INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PROTEASE-ACTIVATED RECEPTOR-2 (PAR-2) is a member of seven transmembrane domain G protein-coupled receptors activated by proteolytic cleavage (6, 32). The pathophysiological role in vivo of (PAR-2) remains poorly understood. PAR-2 is widely distributed in endothelial (11-18) and vascular smooth muscle (11-27) cells. It plays a role in the regulation of blood pressure and vascular tone (4, 5, 9, 12, 18, 27, 36, 37), endotoxic shock (5), and inflammation (22, 33). Recently, we provided the first evidence that PAR-2 is expressed in the heart, and it also contributes to reduce myocardial ischemia-reperfusion injury (28). PAR-2-dependent signaling in the heart has also been confirmed by others (34). To activate PAR-2, we used a short synthetic activating peptide (PAR-2AP) that can activate the receptor in absence of proteolytic cleavage (5, 32).

Widespread interest in the benefits of myocardial ischemic preconditioning (IP) has resulted from several experimental and clinical studies (31). To investigate whether PAR-2 plays a role in the protection afforded by preconditioning, we studied the possible effect of PAR-2 in an experimental model of IP. Because our working hypothesis is that PAR-2 is involved in inflammatory and injury response events (6, 27, 5, 22, 39, 17), we attempted to verify whether PAR-2 plays a more general role in cardiac responses to ischemia and inflammation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated heart preparation. The hearts of 55 male rats (6 mo old; 376 ± 43 g mean body wt; 973 ± 63 mg mean heart wet wt) were perfused in the retrograde Langendorff mode under a constant pressure of 80 mmHg as described previously (2, 28). Coronary flow rate was measured from a collection of coronary sinus effluent and related to heart wet weight (in ml · min¹ · g¹). Arrhythmias were scored according to the Lambeth Convention Guidelines as follows: 0 = no arrhythmias; 1 = single ventricular premature beats; 2 = couplets and salvos; 3 = ventricular tachycardia; 4 = nonsustained ventricular fibrillation; and 5 = sustained ventricular fibrillation (2, 28). The study was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the position of the American Physiology Society.

Protocols. We used the classic ischemia-reperfusion injury (i.e., 20 min of global ischemia induced by cross-clamping the perfusion line following by 40 min of reperfusion) (2, 28) or a preconditioning protocol (preconditioning transient ischemic stimulus for 2 min followed by a 10-min window of reperfusion and then a standard ischemia-reperfusion insult) (1). To reduce experimental confounding variables, we preferred to use this simple protocol of ischemia-reperfusion injury (2, 28) and single preconditioning ischemic stimulus (1). Hearts were maintained at 37°C throughout ischemia by immersion in warm perfusate. Hemodynamic parameters were recorded until 40 min of reperfusion. Peptides (see below) were freshly diluted with the appropriate amounts of perfusion buffer (in this experimental design it was PNM-spikes Krebs) and administered by a Harvard syringe pump through a sidearm in the perfusion apparatus to achieve different final concentrations (30-100 µM). Two different protocols were used. First, infusion of PAR-2AP was started at 5 min of stabilization and continued for 5 min into reperfusion. In the second protocol, infusion of PAR-2AP lasted only during transient ischemia (Fig. 1). The protective effects of PAR-2AP on myocardial ischemia-reperfusion injury after global ischemia alone were previously reported (28). PAR-2AP (SLIGRL-NH₂) and LSIGRL-NH₂ (scrambled control peptide) were synthesized by standard solid-phase 9-fluorenylmethoxycarbonyl chemistry as described (5, 28). At the end of the experiment, the hearts were removed from the perfusion apparatus, blotted, and weighed. The cardiac homogenate was used for malondialdehyde (MDA) assay, glutathione reductase, and peroxidase tissue determinations, whereas Western blot analysis was performed on whole heart homogenate (see Biochemical determinations).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Summary of the experimental protocols used. Standard ischemia-reperfusion (IR) injury was performed using 20 min of ischemia (I) followed by 40 min of reperfusion (R). Preconditioning (Prec) protocol was performed by using 2 min of transient ischemia (TI) followed by 10 min of R before the standard 20 min of I and then 40 min of R. Protocol 1 involves infusion of peptides from the beginning of TI until 5 min of R. Protocol 2 involves infusion of protease-activated receptor-2-activating peptide (PAR-2AP) only during TI preconditioning stimulus.

