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Am J Physiol Heart Circ Physiol 293: H2845-H2852, 2007. First published August 24, 2007; doi:10.1152/ajpheart.00209.2007
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PAR-2 activation at the time of reperfusion salvages myocardium via an ERK1/2 pathway in in vivo rat hearts

Rong Jiang, Amanda Zatta, Hajime Kin, Ningping Wang, James G. Reeves, James Mykytenko, Jeremiah Deneve, Zhi-Qing Zhao, Robert A. Guyton, and Jakob Vinten-Johansen

Department of Cardiothoracic Surgery, Cardiothoracic Research Laboratory, Carlyle Fraser Heart Center of Crawford Long Hospital, Emory University School of Medicine, Atlanta, Georgia

Submitted 16 February 2007 ; accepted in final form 17 August 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Protease-activated receptor-2 (PAR-2) may have proinflammatory effects in some tissues and protective effects in other tissues. The role of PAR-2 in in vivo myocardial ischemia-reperfusion has not yet been determined. This study tested the hypothesis that PAR-2 activation with the PAR-2 agonist peptide SLIGRL (PAR-2 AP) reduces myocardial infarct size when given at reperfusion in vivo, and this cardioprotection involves the ERK1/2 pathway. Anesthetized rats were randomly assigned to the following groups with 30 min of regional ischemia and 3 h reperfusion: 1) control with saline; 2) vehicle (DMSO); 3) PAR-2 AP, 1 mg/kg given intravenously 5 min before reperfusion; 4) scrambled peptide (SP), 1 mg/kg; 5) the ERK1/2 inhibitor PD-98059 (PD), 0.3 mg/kg given 10 min before reperfusion; 6) the phosphatidylinositol 3-kinase inhibitor LY-294002 (LY), 0.3 mg/kg given 10 min before reperfusion; 7) PD + PAR-2 AP, 0.3 mg/kg PD given 5 min before PAR-2 AP; 8) LY + PAR-2 AP, 0.3 mg/kg LY given 5 min before PAR-2 AP; 9) chelerythrine (Chel) alone, 5 mg/kg given 10 min before reperfusion; and 10) Chel + PAR-2 AP, Chel was given 5 min before PAR-2 AP (10 min before reperfusion). Activation of ERK1/2, ERK5, Akt, and the downstream targets of ERK1/2 [P90 RSK and bcl-xl/bcl-2-associated death promoter (BAD)] was determined by Western blot analysis in separate experiments. PAR-2 AP significantly reduced infarct size compared with control (36 ± 2% vs. 53 ± 1%, P < 0.05), and SP had no effect on infarct size (53 ± 3%). PAR-2 AP significantly increased phosphorylation of ERK1/2, p90RSK, and BAD but not Akt or ERK5. Accordingly, the infarct-size sparing effect of PAR-2 AP was abolished by PD (PAR-2 AP, 36 ± 2% vs. PD + PAR-2 AP, 50 ± 1%; P < 0.05) and by Chel (Chel + PAR-2 AP, 58 ± 2%) but not by LY (PAR-2 AP, 36 ± 2% vs. LY + PAR-2 AP, 38 ± 3%; P > 0.05). Therefore, PAR-2 activation is cardioprotective in the in vivo rat heart ischemia-reperfusion model, and this protection involves the ERK1/2 pathway and PKC.

reperfusion injury; infarct size; protein kinases

PROTEASE-ACTIVATED receptors (PARs) are members of the G protein-coupled receptor family with seven helical hydrophobic transmembrane domains. Four receptor subtypes have been identified (PAR-1, -2, -3, and -4), each being activated in a protease-dependent manner. PAR-1 are activated by thrombin after binding to a hirudin-like site. Exposure of the naive PAR-2 to trypsin, mast cell-derived tryptase, the tissue factor/factor (TF/F) VIIa complex (4), or factor Xa proteolytically cleaves the terminal 36 amino acids, thereby revealing a new NH2 terminus that acts like a tethered ligand to activate the self-same receptor (36, 41). Synthetic oligonucleotides that have amino acid sequences identical to the tethered ligand also activate the receptor. Whereas PAR-1 is expressed on human platelets and vascular endothelial cells, PAR-2 is more widely distributed, being expressed on vascular endothelial cells, neutrophils, eosinophils, sensory neurons, vascular smooth muscle cells, and rat jejunum myocytes and in whole homogenates of cardiac muscle (32). Inflammatory stimuli such as IL-1 and TNF-{alpha} can upregulate PAR-2 expression on endothelial cells (35). In addition, lipopolysaccharide, ischemia-reperfusion (32), and angioplasty balloon catheter injury (7) increase the expression of PAR-2. PAR-2 activation has been associated with inflammatory effects such as edema (45) and leukocyte recruitment (44).

