Pentoxifylline attenuates cardiac dysfunction and reduces TNF- level ...

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Apr 15, 2005 - Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Canada. Submitted 22 February 2005; accepted in final ...
Am J Physiol Heart Circ Physiol 289: H832–H839, 2005. First published April 15, 2005; doi:10.1152/ajpheart.00178.2005.

Pentoxifylline attenuates cardiac dysfunction and reduces TNF-␣ level in ischemic-reperfused heart Ming Zhang,1 Yan-Jun Xu,1 Harjot K. Saini,1 Belma Turan,1 Peter P. Liu,2 and Naranjan S. Dhalla1 1

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Center, Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg; and 2Division of Cardiology, Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Canada Submitted 22 February 2005; accepted in final form 12 April 2005

Zhang, Ming, Yan-Jun Xu, Harjot K. Saini, Belma Turan, Peter P. Liu, and Naranjan S. Dhalla. Pentoxifylline attenuates cardiac dysfunction and reduces TNF-␣ level in ischemic-reperfused heart. Am J Physiol Heart Circ Physiol 289: H832–H839, 2005. First published April 15, 2005; doi:10.1152/ajpheart.00178.2005.—Although pentoxifylline (PTXF), a phosphodiesterase inhibitor, has been reported to exert beneficial effects in cardiac bypass surgery, its effect and mechanisms against ischemia-reperfusion (I/R) injury in heart are poorly understood. Because I/R is known to increase the level of tumor necrosis factor (TNF)-␣ in myocardium and PTXF has been shown to depress the production of TNF-␣ in failing heart, this study examined the hypothesis that PTXF may attenuate cardiac dysfunction and reduce TNF-␣ content in I/R heart. For this purpose, isolated rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 2–30 min. Although cardiac dysfunction due to ischemia was not affected, the recovery of heart function upon reperfusion was markedly improved by PTXF treatment. This cardioprotective effect of PTXF was dose dependent; maximal effect was seen at a concentration of 125 ␮M. TNF-␣, nuclear factor-␬B (NF-␬B), and phosphorylated NF-␬B contents were decreased in ischemic heart but were markedly increased within 2 min of starting reperfusion. The ratio of cytosolic-to-homogenate NF-␬B was decreased, whereas the ratio of particulate-to-homogenate NF-␬B was increased in I/R hearts. These changes in TNF-␣ and NF-␬B protein contents as well as in NF-␬B redistribution due to I/R were significantly attenuated by PTXF treatment. The results of this study indicate that the cardioprotective effects of PTXF against I/R injury may be due to reductions in the activation of NF-␬B and the production of TNF-␣ content. tumor necrosis factor; nuclear factor-␬B; heart failure; cardiomyopathy; ischemia; reperfusion

of mortality due to cardiac bypass and open-heart surgery, ischemia-reperfusion (I/R) injury has received a great deal of attention in recent years (5, 11). It has been shown that multiple factors including intracellular Ca2⫹ overload and oxidative stress are involved in the I/R injury that leads to myocardial dysfunction and cell death (2, 10). Recent studies have indicated that I/R increases the production and release of proinflammatory cytokines such as tumor necrosis factor (TNF)-␣, which play a key role in causing contractile dysfunction (4, 12), apoptosis (18), and remodeling in heart (25). Therefore, reducing the production of TNF-␣ can be seen as an efficient therapeutic strategy to prevent I/R-induced injury in heart. IN VIEW OF THE HIGH DEGREE

Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (E-mail: [email protected]). H832

Pentoxifylline (PTXF), a phosphodiesterase inhibitor, has been reported to depress the production of TNF-␣ and produce beneficial effects in heart failure and idiopathic dilated cardiomyopathy (31, 32). Furthermore, PTXF was found to reduce TNF-␣ content and improve cardiac function in hearts subjected to Ca2⫹ depletion and repletion (43). Although PTXF has also been shown to exert beneficial effects in cardiopulmonary bypass and open-heart surgery due to its potent hemorrheological properties (13, 36, 37), it is not clear whether PTXF protects heart from I/R injury. The present study was therefore undertaken to examine whether PTXF prevents cardiac dysfunction due to I/R in isolated heart preparations. Experiments were also carried out to test whether the beneficial effects of PTXF are associated with reduction in TNF-␣ content in I/R hearts. Because nuclear factor-␬B (NF-␬B) is intimately involved in the synthesis of TNF-␣ (3), changes in NF-␬B protein content and distribution in untreated and PTXFtreated I/R hearts were investigated. METHODS