Cardiac tissue homogenization and infarct size. Both the atria and right ventricle were removed, and the left ventricle was homogenized in 9 vol of 1.15% KCl solution (2, 28, 35). To prevent autooxidation of the tissue samples, homogenization was carried out at 4°C in nitrogen-equilibrated solutions, in the presence of 10 µM deferoxamine, 0.04% butyrate hydroxytoluene, and 2% ethanol (2, 28, 35). Protein concentrations were determined by the Lowry method (24). We performed the classic measurement of risk zone as described previously (28). Two-millimeter-thick slices were stained for 20 min in 1% triphenyltetrazolium chloride. The infarct size (measured as mm³ or as percentage of risk zone, e.g., nonfluorescent area under ultraviolet light) was traced on acetate by a computer-assisted imaging analyzer (Ophoto Software, T1-2 version 2.0, Apple).

Biochemical determinations. At various time-selected points, the supernatant was used for the determination of MDA using the thiobarbituric assay (28, 35). Similarly, to measure GSSG release, additional 0.4-ml aliquots of coronary effluent were simultaneously drawn into a syringe containing 100 µl of 10 mM EDTA and 50 mM N-ethyl-maleimide in 100 mM K-phosphate buffer at pH 7.4 (2, 28, 35). Concentrations of total glutathione (i.e., GSSG + GSH) and GSH were measured by the glutathione reductase-5,5'- dithiobis-2-nitrobenzoic acid recirculating assay (2, 28, 35) (expressed as nanomoles of GSH equivalents released per minute per gram wet weight). Total integrated creatine kinase (CK) activity over reperfusion was evaluated as described (28, 29). Tissue glutathione reductase and peroxidase and Mn-superoxide dismutase were determined spectrophotometrically as described (2, 28). Neutrophil accumulation was determined by measuring myeloperoxidase (MPO) content in snap-frozen cardiac tissues. Samples were spun at 11,000 g for 5 min, and the supernatant was collected. Enzymatic detection was performed spectrometrically at 450 nm as described (8). Results were expressed as the determination of the MPO increase after reperfusion. Values are presented after normalization with respect to normal untreated nonischemic controls.

Statistical analysis. Data are presented as means ± SE. Differences among the various groups were tested by repeated measure analysis of variance (ANOVA). When the overall trend was significantly different, comparisons at specific time points were made by Dunnett's corrected t-test considering P < 0.05 as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Left ventricular developed pressure. The hemodynamic effects of PAR-2AP have been extensively described in a previous study (28). IP, as demonstrated in several experimental settings (31), improves heart contractility as measured by developed pressure (Table 1, IP protocol). By using protocol 1 (Fig. 1), 100 µM PAR-2AP caused further improvement of developed pressure (Table 1). PAR-2AP-induced amelioration is dose dependent because 30 µM PAR-2AP, even if less efficacious, still caused a significant effect (Table 1). By using protocol 2 (Fig. 1), only 100 µM PAR-2AP significantly improved ventricular function (Table 1). Thus PAR-2AP induced similar results between protocol 1 and 2 only when we used the 100 µM dose. The scrambled peptide was ineffective (not shown).