In contrast to these proinflammatory effects, PAR-2 activation has been shown to be protective in other tissues including brain, airways, and myocardium undergoing ischemia or lipopolysaccharide challenge (21, 31, 32). PAR-2 may be stimulated by the TF/F VIIa complex and proinflammatory cytokines (26, 29, 30); TF expression is upregulated during reperfusion (20, 22). Napoli et al. (32) recently reported that a synthetic PAR-2 agonist peptide (AP) [Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH2)] administered as a pretreatment before index ischemia decreased infarct size (creatine kinase release) and oxidant generation (GSSH and GSH) after reperfusion and increased postischemic contractile functional recovery in isolated rat hearts with global ischemia. Trypsin, a purported stimulator of PAR-2, modestly decreased creatine kinase release. PAR-2 agonists have also been reported to enhance the cardioprotective effect of preconditioning in an ex vivo rat myocardial ischemia-reperfusion model again when given in advance of ischemia (33). However, it has not been determined whether activation of PAR-2, e.g., by infusion of a PAR-2 AP just before reperfusion rather than a pretreatment, is cardioprotective in vivo. Studies by Kin et al. (25), Tsao et al. (43), and Piper and Schafer (37) suggest that the early moments of reperfusion not only are a trigger point for reperfusion injury but also present an opportunity to intervene in these reperfusion injury mechanisms. In addition, the efficacy of the AP and the involvement of reperfusion injury salvage kinase (RISK) (16) signaling pathways in the in vivo model of ischemia-reperfusion are not known.

In the present study, we tested the hypothesis that activation of PAR-2 with a selective AP administered only during reperfusion reduced reperfusion injury in an in vivo rat model. We also tested the hypothesis that the activation of classic "survival kinases" PKC, phosphatidylinositol 3-kinase (PI3-kinase), and/or extracellular signal-regulated kinase (ERK)1/2 may play a role in PAR-2 agonist-induced cardioprotection in vivo.


   METHODS
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 ABSTRACT
 METHODS
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Animal care. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by National Institutes of Health (NIH Publication No. 85-23, Revised 1996). The experimental protocols were approved by the Emory University Institutional Animal Care and Use Committee.

Chemicals and reagents. Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH2), a specific PAR-2 AP that mirrors the new NH2-terminus sequence after proteolytic cleavage in rodents, and Leu-Ser-Ile-Gly-Arg-Leu-amide (LSIGRL-NH2), a PAR-2-inactive control peptide [scrambled peptide (SP)], were synthesized and purified by high-performance liquid chromatography by Bachem Bioscience (King of Prussia, PA). The specificity of SLIGRL was shown by lack of stimulation of PAR-1 expressed in Xenopus oocytes (2). The ERK1/2 inhibitor PD-98059 (PD) and PI3-kinase selective inhibitor LY-294002 (LY) were purchased from Calbiochem (La Jolla, CA). The PKC inhibitor chelerythrine (Chel) was obtained from Sigma (St. Louis, MO). Antibodies of ERK1/2, Akt, phosphorylated ERK1/2, phosphorylated-Akt, ERK5, phosphor-ERK5, Bcl-2 associated death promoter (BAD), phosphor-BAD, and phosphor-p90RSK for use in Western blot analysis were obtained from Cell Signaling Technology (Beverly, MA).