Perfusion of isolated rat hearts. The experimental protocols in this study were approved by the University of Manitoba Animal Care Committee according to the guidelines of the Canadian Council on Animal Care. The perfusion procedure for the isolated rat heart using the Langendorff technique was similar to that employed in previous studies from our laboratory (26, 35). Each heart was stabilized with perfusion at a constant flow rate of 10 ml/min for 20 min with normal Krebs-Henseleit (K-H) buffer that contained (in mM) 120 NaCl, 4.8 KCl, 1.35 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11 glucose (pH 7.4, 37°C). The perfusion solution was gassed continuously with a 95% O2-5% CO2 mixture. Hearts were stimulated electrically at 300 beats/min using a model 611 stimulator (Phipps & Bird; Richmond, VA). A water-filled plastic balloon was inserted into the left ventricle, and the left ventricular end-diastolic pressure (LVEDP) was adjusted to 9 –10 mmHg at the beginning of the experiment. The left ventricular developed pressure (LVDP), LVEDP, rate of pressure development (⫹dP/dt), and rate of pressure decay (⫺dP/dt) were measured using AcqKnowledge 3.5 for Windows 3.0 (Biopac Systems; Goleta, CA). Data were recorded online through an analog-digital interface (model MP100; Biopac Systems). Experimental protocol for I/R. For the control group, stabilized hearts were perfused with K-H buffer for 60 min, whereas for the I/R group, the control hearts underwent 30 min of global ischemia followed by 30 min of reperfusion with normal K-H medium. For the PTXF-treatment group, PTXF was given for 10 min before ischemia was induced after the stabilization period as well as during the 30-min reperfusion period. Different PTXF concentrations (50, 100, 125, and

