Pflugers Arch - Eur J Physiol (2011) 462:219–233 DOI 10.1007/s00424-011-0970-1 CARDIOVASCULAR PHYSIOLOGY Post-ischemic early acidosis in cardiac postconditioning modifies the activity of antioxidant enzymes, reduces nitration, and favors protein S-nitrosylation Claudia Penna & Maria-Giulia Perrelli & Francesca Tullio & Francesca Moro & Maria Laura Parisella & Annalisa Merlino & Pasquale Pagliaro Received: 3 March 2011 / Revised: 14 April 2011 / Accepted: 18 April 2011 / Published online: 5 May 2011 # Springer-Verlag 2011 Abstract Postconditioning (PostC) modifies the early postischemic pH, redox environment, and activity of enzymes. We hypothesized that early acidosis in PostC may affect superoxide dismutase (SOD) and catalase (CAT) activities, may reduce 3-nitrotyrosine (3-NT) protein levels, and may increase Snitrosylated (SNO) protein levels, thus deploying its protective effects. To verify this hypothesis, we studied the early (7th min) and late (120th min) phases of reperfusion (a) endogenous SOD and CAT activities and (b) 3-NT protein levels and SNO protein levels. Isolated rat hearts underwent 30-min ischemia/120-min reperfusion (I/R) or PostC (5 cycles of 10-s I/R at the beginning of 120-min reperfusion) either with or without exogenous CAT or SOD infused during the initial 3 min of reperfusion. The effects of early reperfusion with acid buffer (AB, pH 6.8) on endogenous antioxidant enzymes were also tested. Pressure, infarct size, and lactate dehydrogenase release were also measured. At the 7th min, PostC C. Penna : M.-G. Perrelli : F. Tullio : F. Moro : A. Merlino : P. Pagliaro Department of Biological and Clinical Sciences, University of Torino, Orbassano, Turin, Italy C. Penna : M.-G. Perrelli : F. Tullio : F. Moro : P. Pagliaro National Institute of Cardiovascular Research (INRC), Bologna, Italy M. L. Parisella Department of Pharmaco-Biology, University of Calabria, Arcavacata di Rende, Cosenza, Italy P. Pagliaro (*) Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Regione Gonzole 10, 10043 Orbassano, Turin, Italy e-mail: pasquale.pagliaro@unito.it induced a significant decrease in SOD activity with no major change both in Mn and Cu/Zn SOD levels and in CAT activity and level. PostC also reduced 3-NT and increased SNO levels. Exogenous SOD, but not CAT, abolished PostC cardioprotection. In late reperfusion (120-min), I/R increased SOD activity but decreased CAT activity and Cu/Zn SOD levels; these effects were reversed by PostC; 3-NT was not affected, but SNO was increased by PostC. AB reproduced PostC effects on antioxidant enzymes. The conclusions are as follows: PostC downregulates endogenous SOD and preserves CAT activity, thus increasing SNO and reducing 3-NT levels. These effects are triggered by early post-ischemic acidosis. Yet acidosis-induced SOD downregulation may limit denitrosylation, thus contributing to PostC triggering. Hence, exogenous SOD, but not CAT, interferes with PostC triggering. Prolonged SOD downregulation and SNO increase may contribute to PostC and AB beneficial effects. Keywords Cardioprotection . Infarction . Ischemia . Postconditioning . Reperfusion injury Introduction Clearly, the only way to limit ischemia damage is organ reperfusion. However, with reperfusion, injury amplification (i.e., reperfusion injury) occurs. In fact, in early postischemic phase, mitochondrial permeability transition pore (mPTP) formation may be the event that would lead to irreversible changes in cellular function and cell death, e.g., [4, 9, 26, 27, 34]. At the onset of a rapid reperfusion, when a large increase in reactive oxygen species (ROS) formation occurs along with pH recovery and Ca2+ overload, mPTP opening is facilitated. This, in turn, favors massive ROS formation by inhibiting the respiratory chain [26, 27, 34]. 