Left ventricular end-diastolic pressure. IP reduced the elevation of diastolic pressure induced by ischemia (Table 1, IP protocol). By using protocol 1 (Fig. 1), recovery of diastolic pressure was significantly improved in hearts that received 100 µM PAR-2AP (Table 1). The dose of 30 µM PAR-2AP caused a significant effect only at the 40-min reperfusion time point (Table 1) compared with control ischemic hearts (Table 1, control and IP protocol). Similarly, when using 100 µM PAR-2AP in protocol 2, there was a significant improvement of diastolic pressure (Table 1). Also in this case, the scrambled peptide was ineffective (not shown).

Coronary flow. In protocol 1, 30 and 100 µM PAR-2AP significantly improved coronary flow at the end of reperfusion (Table 1). With the use of protocol 2 (Fig. 1), only 100 µM PAR-2AP induced a significant improvement of coronary flow. The scrambled peptide was ineffective (not shown).

Heart rate. Baseline values of heart rate were similar among groups (n = 8 for each group, 242 ± 23 beats/min in control ischemia-reperfusion injury, 239 ± 26 beats/min in IP, 245 ± 28 beats/min in 100 µM PAR-2AP protocol 1, and 238 ± 23 beats/min in 100 µM PAR-2AP protocol 2; P = not significant for all comparisons). Similarly, after 5 min of reperfusion, there were no significant differences among groups; results obtained were 131 ± 29, 134 ± 31, 142 ± 36, and 134 ± 27 beats/min for reperfusion injury, IP, and PAR-2AP 100 µM protocol 1 and 2, respectively. These data were not statistically different until 40 min of reperfusion (not shown). The scrambled peptide was ineffective (not shown).

GSSG/GSH on the coronary effluent. The GSSG and GSH release at baseline was negligible in all groups (Fig. 2). IP, by itself, reduced the release of oxidized glutathione (Fig. 2). Infusion of 100 µM PAR-2AP following both protocols significantly further reduced the release of GSSG and GSH (Fig. 2, A and B).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Effects of PAR-2AP administration on oxidized glutathione (GSSG; A) and reduced glutathione (GSH; B) as indexes of myocardial oxidative reperfusion injury. Prot 1 and 2, protocols 1 and 2. * P <0.01 vs. ischemia-reperfusion injury; #P <0.05 vs. preconditioned hearts. Data are expressed as means ± SE; n = 7-8 hearts for each group.

CK activity and cardiac infarct size. IP reduced cardiac damage expressed as CK activity measured after reperfusion (Fig. 3A). Again, 30 and 100 µM PAR-2AP further reduced the release of CK over time by using both protocols, whereas the scrambled peptide was ineffective (Fig. 3A). Similarly, IP significantly reduced the cardiac ischemic risk zone (Fig. 3B). Hearts treated with 30 and 100 µM PAR-2AP, following protocol 1 procedure, had further decreased infarct size (Fig. 3B). Finally, protocol 2 also significantly reduced the risk zone when 100 µM PAR-2AP was used (Fig. 3B). Finally, the scrambled peptide was ineffective (Fig. 3B).

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Effect of PAR-2AP administration (protocols 1 and 2) on cardiac damage evaluated as total integrated creatine kinase (CK) release over reperfusion (A), infarct size (expressed in mm3) (B), or as a percentage of the ischemic risk zone (C). * P <0.05 vs. preconditioned and scrambled peptide-treated hearts; #P <0.01 vs. ischemia-reperfused hearts. Data are expressed as means ± SE, n = 7-8 hearts for each group.

Arrhythmia analysis. The arrhythmic score at reperfusion in control hearts was 4.1 ± 1.3 arbitrary units (AU) but decreased progressively to 3.3 ± 1.1 AU in the IP group (n = 8; P = 0.073) and 2.9 ± 1.2 AU in the 30 µM (P < 0.05) and 2.7 ± 1.0 AU in 100 µM PAR-2AP-treated groups where infusion of the peptide lasted until 5 min reperfusion (n = 8; P < 0.03; protocol 1). The increased score observed in controls was due mainly to ventricular tachycardia and nonsustained ventricular fibrillation. Similarly, in hearts treated with 100 µM PAR-2AP (protocol 2) during transient ischemic stimulus only, the score was significantly reduced (n = 8, 2.8 ± 0.8 AU; P < 0.05). The scrambled peptide (100 µM) did not reduce significantly the arrhythmic score (not shown). Protocol 2 treatment did not affect the arrhythmic score at reperfusion (not shown).

