET and 14,15-EET demonstrate significant cardioprotective effect via activation of cardiac potassium (ATP) channels (30). It has been shown that prostacyclin attenuates myocardial ischemia-reperfusion injury via EP3 prostaglandin receptors by the opening of potassium (ATP) channels (39). Our findings suggest that the active form of the main mediator in postischemic effluent that is obviously metabolized by COX-2 has an activating effect on potassium (ATP) channels, which leads to a decrease in systolic cell shortening and in Ca2+ transients and therefore has a negative inotropic effect. Cellular mediation perhaps takes place in this context, by means of binding to EP receptors.

Study Limitation

Despite our findings, the identity remains unknown of the mediators that influence Ca2+ metabolism and the contractility of cardiomyocytes. The results of the present study suggest that the active form of the main mediator of postischemic effluent is synthesized by COX-2.

Various stress forms induce enhanced expression of COX-2, e.g., growth factors, cytokines, endotoxins, and mechanical stress (21, 31, 32). The fact that we detected COX-2 expression by Western blot analysis in the freshly isolated cardiomyocytes of our study is most likely due to the isolation and preparation process of cells for fluorescence microscopy. A reperfusion period of rat hearts after ischemia leads to intensified COX-2 expression in postischemic and nonischemic (control) hearts, since COX-2 is nonspecifically induced, most probably due to myocardial stress (e.g., myocardial edema formation) caused by perfusion in the Langendorff system.

Further experiments are necessary to clarify whether the main mediator in the postischemic effluent mediates its effect in the cell by binding to EP receptors.

Conclusion

The present study suggests that negative inotropic substance(s) are released from isolated hearts after ischemia during reperfusion and are metabolized to their active form via a COX-2-dependent metabolic pathway. These substances induce a decrease in Ca2+ transients via the opening of potassium (ATP) channels and protect cardiomyocytes against Ca2+ overload.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by the Department of Cardiovascular Medicine at the University of Greifswald.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Felix or A. Staudt, Klinik für Innere Medizin B, Friedrich-Loefflerstr, 23 a, 17475 Greifswald, Germany (e-mail: felix{at}uni-greifswald.de or staudt{at}uni-greifswald.de, respectively)

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.