220 Therefore a vicious cycle is likely to be established in the so-called ROS-induced ROS release (RIRR) [26, 27, 34], thus inducing ROS stress and reperfusion injury (for reviews, see [46, 50]). Survival mechanisms in the heart can be triggered by short, non-lethal periods of ischemia and reperfusion, applied either before (preconditioning) or immediately after (postconditioning, PostC) the index ischemia [3, 10, 20, 25, 33, 44–46, 62]. Both in vivo and in vitro ischemic and pharmacological preconditioning triggering require protective ROS signaling. In fact, either ROS generators or ROS scavengers, given before the index ischemia, trigger or abolish preconditioning [2, 7, 10, 16, 23, 43, 47, 48, 61]. Therefore, ROS are double-edge swords (ROS stress vs. ROS signaling). Also, PostC protection requires ROS signaling and delayed recovery of intracellular pH during initial reperfusion. The mechanisms by which PostC alters the pathophysiology of reperfusion injury involve molecular and physiological mechanisms, such as delaying re-alkalinization of tissue pH [8, 9, 17, 29, 30, 55], triggering the release of ROS and autacoids [13, 46, 50–52, 54, 61], and activation of kinases that impact cellular and subcellular targets or effectors, such as mPTP (e.g., [20, 46, 50, 52]). Importantly, acid buffer (AB) given for the first few minutes of reperfusion is as protective as a PostC protocol [8, 9, 13, 17, 29, 30, 34, 55]. This supports the idea that the delayed recovery of intracellular pH during initial reperfusion is mandatory to limit infarct size in PostC mainly via the prevention of mPTP opening and prevention of RIRR [8, 9, 17, 29, 30, 34, 55]. Both in vitro and in vivo studies demonstrated that large-spectrum ROS scavengers such as N-acetyl-L-cysteine (NAC) and/or N-(2-mercaptopropionyl)-glycine (MPG) given either during the PostC maneuvers [8, 51, 54, 63] or during AB infusion [8–10, 13] prevented their protective effects [13, 51, 54, 63]. Moreover, addition of alkaline buffer in early reperfusion abolishes both AB and PostC protection [8, 9, 17, 29, 55]. Therefore, it is likely that interplays between redox and acid/base conditions in early reperfusion exist. Clearly, the PostC cycles (i.e., intermittent re-oxygenation) and the early acidosis may be responsible of modifications of enzyme activities and chemical reactions, which could lead to produce a few ROS with signaling role instead of ROS stress [13, 51, 53]. PostC protection is also influenced by the interplay between endothelial and cardiac function, with a flow of nitric oxide (NO•) from endothelium to myocardium [20, 46, 50, 67]. Indeed the persistence of acidosis is important for non-enzymatic NO• production [59, 69] so that ROS in concert with NO• and reactive nitrogen species (RNS) may put the heart into a protected state [26, 38, 46, 49–52, 63]. Actually, in addition to activating cyclic guanosine monophosphate/protein kinase G-dependent signaling pathways, NO• and RNS can modify sulfhydryl residues of Pflugers Arch - Eur J Physiol (2011) 462:219–233 protein through S-nitrosylation, which has emerged as an important post-translational protein modification [11, 24, 40, 56, 57]. Nitrosylation has been proposed to be important in the preconditioning cardioprotection, also modifying mitochondrial proteins and limiting oxidative stress [6, 36, 56, 57]. Although PostC requires NO• [28, 29, 33, 49, 52], nitrosylation has not been studied under this condition [56]. Yet, the studies on nitration of proteins with 3-nitrotyrosine concentration (a marker for ONOO− formation) achieved contradictory results: while Kupai et al. [32] reported that