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150 ␮M) were used in this study; the drug was infused into the perfusion medium via a sidearm close to the cannula. For the postischemia-treatment group, 100 ␮M PTXF was given after 30 min of ischemia until the end of reperfusion. Left ventricular function was measured by recording LVDP, LVEDP, ⫹dP/dt, and ⫺dP/dt. Measurement of TNF-␣. Ventricular tissues from control and ischemia as well as 2-, 5-, 10-, and 30-min reperfusion groups with or without PTXF treatment were homogenized in 10 volumes of phosphate-buffered saline that contained 1% Triton X-100 and a protease inhibitor cocktail (Roche; Laval, Quebec, Canada; Ref. 4). The homogenate was centrifuged at 2,500 g for 20 min at 4°C. The supernatant was collected, and the protein concentration was estimated using a modified Bradford method (39). TNF-␣ content was measured using a sandwich ELISA kit for rat TNF-␣ with a 12.5 pg/ml detection limit (R&D Systems; Minneapolis, MN). As indicated previously (43), the assay was performed according to the manufacturer’s instructions. Absorbance of standards and samples was determined spectrophotometrically (SpectraMax Plus384; Molecular Devices; Sunnyvale, CA) at 450 nm. Results were calculated from the standard curve and are reported as picograms per gram of protein. Preparation of tissue extract for NF-␬B determination. Ventricular tissue (50 mg) was homogenated at setting 8 twice for 30 s each (Polytron PT 3000; Brinkmann Instruments; Mississauga, Ontario, Canada) in 1 ml of buffer A, which contained 50 mM Tris 䡠 HCl, 0.25 M sucrose, 10 mM EGTA, 4 mM EDTA, pH 7.5, and protease inhibitor cocktail (Roche). The suspension was sonicated twice for 15 s each and centrifuged at 100,000 g for 60 min in an ultracentrifuge (model L70; Beckman Instruments; Fullerton, CA), and the supernatant was collected as the cytosolic fraction. The pellet was resuspended in 1 ml of buffer B (buffer A plus 1% Triton X-100), incubated on ice for 60 min, and then centrifuged at 100,000 g for 60 min. It is pointed out that both buffer A and buffer B had no phosphatase inhibitors. This supernatant, which contained dissolved membrane protein, was labeled as the particulate fraction. Another piece (50 mg) of ventricular tissue was suspended in buffer B after homogenization (twice for 30 s each) and sonication (twice for 15 s each). The homogenate was incubated on ice for 60 min and then centrifuged at 100,000 g for 60 min. This supernatant was labeled as the homogenate fraction. The protein content was determined using a modified Bradford method, and bovine serum albumin was used as the standard. This method for studying subcellular distribution of an enzyme or protein is the same as was described earlier (39). Analysis of NF-␬B protein content. The analysis of total and phosphorylated NF-␬B (phospho-NF-␬B) was performed by separation of 20 ␮g of protein from homogenate, cytosolic, and particulate fractions from left ventricular tissue on 10% SDS-PAGE and was followed by immunostaining via Western blot assay. The concentration of protein in these samples was adjusted to 1 mg/ml with buffer A or buffer B and SDS-PAGE loading buffer that contained 250 mM Tris 䡠 HCl (pH 6.8), 8% (wt/vol) SDS, 40% glycerol, 200 mM ␤-mercaptoethanol, and 0.4% (wt/vol) bromophenol. Prestained protein marker (Invitrogen Life Technologies; Carlsbad, CA) served as a marker. After electrophoresis, the proteins separated by SDS-PAGE were electroblotted to polyvinylidene difluoride membranes. The membranes were then probed with primary polyclonal antibody against p65 component or phospho-NF-␬B polyclonal antibody at Ser536 (1:1,000 dilution; Cell Signaling Technology; New England BioLabs; Ontario, Canada) and with secondary antibody (1:10,000 dilution of goat anti-rabbit IgG-horseradish peroxidase conjugate). Antigen-antibody complexes were detected using an ECL Plus kit (Amersham-Pharmacia Biotech; Baie d’Urfe, Quebec, Canada). An imaging densitometer (model GS-800; Bio-Rad; Mississauga, Ontario, Canada) was used to scan the protein bands, which were quantified using Quantity One 1-D image-analysis software (BioRad). Protein loading was checked in each experiment by staining the membranes with Ponceau S stain before immunoblotting occurred (3). AJP-Heart Circ Physiol • VOL

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Statistical analysis. The data were expressed as means ⫾ SE. Differences between the control and experimental groups were analyzed using an unpaired Student’s t-test. The data from more than two groups were evaluated by one-way ANOVA followed by the Newman-Keuls test. A P level of ⬍0.05 was considered as the threshold for statistical significance. RESULTS