* K. Birkenmeier and A. Staudt contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase. J Biol Chem 274: 5038–5046, 1999.[Abstract/Free Full Text]
  2. Aimond F, Rauzier JM, Bony C, Vassort G. Simultaneous activation of p38 MAPK and p42/44 MAPK by ATP stimulates the K+ current ITREK in cardiomyocytes. J Biol Chem 275: 39110–39116, 2000.[Abstract/Free Full Text]
  3. Bezugla Y, Kolada A, Kamionka S, Bernard B, Scheibe R, Dieter P. COX-1 and COX-2 contribute differentially to the LPS-induced release of PGE2 and TxA2 in liver macrophages. Prostaglandins Other Lipid Mediat 79: 93–100, 2005.[Web of Science][Medline]
  4. Bolli R, Shinmura K, Tang X, Kodani E, Xuan Y, Guo Y, Dawn B. Discovery of a new function of cyclooxygenase (COX)-2: COX-2 is a cardioprotective protein that alleviates ischemia/reperfusion injury and mediates the late phase of preconditioning. Cardiovasc Res 55: 506–519, 2002.[Abstract/Free Full Text]
  5. Camitta MG, Gabel SA, Chulada P, Bradbury JA, Langenbach R, Zeldin DC. Cyclooxygenase-1 and -2 knockout mice demonstrate increased cardiac ischemia/reperfusion injury but are protected by acute preconditioning. Circulation 104: 2453–2458, 2001.[Abstract/Free Full Text]
  6. Chen J, Capdevila JH, Zeldin DC, Rosenberg RL. Inhibition of cardiac L-type calcium channels by epoxyeicosatrienoic acids. Mol Pharmacol 55: 288–295, 1999.[Abstract/Free Full Text]
  7. Choi S, Yeum OH, Chang IY, You HJ, Park JS, Jeong HS, So I, Kim KW, Jun JY. Activating of ATP-dependent K+ channels comprised of Kir 6.2 and SUR 2B by PGE2 through EP2 receptor in cultured interstitial cells of Cajal from murine small intestine. Cell Physiol Biochem 18: 187–198, 2006.[CrossRef][Web of Science][Medline]
  8. Coetzee M, Haag M, Claassen N, Kruger MC. Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3—E1 osteoblast-like cells. Prostaglandins Leukot Essent Fatty Acids 73: 423–430, 2005.[CrossRef][Web of Science][Medline]
  9. Dos Santos P, Kawaltowski AJ, Laclau MN, Seetharamanau S, Paucek P, Boudina S, Thambo JB, Tariosse L, Garlid KD. Mechanisms by which opening the mitochondrial ATP-sensitive K+ channel protects the ischemic heart. Am J Physiol Heart Circ Physiol 283: H284–H295, 2002.[Abstract/Free Full Text]
  10. Dupouy VM, Ferre PJ, Uro-Coste E, Lefebvre HP. Time course of COX-1 and COX-2 expression during ischemia-reperfusion in rat skeletal muscle. J Appl Physiol 100: 233–239, 2006.[Abstract/Free Full Text]
  11. Esser R, Berry C, Zhengming D, Dawson J, Fox A, Fujimoto RA, Haston W, Kimble EF, Koehler J, Peppard J, Quadros E, Quintavalla J, Toscana K, Urban L, Dazer J, Zhang X, Zhan S, Marshall PJ. Preclinical pharmacology of lumiracoxib: a novel selective inhibitor of cyclooxygenase-2. Br J Pharmacol 144: 538–550, 2005.[CrossRef][Web of Science][Medline]
  12. Faivre JF, Findlay I. Action potential duration and activation of ATP-sensitive potassium current in isolated guinea-pig ventricular myocytes. Biochim Biophys Acta 1029: 167–172, 1990.[Medline]
  13. Felix SB, Stangl V, Frank TM, Harms C, Staudt A, Berndt T, Kastner R, Baumann G. Release of a stable cardiodepressant mediator after myocardial ischemia during reperfusion. Cardiovasc Res 35: 68–79, 1997.[Abstract/Free Full Text]
  14. Felix SB, Stangl V, Pietsch P, Bramlage P, Staudt A, Bartel S, Krause EG, Borschke JU, Wernecke KD, Isenberg G, Baumann G. Soluble substances released from postischemic reperfused rat hearts reduce calcium transient and contractility by blocking the L-type calcium channel. J Am Coll Cardiol 37: 668–675, 2001.[Abstract/Free Full Text]
  15. Ferrari R. The role of mitochondria in ischemic heart disease. J Cardiovasc Pharmacol 28: 1–10, 1996.[CrossRef][Web of Science][Medline]