PostC increases cardiac 3-NT after 5 min of reperfusion, Iliodromitis et al. [28] observed that PostC reduces the myocardial and circulating levels of 3-NT after 10 min of reperfusion. Wang et al. [65] also observed a decrease in ONOO− formation after hypoxic PostC. Moreover, we have been unable to reproduce cardioprotection with ROS generator given during early reperfusion [51]. Since ROS scavengers (NAC or MPG) abolish ABand PostC-induced protection [8, 9, 51, 54, 63], it is likely that the type, the concentration, and/or the compartmentalization of ROS/RNS may play a pivotal role in triggering protection at early reperfusion time. To shed some light on this scenario, we studied antioxidant enzymes, nitration, and S-nitrosylation of proteins. Many endogenous enzymes regulate the homeostasis of ROS/RNS. In the myocardium, three iso-forms of superoxide dismutases (SOD), which convert superoxide (O2−) to hydrogen peroxide (H2O2), have been described. The steady-state levels of H2O2 by conversion to water are regulated by enzymes such as catalase (CAT) and glutathione peroxidase (GPx) [35, 64]. Importantly, the activity and levels of endogenous antioxidant iso-enzymes are differently affected by ischemia/reperfusion (I/R) in the various tissues and compartments [12, 37]. Yet, little is known about the activity of antioxidant enzymes during PostC in the cardiac tissue. While the optimal pH is 7.0 for CAT activity, it is 7.8 for SOD. Intriguingly, SOD is also a de-nitrosylating enzyme in different systems [21, 42, 56]. Therefore, we hypothesized that intracellular acidosis during PostC and AB in early reperfusion may downregulate SOD activity and may lead to modifications of 3-NT and SNO levels (Fig. 1). We also hypothesized that a delicate balance between acidosis, ROS/RNS, the S-nitrosylation, and nitration/oxidation may occur in early reperfusion [52, 56]. It is, thus, likely that the presence of additional specific antioxidant (SOD or CAT) would alter differently the delicate balance induced by PostC maneuvers in early reperfusion. It is also likely that when post-ischemic pH is recovered in late reperfusion, enzyme activity is also recovered. Therefore, in the present study, we assessed: (1) the ability of two specific antioxidant enzymes (exogenous SOD or CAT) given during early reperfusion to abolish Pflugers Arch - Eur J Physiol (2011) 462:219–233 221 PostC (early acidotic reperfusion) O2- Intermittent reintroduction of O2 Early acidosis Hypotheses Flow of reactions Established facts Non en z ym produc atic tion SOD H2 O2 CAT H2O Early acidosis NO. Cysteine oxidation eNOS activation ONOOP-Tyr-3-NO2 Tyrosine nitration NO De N2O3 GS SOD ni tro sy la tio n Secondary reaction P-SNO S-nitrosylation Fig. 1 Suggested schematic chemical relationship among different reactive oxygen species and reactive nitrogen species in PostC and early acidotic reperfusion. The scheme represents a summary of (1) established facts, (2) well-known flow of chemical reactions, and (3) hypotheses; two dotted verticals lines divide the scheme in these three elements. The scheme is built starting from the evidences that in early reperfusion postconditioning (PostC) are characterized by intermittent reintroduction of O2, reactive oxygen species signaling, persistence of acidosis, and nitric oxide (NO•) production by endothelial nitric oxide synthase (eNOS) and non-enzymatic processes [8, 9, 17, 20, 28–30, 46, 50, 55, 56, 65]. Based on superoxide (SOD) optimal pH (7.8) and catalase (CAT) optimal pH (7.0), the scheme suggests the hypotheses of SOD down-regulation (arrow down) and CAT up-regulation (arrow up) by the early acidosis, which may be a key event for PostC cardioprotection [8, 9, 17, 29, 