I/R-mediated alterations of cardiac function. Ischemia for a period of 30 min followed by 30 min of reperfusion caused a marked depression of cardiac function. Figure 1 shows alterations in left ventricular pressures as well as pressure development and decay. There was a 9-fold decrease in LVDP and a 30-fold increase in LVEDP after 30 min of reperfusion. A 10-fold decrease in both ⫹dP/dt and ⫺dP/dt in the I/R group was also observed (Table 1). The data in Fig. 2 and Table 1 also indicate that treatment with PTXF (100 ␮M) 10 min before ischemia and 30 min during the reperfusion period improved cardiac function in the I/R heart; there was ⬃75% recovery of LVDP and ⬃60% recovery of both ⫹dP/dt and ⫺dP/dt. Although a significant decrease in LVEDP was seen after PTXF treatment, LVEDP still remained 10-fold higher compared with the pre-I/R value. The dose responses for the beneficial effects of PTXF on cardiac function against I/R injury showed that 50 ␮M PTXF did not exert any significant action, whereas maximal effects were seen at 125 ␮M PTXF (Fig. 2). Effects of postischemia treatment with PTXF on I/R-induced injury. To investigate the effects of postischemia treatment with PTXF in I/R heart, PTXF (100 ␮M) was given after 30 min of ischemia. Table 2 shows that PTXF improved cardiac performance compared with the untreated I/R group; there was a 3.3-fold increase in LVDP as well as 7.8- and 5.5-fold increases in ⫹dP/dt and ⫺dP/dt, respectively. On the other hand, LVEDP was found to be 2.6-fold lower than that in the I/R group. Because the effect of postischemic treatment with PTXF on the recovery of LVDP was significantly lower than that for preischemic treatment (Tables 1 and 2), all subsequent experiments were carried out by treating the hearts with PTXF for 10 min before starting global ischemia and then treating with PTXF during the reperfusion period. Myocardial TNF-␣ content in I/R-induced injury. Myocardial TNF-␣ contents in ischemic and I/R ventricular tissues are shown in Fig. 3. TNF-␣ levels were decreased in ischemic heart; however, this change was not affected by treatment with PTXF (100 ␮M). On the other hand, a significant increase in TNF-␣ content was detected (from 409 ⫾ 32 to 906 ⫾ 92 pg/g protein) at 2 min of reperfusion, but thereafter, TNF-␣ levels decreased toward the control level. Treatment of hearts with PTXF attenuated the initial increase in TNF-␣ content due to reperfusion (at 2 and 5 min); however, TNF-␣ content in the untreated hearts and the PTXF-treated group remained at control levels at 10 and 30 min of reperfusion and were not different (P ⬎ 0.05) from one another (Fig. 3). It should be pointed out that TNF-␣ levels indicated at each time point of I/R may not represent the true level of TNF-␣, because this cytokine has been reported to be released in the coronary effluent during reperfusion from I/R hearts (4, 23). Because TNF-␣ levels were not significantly different from control and 30 min I/R hearts, no attempt was made to test the effect of PTXF during the reperfusion phase.

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Fig. 1. Original tracings of recordings from heart under ischemia-reperfusion (I/R) without (A) and with (B) pentoxifylline (PTXF) treatment. Effects of PTXF on the alterations of left ventricular developed pressure (LVDP; C) and left ventricular end-diastolic pressure (LVEDP; D) at different time points during I/R are shown. Values are means ⫾ SE of 6 separate experiments in each group. *P ⬍ 0.05 vs. I/R group.

Myocardial NF-␬B protein content in I/R. Figure 4 shows the representative Western blots for NF-␬B and the analysis of protein contents of NF-␬B in homogenate, cytosolic, and particulate fractions. It was observed that the relative protein

contents of NF-␬B in these fractions of I/R hearts were decreased by varying degrees; the largest decrease was evident in the cytosolic fraction, whereas the smallest decrease was seen in the particulate fraction. Furthermore, the ratio of cytosolic-

Table 1. Effects of PTXF on cardiac performance in I/R heart

Group

LV Developed Pressure, mmHg

LV EndDiastolic Pressure, mmHg

⫹dP/dt, mmHg/s

⫺dP/dt, mmHg/s

Control Control with PTXF Ischemia Ischemia with PTXF Ischemia reperfusion Ischemia reperfusion with PTXF

118⫾5.6 103⫾9.7 3.1⫾0.5* 2.2⫾0.8* 15.3⫾2.9* 75.6⫾6.1†

9.0⫾0.8 9.7⫾0.5 48.6⫾1.7* 43.7⫾2.5* 92.3⫾4.8* 40.5⫾5.1†

6,618⫾268 5,883⫾786 107⫾16* 97⫾22* 568⫾150* 4,328⫾328†

4,024⫾203 3,608⫾284 118⫾17* 97⫾25* 397⫾78* 2,490⫾128†

Values are means ⫾ SE; n ⫽ 6 separate experiments/group. Data show alterations of left ventricular (LV) developed pressure and end-diastolic pressure and rates of pressure development and decay (⫹dP/dt and ⫺dP/dt, respectively). PTXF, pentoxifylline. *P ⬍ 0.05 vs. control group; †P ⬍ 0.05 vs. ischemia-reperfusion (I/R) group. AJP-Heart Circ Physiol • VOL