  16. Fornai M, Blandizzi C, Antonioli L, Coluzzi R, Bernardini N, Segnani C, De Ponti F, Del Tacca M. Differential role of cyclooxygenase 1 and 2 isoformes in the modulation of colonic neuromuscular function in experimental inflammation. J Pharmacol Exp Ther 317: 938–945, 2006.[Abstract/Free Full Text]
  17. Fryer RM, Hsu AK, Gross GJ. Mitochondrial KATP channel opening is important during index ischemia and following myocardial reperfusion in ischemic preconditional rat hearts. J Mol Cell Cardiol 33: 831–834, 2001.[CrossRef][Web of Science][Medline]
  18. Futaki N, Takahashi S, Yokoyama M, Arai I, Higuchi S, Otomo S. NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro. Prostaglandins 47: 55–59, 1994.[CrossRef][Web of Science][Medline]
  19. Halestrap AP. Calcium, mitochondria and reperfusion injury: a pore way to die. Biochem Soc Trans 34: 232–237, 2006.[CrossRef][Web of Science][Medline]
  20. Hiraoka M. Pathophysiological functions of ATP-sensitive K+ channels in myocardial ischemia. Jpn Heart J 38: 297–315, 1997.[Medline]
  21. Ishikawa T, Morris PL. Interleukin-1beta signals through a c-Jun N-terminal kinase-dependent inducible nitric oxide synthase and nitric oxide production pathway in Sertoli epithelial cells. Endocrinology 147: 5424–5430, 2006.[Abstract/Free Full Text]
  22. Kishimoto Y, Yashima K, Morisawa T, Shiota G, Kawasaki H, Hasegawa J. Effects of cyclooxygenase-2 inhibitor NS-398 on APC and c-myc expression in rat. Colon carcinogenesis is induced by azoxymethane. J Gastroenterol 37: 186–193, 2002.[CrossRef][Web of Science][Medline]
  23. Kodani E, Shinmura K, Xuan YT, Takano H, Auchampach JA, Tang XL, Bolli R. Cyclooxygenase-2 does not mediate late preconditioning induced by activation of adenosine A1 or A3 receptors. Am J Physiol Heart Circ Physiol 281: H959–H968, 2001.[Abstract/Free Full Text]
  24. Laneuville O, Breuer DK, Dewitt DL, Hla T, Funk CD, Smith WL. Differential inhibition of human prostaglandin endoperoxide H synthase-1 and -2 by nonsteroidal anti-inflammatory drugs. J Pharmacol Exp Ther 271: 927–934, 1994.[Abstract/Free Full Text]
  25. Lau WM. Effects of potassium channel blockers on the negative inotropic responses induced by cromakalim and pinacidil in guinea pig heart. Pharmacology 45: 9–16, 1992.[Medline]
  26. Lu T, Hoshi T, Weintraub NL, Spector AA, Lee HC. Activation of ATP-sensitive K+ channels by epoxyeicosatrienoic acids in rat cardiac ventricular myocytes. J Physiol 537: 811–827, 2001.[Abstract/Free Full Text]
  27. Mobert J, Becker BF. Cyclooxygenase inhibition aggravates ischemia-reperfusion injury in the perfused guinea pig heart: involvement of isoprostanes. J Am Coll Cardiol 3: 1687–1694, 1998.
  28. Muller-Ehmsen J, Brixius K, Hoischen S, Schwinger RH. Inotropic and coronary vasodilatory actions of the K-adenosine triphosphate channel modulator nicorandil in human tissue. J Pharmacol Exp Ther 279: 1220–1228, 1996.[Abstract/Free Full Text]
  29. Nithipatikom K, DiCamelli RF, Kohler S, Gumina RJ, Falck JR, Campbell WB, Gross GJ. Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs. Anal Biochem 292: 115–124, 2001.[CrossRef][Web of Science][Medline]
  30. Nithipatikom K, Moore JM, Isbell MA, Falck JR, Gross GJ. Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury. Am J Physiol Heart Circ Physiol 291: H537–H542, 2006.[Abstract/Free Full Text]
  31. Norvell SM, Ponik SM, Bowen DK, Gerard R, Pavalko FM. Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules. J Appl Physiol 96: 957–966, 2004.[Abstract/Free Full Text]
  32. Oh PS, Lee SJ, Lim KT. Glycoprotein isolated from Rhus verniciflua stokes inhibits inflammation-related protein and nitric oxide production in LPS-stimulated RAW264.7 cells. Biol Pharm Bull 30: 111–116, 2007.[CrossRef][Web of Science][Medline]
  33. Oshima M, Oshima H, Taketo MM. Hypergravity induces expression of cyclooxygenase-2 in the heart vessels. Biochem Biophys Res Commun 330: 928–933, 2005.[CrossRef][Web of Science][Medline]