30, 55]. In the excess of NO•, the secondary reaction between ONOO− and NO• may increase [60, 66] and this might favor S-nitrosylation; SOD down-regulation may limit denitrosylation. Therefore, a reduction in tyrosine nitration (arrow down) and an increase in S-nitrosylation (arrow up) are also suggested. In the scheme, the processes, the enzyme activities and the reactions hypothetically decreased/reduced by PostC/acidosis are in dashed lines. GSNO S-nitrosoglutathione (see text for further explanation) PostC triggering, (2) the activity and the level of endogenous SOD and CAT in early and late reperfusion either after PostC or early AB infusion, and (3) the effects of PostC with and without exogenous SOD on the levels of 3-NT and SNO proteins in early and late reperfusion. Male Wistar rats (5–6 months old) (Janvier S.A.S., St Berthevin Cedex, France) received humane care in compliance with Italian law (DL-116, Jan. 27, 1992) and in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). retrogradely perfused with oxygenated Krebs–Henseleit buffer (127 mM NaCl, 17.7 mM NaHCO3, 5.1 mM KCl, 1.5 mM CaCl2, 1.26 mM MgCl2, 11 mM D-glucose (Sigma-Aldrich Corp., St. Louis, MO, USA), and gassed with 95% O2 and 5% CO2). A constant flow was adjusted with a pump to obtain a perfusion pressure of 80–85 mmHg during stabilization. Thereafter, the same flow level (9± 1 ml/min/g) was maintained throughout the experiment. A balloon was placed into the left ventricle and connected to an electromanometer to record left ventricular pressure (LVP). The balloon was filled with saline to achieve an end-diastolic LVP (EDLVP) of 5 mmHg. Coronary perfusion pressure, coronary flow, EDLVP, and developed LVP (DevLVP) were monitored to assess the preparation conditions. The hearts were electrically paced at 280 bpm and kept in a temperature-controlled chamber (37° C). Isolated heart perfusion Experimental protocols (Fig. 2) The methods were similar to those previously described [45, 50–54]. In brief, each animal was anesthetized. Then, 10 min after heparin treatment, the heart was rapidly excised, weighed, attached to the perfusion apparatus, and After 30 min of stabilization, hearts were subjected to a specific protocol, which was the same for all groups and included 30 min of global, normothermic ischemia and 120 min of reperfusion followed by 30 min of ischemia (see Materials and methods Animals 222 Fig. 2 Experimental design. The isolated, Langendorff-perfused hearts were stabilized for 30-min (Stab) and then subjected to 30 min of normothermic, global ischemia (I) followed by 120-min of reperfusion (R). Postconditioning (PostC) protocol (5 cycles 10-s ischemia/reperfusion) is indicated by vertical lines at the beginning of the reperfusion period. Treatment with exogenous active or inactivated antioxidant enzymes: either catalase (CAT, CATi) or superoxide dismutase (SOD, SODi) has been infused for 3 min during early reperfusion, as indicated by horizontal black lines. In additional experiments (Sham, I/R, PostC, and acid buffer), the activity of CAT and SOD was tested at specific time-points (baseline, 7 min, and 120 min after the beginning of reperfusion; Sham hearts were tested at corresponding time-points of perfusion only as indicated by the arrows; for further explanation, see text) Pflugers Arch - Eur J Physiol (2011) 462:219–233 Experimental Design I/R, Group 1 Stab I (30 min) Stab I (30 min) R (120 min) PostC, Group 2 R (120 min) PostC+CAT, Group 3; PostC+SOD, Group 4; Stab I (30 min) R (120 min) I/R+CAT, Group 5; I/R+SOD, Group 6. Stab I (30 min) R (120 min) PostC+ CATi, Group 7; PostC+ SODi, Group 8 Stab I (30 min) R (120 min) 3 min Additional experiments Sham Buffer Perfusion I/R Stab