Surgical preparation. Male Sprague-Dawley rats weighing 270–370 g were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). After endotracheal intubation with a 14-gauge angiocatheter, the animals were ventilated with oxygen-enriched room air by a rodent respirator (Harvard Rodent Ventilator Model 683 set initially at 30–40 breaths/min, and tidal volume set to 1.0 ml/100 g body wt). Normal blood gas levels and acid-base status were maintained by adjusting the rate and tidal volume or by intravenous administration of sodium bicarbonate, as necessary to correct for acidemia. The left carotid artery was cannulated with a 24-gauge angiocath connected to a fluid-filled pressure transducer to monitor mean arterial blood pressure (MAP) and heart rate (HR). The right external jugular vein was cannulated for drug and fluid administration. The chest was opened via a left thoracotomy through the fourth or fifth intercostal space, and the ribs were gently retracted to expose the heart. After pericardiotomy, a 6-0 proline (Ethicon, NJ) ligature was placed under the left coronary artery (LCA), and the ends of the tie were threaded through a small plastic (PE-50) tube to form a snare for reversible occlusion. Body temperature was monitored by rectal thermometer and maintained constant between 37° and 37.5°C by a heating pad. Hemodynamic data were captured and analyzed by IOX software from EMKA Technologies (Falls Church, VA).

Experimental protocol. Following the stabilization of hemodynamics and blood gases, the LCA was occluded for 30 min and then reperfused by loosening the ligature for 3 h. Rats were randomly assigned to one of the following ten groups based on the intervention (n = 8 in each group): 1) control: saline (1 ml/kg) was administered 5 min before reperfusion; 2) PAR-2 AP: SLIGRL-NH2 (1 mg/kg) was administered at a bolus injection 5 min before reperfusion; 3) PAR-2 SP: Leu-Arg-Gly-Ile-Leu-Ser-amide (LRGILS-NH2; 1 mg/kg) was administered 5 min before reperfusion; 4) the vehicle for PD and LY, dimethyl sulfoxide (DMSO < 300 µl/kg), was administered 10 min before reperfusion; 5) PD alone (0.3 mg/kg) was administered 10 min before reperfusion; 6) LY alone (0.3 mg/kg) was administered 10 min before reperfusion; 7) PAR-2 AP was administered 5 min after infusion of the selective blocker of ERK1/2 PD (0.3 mg/kg) or (group 8) PI3-kinase inhibitor LY (0.3 mg/kg), both administered 10 min before reperfusion; 9) Chel (5 mg/kg) given 10 min before reperfusion; and 10) Chel (5 mg/kg) given 10 min before reperfusion plus PAR-2 AP 5 min before reperfusion. All drugs were administered intravenously as a slow bolus injection. The dose of PAR-2 AP used in this study was based on dose-response pilot studies completed in our laboratory that looked at 0.1 mg/kg and 1.0 mg/kg on infarct size. The dose of PD and LY was determined from previous studies (15, 47). PAR-2 AP and PAR-2 SP were dissolved in saline with a concentration of 1 mg/ml. PD, LY, and Chel were dissolved in DMSO.

Area at risk and infarct size. At the end of the experiment, the LCA was religated at the original site, and the area at risk (AAR) was determined by in vivo injection of 1 ml of 20% Unisperse blue dye via the external jugular vein. Extra left ventricular tissue was removed, and the left ventricle was sliced transversely into five to six slices. The nonstained AAR was separated from the blue-stained nonischemic zone myocardium, and the AAR was incubated in a 37°C 1% solution of buffered (pH 7.4) triphenyltetrazolium chloride (TTC) for 15 min to identify the area of necrosis (AN) within the AAR. The AAR was expressed as a percentage of the left ventricular mass (AAR/LV), and the AN was expressed as a percentage of the AAR (AN/AAR), the mass of each area being determined gravimetrically.

Isolated neonatal rat cardiomyocytes and cell culture. Primary culture of neonatal rat cardiomyocytes were prepared as described previously (42). Briefly, the hearts from 1–3-day-old Wistar rats were minced and dissociated with 0.08% trypsin. The dispersed cells were then plated to a field density of 2 x 105 cells/cm2 on 60-mm culture dishes with double minimum essential medium (DMEM) supplemented with 10% fetal bovine serum. After 24 h in a 5% CO2-95% air incubator at 37°C, the culture medium was changed to DMEM with 10% fetal bovine serum containing cytosine arabinoside (Ara C, 10 µM) to eliminate noncardiomyocytes. H9c2 cardiac muscle cells (American Type Culture Collection) were cultured for protein extraction and Western blot analysis.