  34. Rebsamen MC, Capoccia R, Vallotton MB, Lang U. Role of cyclooxygenase 2, p38 and p42/44 MAPK in the secretion of prostacyclin induced by epidermal growth factor, endothelin-1 and angiotensin II in rat ventricular cardiomyocytes. J Mol Cell Cardiol 35: 81–89, 2003.[CrossRef][Web of Science][Medline]
  35. Rosolowsky M, Falck JR, Willerson JT, Campbell WB. Synthesis of lipoxygenase and epoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries. Circ Res 66: 608–621, 1990.[Abstract/Free Full Text]
  36. Satoh E, Yanagisawa T, Taira N. Effects of potassium channels blockers on the negative inotropic responses induced by cromakalim and pinacidil guinea pig atrium. Pharmacology 45: 9–16, 1992.[Medline]
  37. Schrör K. Eicosanoids and myocardial ischemia. Basic Res Cardiol 82: 235–243, 1987.[Web of Science][Medline]
  38. Shibata R, Sato K, Pimentel DR, Takemura Y, Kihara S, Ohashi K, Funahashi T, Ouchi N, Walsh K. Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms. Nat Med 11: 1096–1103, 2005.[CrossRef][Web of Science][Medline]
  39. Shinmura K, Tamaki K, Sato T, Ishida H, Bolli R. Prostacyclin attenuates oxidative damage of myocytes by opening mitochondrial ATP-sensitive K+ channels via EP3 receptor. Am J Physiol Heart Circ Physiol 288: H2093–H2101, 2005.[Abstract/Free Full Text]
  40. Shinmura K, Tang XL, Wang Y, Xuan YT, Liu SQ, Bhatnagar A, Bolli R. Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits. Proc Natl Acad Sci USA 97: 10197–10202, 2000.[Abstract/Free Full Text]
  41. Smith CJ. Pharmacological analysis of cyclooxygenase-1 in inflammation. Proc Natl Acad Sci USA 95: 13313–13318, 1998.[Abstract/Free Full Text]
  42. Stangl V, Felix SB, Meyer R, Berndt T, Kastner R, Wernecke KD, Baumann G. Cardiodepressive mediators are released after ischemia from an isolated heart: role of coronary endothelial cells. J Am Coll Cardiol 29: 1390–1396, 1997.[Abstract]
  43. Szenczi O, Kemecsei P, Miklos Z, Ligeti L, Snoeckx LH, Van Riel NA, Op den Buijs J, Van der Vusse GJ, Ivancis T. In vivo heat shock preconditioning mitigates calcium overload during ischemia/reperfusion in the isolated, perfused rat heart. Pflügers Arch 449: 518–525, 2005.[CrossRef][Web of Science][Medline]
  44. Tacconelli S, Capone ML, Patrignani P. Clinical pharmacology of novel selective COX-2 inhibitors. Curr Pharm Des 10: 589–601, 2004.[CrossRef][Web of Science][Medline]
  45. Tripp CS, Blomme EA, Chinn KS, Hardy MM, La Celle P, Pentland AP. Epidermal COX-2 induction following ultraviolet irradiation: suggested mechanism for the role of COX-2 inhibition. J Invest Dermatol 121: 853–861, 2003.[CrossRef][Web of Science][Medline]
  46. Uemura Y, Kobayashi M, Nakata H, Kubota T, Saito T, Bandobashi K, Taguchi H. Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on lung cancer: roles of cyclooxygenase-2. Oncol Rep 17: 955–961, 2007.[Web of Science][Medline]
  47. Wang L, Cherednichenko G, Hernandez L, Halow J, Camacho SA, Fiueredo V, Schaefer S. Preconditioning limits mitochondrial Ca2+ during ischemia in rat hearts: role of KATP channels. Am J Physiol Heart Circ Physiol 280: H2321–H2328, 2001.[Abstract/Free Full Text]
  48. Wang Y, Kodani E, Wang J, Zhang SX, Takano H, Tang XL, Bolli R. Cardioprotection during final stage of the late phase of ischemic preconditioning is mediated by neuronal NO synthase in concert with cyclooxygenase-2. Circ Res 95: 84–91, 2004.[Abstract/Free Full Text]
  49. Yanagisawa T, Hashimoto H, Taira N. The negative inotropic effect of nicorandil is independent of cyclic GMP changes: a comparison with pinacidil and cromakalim in canine atrial muscle. Br J Pharmacol 95: 393–398, 1988.[Web of Science][Medline]




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