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Fig. 2. Dose responses of PTXF on cardiac performance under I/R. Hearts were exposed to PTXF at 0, 50, 100, 125, and 150 ␮M concentrations. Alterations of LVDP (A), rate of pressure development (⫹dP/dt; B), LVEDP (C), and rate of pressure decay (⫺dP/dt; D) at different doses are presented. *P ⬍ 0.05 vs. I/R group.

to-homogenate NF-␬B contents was decreased, whereas that for the particulate-to-homogenate contents was increased. PTXF treatment was found to attenuate the I/R-induced changes in homogenate and cytosolic NF-␬B content as well as in ratios for the cytosolic-to-homogenate and particulate-tohomogenate NF-␬B contents (Fig. 4). Preliminary experiments using two hearts at 2 and 30 min of reperfusion in the 30-min ischemic hearts also showed similar changes in the homogenate as well as the redistribution of NF-␬B in the cytosolic and particulate fractions. To investigate the activation of NF-␬B in the I/R hearts, the phospho- and total NF-␬B contents were measured in the homogenate. As can be seen from Fig. 5, 30 min of ischemia with or without PTXF treatment decreased the protein content significantly. However, at 2 min of reperfusion, the protein contents of phospho-NF-␬B as well as total NF-␬B were significantly increased; this increase was attenuated by PTXF treatment. In view of the maximal recovery of cardiac function in I/R hearts due to pretreatment with PTXF, no experiments were carried out to determine the effects of postischemic treatment with PTXF on NF-␬B translocation.

DISCUSSION

Depressed cardiac function as a consequence of I/R injury has been reported clinically (15) and experimentally (26, 35). Our results showing depression in cardiac function in I/R hearts are in agreement with previous reports (26, 35). Although depressed cardiac performance due to I/R is considered to be a consequence of intracellular Ca2⫹ overload and free radical generation (2, 10, 11), it has been suggested that the production of some cytokines, such as TNF-␣, may be an

Table 2. Effects of postischemia treatment with PTXF on cardiac performance in I/R heart

Control

LV developed pressure, mmHg LV end-diastolic pressure, mmHg ⫹dP/dt, mmHg/s ⫺dP/dt, mmHg/s

I/R

115⫾4.4 13.0⫾1.7* 9.4⫾0.9 95⫾5.1* 6,395⫾253 393⫾99* 3,943⫾221 322⫾67*

Postischemia Treatment With PTXF

43⫾14† 37⫾10† 2,979⫾778† 1,697⫾387†

Values are means ⫾ SE; n ⫽ 6 separate experiments/group. Data show alterations of LV developed and end-diastolic pressures and rates of pressure development and decay at 30 min of reperfusion. *P ⬍ 0.05 vs. control group; †P ⬍ 0.05 vs. I/R group. AJP-Heart Circ Physiol • VOL

Fig. 3. Tumor necrosis factor (TNF)-␣ protein levels in myocardium subjected to I/R after 30 min of ischemia and 2, 5, 10, and 30 min of reperfusion with or without PTXF treatment are shown. *P ⬍ 0.05 vs. control group; #P ⬍ 0.05 vs. I/R group without PTXF treatment.

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Fig. 4. Western blotting analysis shows alterations in protein content of nuclear factor (NF)-␬B in homogenate (A), cytosolic (B), and particulate (C) fractions of I/R heart with or without PTXF (100 ␮M) treatment. Ratios of protein content of NF-␬B in cystolic-tohomogenate (D) and particulate-to-homogenate (E) fractions are shown. *P ⬍ 0.05 vs. control group; #P ⬍ 0.05 vs. I/R group.