Western blot analysis. Additional experiments (n = 5 per group) were performed to quantify the extent of ERK1/2 and Akt phosphorylation in the presence of PAR-2 agonist, SP, saline vehicle, or the inhibitors PD or LY. Five minutes after ischemia and reperfusion, hearts were excised, and 200 mg AAR tissue was separated and homogenized in lysis buffer [50 mmol/l HEPES, 150 mmol/l NaCl, 1.5 mmol/l MgCl2, 1 mmol/l EGTA, 1% Triton X-100, 10% glycerol plus 8.6 µmol/l leupeptin, 5.8 µmol/l pepstatin A, 4 mmol/l phenylmethylsulfonyl fluoride (PMSF), 0.6 µmol/l aprotinin, 4 mmol/l sodium fluoride, and 0.8 mmol/l sodium orthovanadate] and then centrifuged at 10,000 g for 15 min to remove nuclei and debris. Protein concentration of the supernatants was determined by the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). Samples (60 µg) were separated by 10% SDS-PAGE, and the protein was transferred to nitrocellulose (Hybond ECL, Amersham, Piscataway, NJ). After the blot was blocked and washed, it was incubated overnight at 4°C with primary antibodies and incubated with a secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. After immunoblots were washed, they were incubated with an enhanced chemiluminescence detection system (Amersham). Densitometry was quantitated using NIH ImageJ software.

Detection of PAR-2 in rat myocardium with immunohistochemistry. The left ventricular tissue blocks were embedded in optimal cutting temperature compound (OCT; Tissue-TeK) and frozen in liquid nitrogen. The tissue blocks were cut using a Microtome cryostat HM 505E, and the cryosections (7 µm thick) were mounted on coated Superfrost/Plus Microscope slides (Fisher Scientific), refrozen, and stored at –70°C until used. The cryostat sections were then fixed in 100% acetone for 5 min and blocked in 1% gelatin/PBS for 20 min. The slides were subsequently incubated with monoclonal mouse anti-PAR-2 antibody (SAM11, Santa Cruz Biotechnology), washed in PBS, and incubated with a biotinylated horse anti-mouse IgG (Vector). The slides were further stained by the ABC-AP kit (Vector) and substrated with alkaline phosphatase substrate (Vector). Counterstaining was performed with and without hematoxylin. Localization of PAR-2 on myocardial tissue sections was carried out with an Olympus IX-50 fluorescence microscope, and the images were captured by a Micro Publisher 3.3 real-time viewing digital imaging camera. The quality of immunohistochemistry assay was controlled by elimination of the primary antibody.

Statistical analysis. All data are expressed as means ± SE. All data were analyzed using SigmaStat 2.0 for Windows statistical software package (SPSS, Chicago, IL). A one-way analysis of variance (ANOVA) (infarct size, AAR) and ANOVA for repeated measures (hemodynamics) were used as appropriate with post-hoc analysis between groups determined by the Student-Newman-Keuls test correcting for multiple comparisons. Infarct size was analyzed for all groups together, independent of separation of groups in the results section. A P value <0.05 was considered significant.


   RESULTS
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The presence of PAR-2 in rat heart. To confirm the expression of PAR-2 in the rat heart, Western blot analysis showed that PAR-2 protein is expressed in left ventricular tissue homogenates, neonatal cardiomyocytes, and H9c2 cells (Fig. 1). In addition, there were cells in left ventricular tissue sections obtained from the myocardium that expressed PAR-2. From Fig. 2, the PAR-2 antigen is expressed on the membrane surface and cell junctions of rat cardiomyocytes and on vascular endothelial cells and smooth muscle cells of vessels (Fig. 2).


Figure 1
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  		Fig. 1. Representative Western blot analysis of protease-activated receptor-2 (PAR-2) expression in (left to right) neonatal cardiomyocytes, lane 1; H9c2 cells, lane 2; and rat left ventricular (LV) myocardium homogenates, lanes 3 and 4. PAR-2 was expressed in all cell types, and tissue was sampled.

 

Figure 2
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  		Fig. 2. PAR-2 in rat myocardium identified by immunohistochemistry. PAR-2-antibody positive cells (red) in vascular smooth muscle cells and endothelium (arrow A) and on the sarcolemma of cardiomyocytes (arrow B) (magnification, x200).