I (30 min) R (120 min) PostC Stab I (30 min) R (120 min) AB Stab I (30 min) baseline below). Pacing was discontinued on initiation of ischemia and restarted after the 3rd minute of reperfusion in all groups [44, 50–54]. After stabilization, the hearts of the control group (I/R, group 1, n=12) were exposed to 30 min of ischemia and then to 120 min of reperfusion only. In group 2 (PostC group; n=12), after the 30-min ischemia, the hearts immediately underwent a protocol of PostC (i.e., 5 cycles of 10-s reperfusion and ischemia) [50–54]. Antioxidants, either CAT or SOD (Sigma-Aldrich Corp., USA), were given at the beginning of reperfusion for 3 min, with and without PostC, at doses previously used in isolated rat hearts [1]. In particular, group 3 (PostC+CAT, n=10) and group 4 (PostC+SOD, n=10) hearts underwent I/R plus PostC, in the presence of CAT (100 U/ml) or SOD (10 U/ml), respectively; group 5 (I/R+CAT, n=9) and group 6 (I/R+SOD, n=9) hearts underwent I/R only in the presence of CAT (100 U/ml) or SOD (10 U/ml), respectively [1]. For comparative purposes, the hearts were also perfused in the presence of PostC maneuvers with heat-inactivated CAT (100 U/ml; PostC+CATi, group 7, n=6) or heat- R (120 min) 7th 120th min inactivated SOD (10 U/ml; PostC+SODi, group 8, n=6). The inactivated enzymes were infused at the beginning of reperfusion for 3 min. Heat inactivation was obtained as previously described and confirmed by spectrophotometer analysis. Non-heated exogenous enzymes were normally active [14, 58]. In other experiments (n=4 for each group), hearts underwent either I/R or PostC in the presence of SOD (10 U/ml) plus CAT (100 U/ml) co-infused for an initial 3 min of reperfusion (not shown in Fig. 2). CAT and SOD activities and levels Additional hearts (Fig. 2; n=6 for each group) were subjected to 180-min perfusion only (i.e., Sham), I/R only, PostC, or AB (pH 6.8) in early reperfusion. AB method was identical to that reported by Rodríguez-Sinovas et al. [55]. In these hearts, the activities and levels of endogenous CAT and SOD were tested at specific time-points (baseline, 7 min, and 120 min after the beginning of reperfusion; Sham hearts were tested at corresponding time-points of Pflugers Arch - Eur J Physiol (2011) 462:219–233 223 Homogenization protocol: in order to check the protein levels, samples of Sham, I/R, PostC, and AB hearts (n=6 for each group) were homogenized on ice in RIPA lysis buffer (Santa Cruz Biotechnology) using a polytron tissue grinder. The homogenate was centrifuged at 4°C for 30 min at 13,000 g, and the supernatant was collected to quantify proteins with Bradford’s method [5]. Western blot analyses were performed as previously described [53]. with Bradford’s method [5], and the biotin switch assay with or without ascorbate (1 mM) was performed as previously described [31]. In particular, blocking buffer (225 mmol l−1 Hepes, pH 7.7, 0.9 mmol l−1 EDTA, 0.09 mmol l−1 neocuproine, 2.5% SDS, and 20 mmol l−1 MMTS) and HENS buffer (250 mmol l−1 Hepes, pH 7.7, 1 mmol l−1 EDTA, 0.1 mmol l−1 neocuproine, and 1% SDS) were used (Sigma-Aldrich Corp) [31]. To detect biotinylated proteins by Western blot, samples from the biotin switch assay were separated on 12% SDS– PAGE gels, transferred to PVDF membranes (GE Healthcare Biosciences Piscataway, NJ, USA), blocked with non-fat dried milk (Santa Cruz, CA, USA), and incubated with streptavidin peroxidase, diluted at 1/10,000 for 1 h. In order to ascertain that variations of nitrosylation occur within cells, Snitrosylations of cardiac electron transfer flavoprotein α,β (ETFAα,β) and von Willebrand Factor (vWF) were tested in separated assays (two samples, for two hearts for each group): after detection of