important factor involved in I/R injury (4, 23). On the other hand, different investigators have demonstrated that TNF-␣ may produce cardioprotection by enhancing myocardial tolerance to ischemia (27, 33, 34, 44). Such conflicting reports regarding the role of TNF-␣ in I/R are probably dependent on the absolute levels of TNF-␣ during the I/R period, because high levels of this cytokine may produce deleterious alterations caused by I/R and thereby decrease contractile function and induce apoptosis (23, 28). Other studies have demonstrated that TNF-␣ may serve as an initiator for the production of cardiodepressant cytokines such as IL-1, -2, and -6 (28). In this regard, it has been demonstrated that as TNF-␣ was being washed out from coronary vessels, the newly produced cytokines further reduced the peak systolic Ca2⫹ transients as well as the contractility by increasing both the production of NO and the subsequent cGMP-mediated decrease in L-type Ca2⫹ channel currents (15, 22, 24, 27). Consistent with a previous report by Gurevitch et al. (14), our results demonstrated that TNF-␣ levels were elevated during 2–5 min of reperfusion. Such an increase in TNF-␣ may trigger the synthesis of other deleterious cytokines in heart (38) and thus may worsen I/R-induced myocardium injury. The decrease in TNF-␣ levels in the I/R heart after 2 min of reperfusion may be due to coronary washing out as a result of cell necrosis and lesions in the cell membrane. This view is in agreement with others who have reported a marked increase in creatine kinases and other proteins in the perfusate during the reperfusion period (14). Because TNF-␣ is known to depress contractile function in isolated hamster, rat, and human myocardia (4, 12, 27), it is possible that the observed increase in TNF-␣ protein level at 2 min of reperfusion may play an important role in the depression of cardiac function in I/R heart. AJP-Heart Circ Physiol • VOL

PTXF is considered to produce beneficial effects in heart failure as well as in idiopathic dilated cardiomyopathy due to its inhibitory effect on TNF-␣ synthesis (31, 32). Furthermore, it has been documented that PTXF protected heart and lungs during cardiopulmonary bypass surgery and protected the skeletal muscle from ischemia (17). PTXF has also been reported to exhibit hemorrheological properties as it improved red blood cell deformability, decreased red blood cell aggregation, and inhibited neutrophil adhesion (7). Treatment with PTXF in vivo was shown to render protection against cardiac dysfunction and provide an overall increase in survival (41). In the present study, treatment with PTXF protected the heart under in vitro conditions from I/R injury, because it improved cardiac performance compared with the I/R group. These results are consistent with a previous study (19) that showed similar effects of PTXF against acute myocardial infarction. Inhibition of phosphodiesterase by PTXF has been reported to increase the contractile force in isolated atrial muscle in dogs (42). However, in isolated rat heart, perfusion with PTXF produced no changes in the left ventricular volume and LVDP relationship (40). Similarly in our study, perfusion of hearts with PTXF resulted in no changes in cardiac performance. Such different responses to PTXF may be due to selection of species and tissue type. Nonetheless, the possibility of postischemic improvement in cardiac function due to the positive inotropic effect of PTXF treatment cannot be ruled out. Because PTXF treatment depressed TNF-␣ synthesis at 2 min of reperfusion, it can also be assumed that PTXF depressed the occurrence of the first peak of TNF-␣ elevation and in turn may have blocked the direct deleterious effect of TNF-␣ in addition to blocking the synthesis of other cytokines (38). From the present study, we have also found that TNF-␣ protein levels were maintained

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Fig. 5. Western blot analysis shows the alterations in protein content of phosphorylated (phospho)-NF-␬B (A) and total NF-␬B (B) in homogenate fraction of ischemic (I) and I/R hearts with or without PTXF (100 ␮M) treatment. Representative bands (A and B, top) indicate the analysis of the relative protein contents of phospho- and total NF-␬B in the homogenate fraction. Values are means ⫾ SE of 6 hearts/group. *P ⬍ 0.05 vs. control group; #P ⬍ 0.05 vs. I/R group.