 
Hemodynamic data. PAR-2 AP infusion had no effect on HR but produced a 40 ± 4 mmHg reduction (P < 0.05 vs. control) in MAP, which returned to pretreatment level within 2 min after injection, i.e., before the onset of reperfusion (Fig. 3). Hemodynamic variables recorded 5 min after onset of reperfusion in the PAR-2 AP group were comparable with those in the control group (Table 1). In contrast, PAR-2 SP had no effect on either HR or MAP. These results are consistent with previous studies in which in vivo activation of PAR-2 caused transient hypotension (5, 10). PD, LY, or Chel alone had no effect on HR and MAP. Pretreatment with PD, LY, or Chel had no effect on transient hypotension induced by PAR-2 AP administration. There were no significant differences observed between the control groups (control and DMSO) and treatment groups for HR and MAP at 5, 60, and 180 min of reperfusion. The hemodynamic data are summarized in Table 1.


Figure 3
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  		Fig. 3. The effect of PAR-2 agonist peptide SLIGRL (PAR-2 AP) infusion on mean arterial blood pressure (MAP). PAR-2 AP infusion produced a 40 ± 4 mmHg reduction (P < 0.05 vs. control) in MAP, which returned to pretreatment level within 2 min after injection. PD-98059 (PD) did not attenuate the PAR-2 AP-induced MAP reduction. *P < 0.05 for PAR-2 AP and PD + PAR-2 AP vs. the control group.

 

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  		Table 1. Hemodynamic variables during the course of the experiment

 
Effect of PAR-2 AP on infarct size. The area placed at risk by LCA occlusion, expressed as a percentage of left ventricular mass (AAR/LV), was comparable among groups (~30–36%, Table 2). Infarct size, expressed as a percentage of AAR (AN/AAR), was significantly reduced in the PAR-2 AP group (36 ± 2%) compared with the control or SP group (53 ± 1% and 53 ± 3%, respectively, P < 0.001; Fig. 4A). Therefore, the reduction in infarct size by PAR-2 AP is specific to agonist effects of the peptide since an equal dose of the SP had no effect on infarct size compared with control.


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  		Table 2. Infarct size data

 

Figure 4
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  		Fig. 4. A: the effect of PAR-2 AP on infarct size. Infarct size is expressed as a percentage of the area at risk in rats treated with saline (control), PAR-2-scrambled peptide (PAR-2 SP), or PAR-2 AP 5 min before reperfusion (n = 8 per group). *P < 0.05 vs. control and PAR-2 SP. Values represent means ± SE. B: the effect of ERK1/2, PKC, and phosphatidylinositol 3-kinase (PI3-kinase)/Akt inhibitors on infarct size with and without PAR-2 AP. The protective effect of PAR-2 AP was unaffected by the PI3-kinase inhibitor LY-294002 (LY) but abolished by the ERK1/2 inhibitor PD and PKC inhibitor chelerythrine (Chel). LY, PD, and Chel alone had no effect on infarct size. Dimethylsulfoxide (DMSO) represents the vehicle control group. Values represent means ± SE. *P < 0.05 vs. DMSO.

 
The effect of ERK1/2, PI3-kinase, and PKC inhibitors on infarct size. In the second series of experiments, we investigated the effect of PI3-kinase, ERK1/2, and PKC inhibitors on infarct size to determine whether PAR-2 AP was acting via any of these pathways. Interestingly, when PAR-2 AP was administered after the injection of PI3-kinase inhibitor LY, infarct size was similar to that of the PAR-2 AP group (LY + PAR-2 AP, 38 ± 3% vs. PAR-2 AP, 36 ± 2%, P > 0.05; Fig. 4B). However, the infarct-sparing effect of PAR-2 AP was completely abolished with the ERK1/2 inhibitor PD (PD + PAR-2 AP, 50 ± 1% vs. PAR-2 AP, 36 ± 2%, P < 0.05; Fig. 4B). The protection of PAR-2 AP was completely lost in the presence of the PKC inhibitor Chel (58 ± 2% vs. 36 ± 2%). None of the inhibitors had any effect on infarct size when given alone (LY, 58 ± 2%; PD, 54 ± 1%; Chel, 58 ± 2% vs. DMSO, 56 ± 2%; Fig. 4B). These results suggest that the ERK1/2 and PKC but not the PI3-kinase signaling pathways are involved in the protection exerted by PAR-2 AP treatment at reperfusion.