Tissue MDA levels. In preliminary experiments, we established that MDA content in normally perfused hearts at baseline averaged 0.95 ± 0.1 nmol/mg protein. This content increased slightly after 20 min of ischemia (1.20 ± 0.16 nmol/mg protein, P = not significant vs. baseline; n = 8), whereas it significantly increased in reperfused hearts (1.42 ± 0.2 nmol/mg protein, P < 0.05 vs. baseline; n = 7). IP protocol per se reduced lipid peroxidation (1.25 ± 0.18 nmol/mg protein, P < 0.05 vs. baseline, n = 8). At the end of reperfusion by using protocol 1 (Fig. 1), where infusion peptide lasted until 5 min reperfusion, this increase in peroxidation was significantly further reduced with treatment with 100 µM PAR-2AP (1.05 ± 0.1 nmol/mg protein, P < 0.04 vs. controls and P < 0.05 vs. IP protocol; n = 8 each group) as well as in hearts treated with 30 µM PAR-2AP (1.11 ± 0.1 nmol/mg protein, P < 0.05 vs. controls and IP protocol; n = 7 each group). Similarly, 100 µM PAR-2AP administered only during transient ischemic stimulus reduced MDA levels (1.16 ± 0.1 nmol/mg of protein, P < 0.05 vs. controls and IP protocol; n = 8 each group). Finally, the scrambled peptide (100 µM) did not reduce significantly the amount of MDA during reperfusion (not shown).

Tissue activities of glutathione metabolism enzymes and Mn-superoxide dismutase. Cardiac tissue activities of gluthatione peroxidase and reductase are showed in Table 2. These levels are significantly reduced by IP, but 30 and 100 µM PAR-2AP with both protocols of administration did not further improve these indexes compared with preconditioning itself. In contrast 30 and 100 µM PAR-2AP, administered with protocol 1, significantly increased the Mn-superoxide dismutase compared with control ischemic reperfused and IP hearts (Table 2). This effect was also present when the heart was exposed to 100 µM PAR-2AP with protocol 2 (Table 2).

Neutrophil accumulation. The effects of PAR-2AP (30 and 100 µM) on neutrophil accumulation in hearts was determined by measuring the MPO content after reperfusion (Table 3). PAR-2AP infusion significantly further reduced neutrophils accumulation compared with IP hearts (P < 0.05). The presence of neutrophils was also detected by immunohistochemistry using the antimurine neutrophil monoclonal antibody GR-1, which confirmed the relative accumulation of MPO [R = 0.56 (P < 0.01) between MPO activity and GR-1-positive sections].