biotin, the membranes were stripped and reincubated with polyclonal antibodies against ETFAα,β (Santa Cruz, CA, USA) and vWF (Dako, Denmark) [18, 19]. vWF was tested because of its localization within endothelial cells and ETFAα,β was tested because the mitochondrial proteins extracted from whole hearts originate mainly from cardiomyocytes. To confirm equal protein loading, membranes were incubated with an anti-β-actin antibody (Sigma-Aldrich Corp). The S-immunoblotted proteins were visualized by using Immuno-Star HRP Substrate Kit (Bio-Rad Laboratories) and quantified by Kodak Image Station 440CF. Image analyses were performed by the Kodak 1D 3.5 software. S-nitrosylated and 3-nitrotyrosine myocardial protein levels 3-Nitrotyrosine assay Hearts (n=6 for each group) subjected to I/R only, PostC, PostC+SOD, or perfusion only (i.e., Sham) were used to assess the levels of S-nitrosylated and 3-NT at baseline, 7 min after the beginning of reperfusion and at the end of reperfusion; Sham hearts were tested at corresponding timepoints of perfusion only. The protein concentrations of the samples were quantified with Bradford’s method [5]. Nitrotyrosine, as a marker of nitrosative stress, was measured with a competitive enzyme immunoassay: the levels of 3-NT were measured according to the manufacturer’s directions using Cell Biolabs kit (Cell Biolabs, Inc., San Diego, CA, USA). The detection sensitivity range was 20 nM to 8 μM of 3-nitrotyrosine-BSA equivalent. perfusion only). The 7-min reperfusion time-point was employed on the basis of previous studies on PostC triggering [4, 39, 62]. CAT and SOD activities by spectrophotometric analysis Activity was analyzed according to the manufacturer’s directions using Cayman chemical kits (Ann Arbor, MI, USA). Briefly, tissues from the heart of rats were homogenized, cell debris was pelleted, and the resulting supernatants were used for the enzyme activity assays. CAT and SOD activities were measured at 540- and 450-nm wavelengths, respectively. A unit of CAT activity was defined as the amount of enzyme that caused the formation of 1.0 nmol of formaldehyde per minute at 25°C. A unit of SOD activity was defined as the amount of SOD needed to exhibit 50% dismutation of the produced superoxide radical at 25°C. The final enzymatic activities were calculated by normalizing the results to the total protein concentration of the whole extract. CAT and SOD levels by Western blot analysis Biotin switch assay and Western blot for the detection of S-nitrosylated proteins Assessment of myocardial injury All preparative procedures were performed in the dark to prevent light-induced cleavage of nitrosylations [31]. Heart samples, collected at specified time-points, were homogenized and centrifuged on ice in appropriate buffers (20 mmol l−1 Tris, pH 7.5, 150 mmol l−1 NaCl, 1% Igepal CA 630, 0.5% sodium deoxycholate, 1 mmol l−1 EDTA, 0.1% SDS, 200 mmol l−1 sodium orthovanadate, and protease inhibitor cocktail (Sigma-Aldrich Corp.)) using a polytron tissue grinder. Proteins were quantified Since in isolated hearts the pre- and postconditioning are known to reduce the release of lactate-dehydrogenase (LDH) during reperfusion, the release of LDH was measured during the 2 h of reperfusion as previously described [45, 49, 51, 53]. Infarct areas were also assessed at the end of the experiment with the nitro-blu-tetrazolium technique (Sigma-Aldrich) as described [45, 49, 51, 53, 54]. The necrotic mass was expressed as a percentage of total left ventricular mass (i.e., risk area). 