Western blot analysis. To determine whether PAR-2 AP activates classical RISK pathways, Western blot analysis was performed to evaluate ERK1/2 pathway activation using an antibody specific for the phosphorylated form of ERK1/2 in the hearts harvested at 5 min of reperfusion from control, SP, and PAR-2 AP with or without PD or LY groups. PAR-2 AP administered at reperfusion was associated with significantly greater phosphorylated ERK1/2 compared with control and SP groups (Fig. 5A). In addition, p90RSK and BAD as downstream targets of ERK1/2 were also significantly phosphorylated in the PAR-2 AP group (Fig. 5B). P90RSK and BAD phosphorylation was reduced by PD (Fig. 5B). However, there was no significant difference in expression of phosphor-ERK5 among all groups (Fig. 5D). These results suggest that PAR-2 AP specifically activates the ERK1/2 pathway in the in vivo rat hearts, which is consistent with the infarct size data that the ERK1/2 inhibitor PD blocked PAR-2 AP-induced infarct size reduction. In contrast, PAR-2 activation did not alter phosphorylated Akt band density in the AAR myocardium (Fig. 5C). The results are consistent with a lack of reversal of infarct size by the PI3-kinase inhibitor LY given before reperfusion.


Figure 5
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  		Fig. 5. Representative Western blot analysis of heart tissue samples acquired from the ischemic zone 5 min after reperfusion in rats treated with saline (control, n = 5), PAR-2 SP (n = 5), PAR-2 AP (n = 5), PD + PAR-2 AP, and PD alone. The blots were probed for phosphorylated ERK1/2, Akt, p90RSK, bcl-xl/bcl-2-associated death protein (BAD), and total ERK1/2. A: phosphorylated ERK1/2 and total ERK1/2 for ischemic zone tissue at 5 min of reperfusion, and absolute densitometries of phosphorylated ERK1/2 for ischemic zone tissue at 5 min of reperfusion. *Significant differences vs. control (P < 0.05). **P < 0.05 vs. PD + PAR-2 AP. Values represent means ± SE. B: phosphorylated p90RSK and BAD for ischemic zone tissue at 5 min of reperfusion, and absolute densitometries of phosphorylated p90RSK and BAD for ischemic zone tissue at 5 min of reperfusion. *Significant differences vs. control (P < 0.05). **P < 0.05 vs. PD + PAR-2 AP. Values represent means ± SE. C: phosphorylated Akt for ischemic zone tissue at 5 min of reperfusion, and absolute densitometries of phosphorylated Akt for ischemic zone tissue at 5 min of reperfusion. There were no significant differences among groups. Values represent means ± SE. D: phosphorylated ERK5 and total ERK5 for ischemic zone tissue at 5 min of reperfusion, and absolute densitometries of phosphorylated ERK5 for ischemic zone tissue at 5 min of reperfusion. There were no significant differences among groups. Values represent means ± SE.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
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In the present study, we demonstrated that a PAR-2 AP, SLIGRL, administered before the onset of reperfusion in an in vivo rat coronary artery occlusion reperfusion model was associated with a 32% reduction in myocardial infarct size relative to that observed in untreated controls and vehicle groups. The PAR-2 AP (SLIGRL-NH2) for rodents has previously been reported to be specific for activation of PAR-2 in PAR-2 knockout mice (6). In the present study, the specificity of PAR-2 AP was confirmed by the observation that the SP (LRGILS) had no effect on infarct size and activation of signaling transduction pathways. The infarct-sparing effects of SLIGRL administered at reperfusion extend the observations from previous studies, which demonstrated a protective effect of PAR-2 activation by a PAR-2-selective AP against ischemia-reperfusion-induced myocardial injury when administered as a pretreatment before ischemia and covering the first 5 min of reperfusion (32, 33). The PAR-2 AP was associated with an increased phosphorylation of the survival kinase ERK1/2; the ERK1/2 inhibitor PD abrogated the infarct-sparing effect of PAR-2 AP, thereby suggesting a functional link with its infarct sparing effect. In contrast, PAR-2 AP given at reperfusion was not associated with phosphorylation of the downstream target of PI3-kinase Akt. The lack of reversal of infarct sparing by PAR-2 with LY is consistent with the lack of detectable change in expression of myocardial phosphor-Akt levels.