Western blot. As previously demonstrated (28), semiquantitative scanning of blots showed an increased PAR-2 expression in hearts treated with 100 µM PAR-2AP versus scrambled peptide-treated IP hearts (40 min of reperfusion; 8.4 ± 2.1 vs. 2.6 ± 1.8 AU; n = 4; P < 0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IP triggers a protective mechanism against myocardial ischemia and reperfusion injury through the release of endogenous factors (31). Despite the difficulties with making measurements in patients, there is no reason to think that the fundamental biology of IP is substantially different between humans and experimental models (31). After reperfusion injury, cardiac damage is characterized by oxidative injury as well as by activation of a cascade of signaling events related to inflammation (13). Furthermore, it is activated complement cascade and it is promoted neutrophil and monocyte targeting (13). Myocardial ischemia is severe enough to initiate inflammation at reflow, but reperfusion injury reduces the amount of net benefit achieved by restoration of blood flow and, more importantly, IP-mediated cardioprotection. Thus IP may represent an ideal framework to study PAR-2 in cardiac ischemia and inflammation. Indeed, in vivo studies have pointed to a role for PAR-2 in the regulation of cardiovascular functions (4, 5, 9, 11, 27, 28, 34) and in the inflammatory response (6, 17). Here, we have demonstrated that IP significantly decreases metabolic and inflammatory indexes due to cardiac ischemia in our experimental conditions. These parameters (i.e., left ventricular developed pressure, left ventricular end-diastolic pressure, coronary flow, infarct size, oxidative injury, and neutrophil accumulation at reflow) were significantly further improved after stimulation of PAR-2. The specificity of the PAR-2AP action was confirmed by the lack of any effect of the scrambled peptide. Other chemical agents that improved efficiency of IP are reviewed in Ref. 31. More importantly, the effects of IP were also evident after a brief stimulation with PAR-2AP as demonstrated by the results obtained by applying protocol 2 (i.e., only during the transient ischemic stimulus). These data imply that PAR-2-dependent signaling events are involved during IP stimulus. It would be interesting to know whether PAR-2AP improves the efficiency of multiple episodes of IP. In our hands, IP also reduces neutrophil accumulation after ischemic injury, and exposure to PAR-2AP significantly enhances these protective effects. In addition, because modulation of reperfusion flow during IP can protect the heart from reperfusion injury (31), PAR-2 may exert a protective effect on myocardial tissue by enhancing coronary flow at reperfusion time.

Several studies have established that GSSG is a reliable indicator of oxidative stress in the heart (19, 20). In the glutathione cycle, oxidative compounds are partially metabolized in a glutathione peroxidase-catalyzed reaction with GSH, which is present in large amounts inside the cells. GSSG formation is subsequently reduced to restore GSH. However, when the cells are exposed to a large amount of oxidants, GSSG formation may exceed the rate of metabolism, resulting in a condition of "oxidative stress" (3, 16). Besides, activation of PAR-2 induces a significant decrease in GSH and GSSG release but does not interfere substantially with tissue activities of glutathione peroxidase and reductase. These results imply that following PAR-2 activation there is a direct effect on the heart because glutathione peroxidase and glutathione reductase, enzymes specifically involved into the metabolism of GSH and GSGG, were not inhibited.

Indeed, to counteract the effects of toxic oxygen metabolites, the cells are endowed with radical scavenging systems. As described Napoli et al. (28), hearts exposed to PAR2-AP have significantly more elevated activity of Mn-superoxide dismutase. The above results also fit well with the dose-dependent reduction in peroxidation and cardiac postischemic tissue damage. Further studies should investigate whether the PAR-2AP-induced protective effects are mediated by cytokine release, nitric oxide, potassium ATP channels, and/or protein kinase C (6, 31).

In addition, as also previously described (28), hearts exposed to PAR2-AP have shown significantly increased Mn-superoxide dismutase activity. These findings fit well with the dose-dependent reduction in peroxidation and the observed cardiac postischemic tissue damage. Our results are also in agreement with a recent study where it has been shown that IP reduces neutrophil accumulation in humans (21). The involvement of PAR-2 in cardiac inflammation also fits the hypothesis that the infection-inflammation triggers acute coronary syndromes (6, 10, 23, 25, 38). Bacterial proteinases also activate PAR-2 on neutrophils (23), and it is likely that PAR-2 may represent one of the first alarm protective mechanisms. Overall, our results indicate that activation of PAR-2 is an early signaling event associated with the cardiac balance involved in the response to cardiac ischemia and endogenous protective effect exerted by IP (31).

Further studies are needed to evaluate the effect of PAR-2AP infusion after myocardial ischemia in a possible stimulation of a clinical scenario in which this treatment is associated with thrombolysis and/or primary angioplasty. Modulation of PAR-2 may represent a clinically relevant therapeutically target for controlling cardiac response to ischemic injury.