224 Pflugers Arch - Eur J Physiol (2011) 462:219–233 Statistical analysis All data are expressed as means±S.E.M. One-way ANOVA and Newman–Keuls multiple comparison test (for postANOVA comparisons) were used to evaluate the statistical significance among groups. A p value <0.05 was considered as statistically significant. Results PostC improvement of cardiac function was abolished by exogenous SOD Table 1 shows I/R (group 1) as having markedly increased CPP and EDLVP and drastically reduced DevLVP. PostC (group 2) limited CPP increase and improved the recovery of DevLVP by attenuating the increase in EDLVP during reperfusion. While CAT (group 3) did not affect the PostCinduced improvement of function, SOD (group 4) abolished these protective effects of PostC. The treatment with either CAT or SOD during the first 3 min of reperfusion in the I/R+CAT or I/R+SOD hearts (groups 5 and 6, respectively) did not significantly change the deleterious effects of I/R on mechanical function. Moreover, the treatment with inactivated enzymes (CATi or SODi) in the PostC+CATi or PostC+SODi hearts (groups 7 and 8, respectively) did not significantly change the beneficial effects of PostC on postischemic function. PostC reduction in infarct size and LDH release were reversed by exogenous SOD Infarct size (Fig. 3a), expressed as a percentage of risk area, was 61 ± 5% in control (group 1); PostC (group 2) significantly reduced the infarct size to 34±5% (p<0.01 vs. control). PostC+CAT (group 3) induced a significant reduction of infarct size to 37±5% (p<0.01 vs. control; NS vs. PostC; Fig. 3a). In PostC+SOD (group 4), the infusion of exogenous SOD abolished the cardioprotection by PostC (infarct size 73±8%, p = NS vs. control). The treatment with either CAT or SOD in the I/R+CAT or I/ R+SOD hearts (groups 5 and 6, respectively) did not significantly change the infarct size (71±9% and 77±5%, respectively) with respect to the control (Group 1). In hearts of Groups 7 and 8 with inactivated enzymes (PostC+CATi and PostC+SODi, respectively), PostC maneuvers still induced cardioprotection. In particular, infarct size was 36±11% in PostC+CATi and 22±8% of risk area in PostC+SODi (p = NS vs. each other and vs. PostC, for both). The infarct size data are corroborated by LDH release during reperfusion (Fig. 3b). In fact, LDH release into the coronary venous effluent was 969±53 U/g wet weight in the control (group 1). LDH release was significantly reduced by PostC (group 2; p<0.05 vs. control). Also in PostC+CAT (group 3), a marked reduction of LDH release (p<0.01 vs. control; p = NS vs. PostC) was observed. However, in PostC+SOD (group 4), LDH release was not different from that observed in control. In I/R+CAT (group 5) or I/R+SOD (group 6), LDH release was also similar to that of control. In hearts of groups 7 and 8 (PostC+CATi and PostC+SODi, respectively), PostC maneuvers still reduced LDH release (p = NS vs. each other and vs. PostC, for both; Fig. 3b). In the experiments with the co-infusion of CAT+SOD for an initial 3 min, either with or without PostC maneuvers, both infarct size (78±5% and 69±4%, respectively) and LDH release (858±185 or 931±196 U/g, respectively) resulted similar to those of control group (data not reported in Fig. 3). Notably, once again, the Table 1 Cardiac function parameters Group Before ischemia CPP mmHg At the end of reperfusion LVEDP mmHg DevLVP mmHg CPP mmHg LVEDP mmHg DevLVP mmHg Group 1 (I/R) 83±3 5±1 82±4 178±5 49±2 26±3 Group 2 (PostC) 82±3 5±2 84±5 115±7* 29±2* 45±3* Group 3 (PostC+CAT) 80±2 6±2 74±5 125±9* 30±3* 46±2* Group 4 (PostC+SOD) 85±4 4±1 73±1 180±3 47±2 22±1 Group 5 (I/R+CAT) 84±3 6±1 82±1 151±5 52±1 21±1 Group 6 (I/R+SOD) 86±3 5±1 77±9 170±4 45±2 26±1 Group 7 (PostC+CATi) 80±5 6±2 75±52 110±4* 31±5* 42±5* Group 8 (PostC+SODi) 86±4 5±1 77±5 127±5* 30±2* 41±3* There are no differences among groups before ischemia. At the end of