Intravenous administration of PAR-2 AP just before reperfusion caused a rapid and transient hypotension that was resolved within 2 min after administration and before the initiation of reperfusion. The hypotensive effects of SLIGRL are consistent with previous in vivo rat studies (5, 10). This hypotension may be the result of vasodilation rather than a negative inotropic effect and decreased cardiac output; McLean et al. (28) observed an endothelium-dependent but nitric oxide (NO)- and prostanoid-independent vasodilation on the coronary vasculature when administering SLIGRL, an effect that was also observed for trypsin. This vasodilation of coronary arteries in isolated rat hearts was dependent on the endothelium-derived hyperpolarizing factor. The vasodilatory effect is absent in PAR-2 null mice (6). It is not clear whether the transient hypotension influenced infarct size since it was resolved within 2 min of administration. However, the hypotension persisted after ERK1/2 blockade while infarct size was increased to control levels, suggesting that the infarct-sparing effect of PAR-2 AP was independent of this transient hypotension.

Activation of PARs has opposing actions in the setting of myocardial ischemia-reperfusion. Activation of PAR-1 has been implicated in the pathogenesis of ischemia-reperfusion injury, specifically in the pathogenesis of myocardial infarction (38, 39). Thrombin, as an activator of PAR-1, stimulates surface expression of P-selectin on endothelial cells (23), which facilitates the recruitment and adherence of neutrophils to endothelial cells (14). Neutrophil recruitment to the coronary vascular endothelium triggers subsequent processes that are implicated in the pathogenesis of myocardial infarction (46). In addition, thrombin can directly stimulate the Na+-H+ exchanger in ventricular myocytes (48), which may exacerbate intracellular calcium accumulation during reperfusion. In some tissues, PAR-2 activation has been shown to be proinflammatory (13, 24, 34) and linked with tissue injury, similar to the deleterious effects of PAR-1 activation. Kawabata et al. (24) first showed that PAR-2 AP induces inflammation in the rat paw, and Ferrell et al. (13) demonstrated an essential role of PAR-2 in mediating chronic inflammation in joints. Activation of PAR-2 expressed on neutrophils (18) by receptor APs has been shown to increase neutrophil-endothelial cell interactions (adhesion, rolling) and extravasation, possibly linked to a mechanism involving platelet activating factor, and would be consistent with a proinflammatory function with potentially deleterious consequences.

In contrast, activation of PAR-2 may be protective in the heart and brain. PAR-2 expression is increased by ischemia-reperfusion in both myocardium (32) and the brain (21), although it is not clear whether the increased expression that occurred in myocardium was specific in cardiomyocytes. Studies have not yet been conducted to determine the time course of PAR-2 expression activity and localization in myocardial tissue following ischemia and reperfusion. Napoli et al. (32) reported that infusion of the PAR-2 agonist SLIGRL-NH2 at 100 µM administered before ischemia and continuing for 5 min into reperfusion in isolated perfused rat hearts subjected to global ischemia was associated with significant recovery of systolic function and decreased creatine kinase activity (necrosis) in coronary effluent. Napoli et al. (33) also reported that PAR-2 activation with an AP could increase the cardioprotective effects of ischemic preconditioning. In this study by Napoli et al. (33), however, it was not clear whether protection was exerted during ischemia or during reperfusion. The present study suggests that the cardioprotective effects of PAR-2 activation by systemically administered AP are exerted at reperfusion.

PKC, PI3-kinase, and ERK1/2 have been reported to be involved in intracellular signaling of many cardioprotective therapies. These kinases are integral parts of the RISK pathway (16, 17) that is triggered by pharmacological agents (adenosine, urocortin, opioids, and statins), preconditioning, and postconditioning (8, 19, 47). Ultimately, these kinases inhibit opening of the mitochondrial transition pore and reduce both apoptosis and necrosis (3, 17). It has been previously reported that incubation of neonatal rat cardiomyocytes with PAR-2 AP in increasing concentrations and for different periods of time activates the ERK1/2 and, to a lesser extent, P38 MAPK but not PI3-kinase/Akt (40). The reversal of infarct sparing by ERK1/2 inhibitor PD would be consistent with this link between kinase phosphorylation and infarct size reduction. Accordingly, Western blot analysis showed that PAR-2 AP administration significantly increased the phosphorylation of ERK1/2 and its downstream products p90RSK and BAD in AAR myocardium; however, PAR-2 AP administration had no effect on the phosphorylation of Akt or ERK5. Accordingly, blocking the PI3-kinase pathway with LY before the administration of PAR-2 AP did not inhibit the protection exerted by PAR-2 AP. These results support the notion that ERK1/2 but not PI3-kinase/Akt activation is involved in the cardioprotection with PAR-2 activation in the in vivo rat model of ischemia-reperfusion.