in the control and PTXF-treatment groups when TNF-␣ might have been washed out from I/R heart due to cell necrosis and membrane lesions. Because the previous study (6) has shown that PTXF reduces capillary membrane injury and subsequent protein leakage, it is likely that PTXF treatment might block TNF-␣ washout after 2 min of reperfusion. A slight but insignificant increase in the level of TNF-␣ in PTXF-treated hearts at 10 and 30 min of reperfusion may be due to such an effort of this agent on membrane permeability. It can be argued that the difference in TNF-␣ level in early reperfusion that was observed between I/R and PTXF-treated groups may be due to more rapid washout of this cytokine in PTXF-treated groups. However, this may not be the case; in the present study, the flow rate was kept constant for both treated and untreated groups during the perfusion period. Nonetheless, we have not measured the TNF-␣ level in the perfusate, and thus no conclusive statement can be made in this regard. In addition, it is pointed out that PTXF may also have some TNF-␣-independent effect in I/R heart injury after 10 and 30 min of reperfusion, because an improvement in cardiac performance was observed in the treated group in contrast with the untreated I/R AJP-Heart Circ Physiol • VOL

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group despite no significant difference in TNF-␣ level. PTXFmediated oxygenation of ischemic areas and lowering of the amount of metabolic derangements associated with I/R injury (41) may be the mechanism for such an effect. Because NF-␬B inhibition is generally associated with depression of proinflammatory factors, gene expression, and cell apoptosis (21), it is possible that the activation of NF-␬B may be implicated in I/R-induced heart injury. It was reported (8) that PTXF blocked the translocation of NF-␬B in vascular smooth muscle cells. In the present study, we also obtained a similar effect of PTXF on the activation of NF-␬B in I/R heart. Our data show marked alterations of NF-␬B protein levels in the cytosolic and particulate fractions in myocardium subjected to I/R injury. In view of the fact that NF-␬B is activated in response to diverse stimuli and then translocated to the nucleus and bound to a promoter that leads the gene expression of proinflammatory cytokines (21), it is possible that this change in the distribution of NF-␬B in I/R heart may indicate NF-␬B activation and translocation. It may also be noted that PTXF treatment partially reversed this redistribution of NF-␬B protein content in particulate and cytosolic fractions compared with the I/R group. We also found that PTXF significantly decreased the phosphorylation of NF-␬B at 2 min of reperfusion, which is the time point of the maximal TNF-␣ production. However, the direct protective effect of PTXF by inhibition of NF-␬B-mediated TNF-␣ protein synthesis cannot be explained on the basis of the present study, because the changes in TNF-␣ are seen only during early periods of reperfusion, and NF-␬B may be altered by other mechanisms such as oxidative stress (21). Although some investigators have shown that ischemia alone induces TNF-␣ gene expression and protein synthesis in myocardium (30), others have detected only a minimal expression of TNF-␣ protein 2 h after reperfusion (9). The effects of PTXF on NF-␬B redistribution and phosphorylation at 2 min of reperfusion suggest that PTXF may possibly inhibit NF-␬B activation in response to I/R injury. Although PTXF has been reported to inhibit NF-␬B activity in tumor cells (1), intestinal epithelial cells (29), T lymphocytes, and hepatic satellite cells (16, 20), the data presented here show for the first time that PTXF blocked the redistribution of NF-␬B in myocardium when subjected to I/R injury; this is also the first report that PTXF could inhibit the activation of NF-␬B at 2 min of reperfusion after 30 min of ischemia. Therefore, it is proposed that changes in NF-␬B may be an important factor involved in the mechanism of the beneficial effects of PTXF for improvement of cardiac performance in I/R heart injury. GRANTS The research in this study was supported by a grant from Canadian Institute of Health Research as well as a Canadian Heart Failure Network/ Interdisciplinary Health Research Team program grant from the Institute of Circulatory and Respiratory Health. H. K. Saini is a predoctoral fellow of the Heart and Stroke Foundation of Canada. B. Turan was a visiting professor from the Department of Biophysics, School of Medicine, Ankara University, Ankara, Turkey. P. P. Liu holds the Heart and Stroke Polo Chair, Professor of Medicine and Physiology at the University Health Network, University of Toronto.

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