The involvement of kinase pathways in infarct reduction by PAR-2 activation is consistent with other studies of the role of PAR-2 in ischemia-reperfusion in the heart (32) and brain (21). The protection observed in brain ischemia-reperfusion studies also involved the ERK1/2 pathway but not the PI3-kinase pathway in the in vivo setting, as has been shown for other cardioprotective maneuvers applied at reperfusion such as postconditioning (8, 17).

In vitro studies demonstrate that PAR-2 AP activates PKC, which is a protective kinase in both preconditioning (1) and postconditioning (49). The present study showed that cardioprotection of PAR-2 AP was dependent on PKC. However, we did not identify which isoform of PKC is activated by PAR-2 AP.

Other mechanisms may be involved in the cardioprotection at reperfusion observed in the present study. PAR-2 activation was shown to reduce oxidant generation at reperfusion. It is not clear whether increasing coronary perfusion as reported by Napoli et al. (32) is cardioprotective per se or a manifestation of reduced injury (i.e., no reflow). Preservation of the vascular endothelium after ischemia-reperfusion is related to retaining the ability to generate and release NO. NO is cardioprotective by decreasing the recruitment and adherence and activation of neutrophils. Although the study by McLean et al. (28) suggests preserved coronary function with SLIGRL after ischemia-reperfusion, their results suggest a loss of ability to generate NO. It is not clear how PAR-2-induced stimulation of neutrophil recruitment observed by Vergnolle (44) relates to an overall cardioprotective effect since the activation and the ensuing interaction with coronary vascular endothelium and release of proinflammatory and oxidant products is a purported mechanism of reperfusion injury (9, 46).

Although an exogenously applied PAR-2 AP protects the heart from reperfusion injury, little is known of the cardioprotective role of PAR-2 stimulated by endogenous factors. Indeed, the observation reported by Jin et al. (21) that the absence of PAR-2 increases focal postischemic brain injury in PAR-2 knockout mice suggests that PAR-2 may have a role in endogenous protection. Several of the known stimulators of PAR-2 are upregulated or increased by ischemia-reperfusion, including cytokines (TNF-{alpha}) (29, 30) and tissue factor (11, 12, 20, 22). In addition, PAR-2 may be activated by endogenous proteases, most likely mast cell tryptase released in the AAR myocardium, thereby exerting local cardioprotection (27).

In summary, we have shown that PAR-2 is expressed in H9c2 cells and cultured cardiomyocytes, but in the in vivo rat myocardium PAR-2 was weakly expressed. In addition, PAR-2 activation with an exogenous AP during reperfusion reduces infarct size. However, the specific site of action (cardiomyocytes, endothelium) was not determined. This protection appears to involve ERK1/2 activation, its downstream targets p90RSK and BAD, and is PKC dependent but does not involve PI3-kinase. This study suggests that PAR-2 may be a target for reperfusion therapeutics. PAR-2 APs may be used to pharmacologically attenuate reperfusion injury. However, the peptide may have to be delivered locally to the AAR since it may be proinflammatory in other tissues.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by NIH Grant HL-069487 to J. Vinten-Johansen and HL-064886 to Z-Q Zhao, and by support from the Carlyle Fraser Heart Centre of Emory Crawford Long Hospital.


   ACKNOWLEDGMENTS
 
R. Jiang and A. J. Zatta are recipients of a postdoctoral fellowship from the American Heart Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Vinten-Johansen, Cardiothoracic Research Laboratory, Crawford Long Hospital, 550 Peachtree St. NE, Atlanta, GA 30308-2225 (e-mail: jvinten{at}emory.edu)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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