reperfusion, all parameters are significantly (p<0.05 for all) different from those observed before ischemia CPP coronary perfusion pressure, LVEDP left ventricular end diastolic pressure, DevLVP developed left ventricular pressure *p<0.05 vs. I/R group 1 at the end of reperfusion Pflugers Arch - Eur J Physiol (2011) 462:219–233 Infarct size 100 p = NS 75 ** PostC+CATi I/R+SOD I/R+CAT PostC+SOD PostC 25 I/R ** ** ** PostC+ SODi 50 PostC+CAT a % of risk area 8 Gr Gr ou p ou p7 ou p Gr Gr Gr 6 5 ou p 4 ou p 3 ou p 2 Gr LDH release p = NS p = NS 1500 p = NS ** addition of exogenous SOD in reperfusion abolished PostC protection. Both PostC and AB reversed the increase in SOD activity induced by I/R in early reperfusion In the additional experiments, the basal activity of endogenous CAT and SOD, detected after 30 min of stabilization, was 106±2.4 mol/min/ml and 1.51±0.1 U/ ml, respectively. In Fig. 4, data are represented as percent variation with respect to baseline level. No appreciable changes in CAT and SOD activities with respect to baseline level are observed on samples of Sham hearts collected after a further 37 or 150 min of perfusion. These timepoints in Sham hearts correspond to the 7th and 120th min of reperfusion in I/R and PostC hearts, i.e., the time-points at which in the I/R, PostC, and AB groups the analysis of enzyme activity was also measured. As can be seen in the I/R samples, at the 7th minute of reperfusion (Fig. 4a, b), the CAT activity was 98±2% 8 PostC+ SODi PostC+CATi ** ou p Gr Gr ou p 7 I/R+SOD ou p6 ou p Gr Gr 5 I/R+CAT PostC+SOD 3 ou p Gr ou p Gr 4 PostC 2 I/R Gr ou p 1 0 * PostC+CAT * 500 ou p U/g ww 1000 Gr b Gr ou p ou p 1 0 Gr Fig. 3 Analysis of ischemia/ reperfusion injury and cardioprotective effects on infarct size and LDH release. a Infarct size (percent of risk area). The amount of necrotic tissue is expressed as percent of the left ventricle, which is considered as the risk area. b LDH release. The amount of LDH released during reperfusion is expressed as an international unit for grams (wet weight) of hearts. Groups are as in Fig. 1. * p<0.05, **p<0.01 vs. Control groups. NS non-significant. Groups 1 and 2, n=12 each; Groups 3 and 4, n=10 each; Groups 5 and 6, n=9 each; Groups 7 and 8, n=6 each 225 (p = NS vs. baseline and Sham) and SOD activity was 139±17% (p<0.05 vs. both baseline and Sham). Yet, in the PostC samples, the CAT activity was 126±20% (p = NS vs. its baseline and other groups) and SOD activity was 78± 4% of baseline (p<0.05 vs. baseline, Sham and I/R). Notably, SOD activity resulted to about 50% lower than that observed in I/R group. A similar trend in CAT and SOD activity variation was observed after AB infusion (Fig. 4a, b). In summary, at early reperfusion, PostC and AB were associated with significant decreases in total SOD activity with respect to I/R and with no significant change in catalase activity. Both PostC and AB prevented the decrease of CAT activity and attenuated the increase in SOD activity induced by I/R in late reperfusion At the 120th minute of reperfusion (Fig. 4c, d), in the I/R samples, the CAT activity was 50±10% (p<0.05 vs. baseline, Sham and the 7th min) and SOD activity was 176±6% 226 Antioxidant Enzyme Activity a c Activity of catalase at 7th min Activity of catalase at 120th min p = NS 150 150 # % of basal % of basal Fig. 4 Antioxidant enzyme activity in Sham, ischemia/reperfusion (I/R), postconditioning (PostC) and acid buffer (AB) hearts. a, b Catalase and superoxide dismutase (SOD), respectively, at the 7th min of reperfusion. c, d Catalase and superoxide dismutase (SOD), respectively, at the 120th min of reperfusion. Data are presented as percent variation of baseline level. §p<0.05, §§p<0.01 vs. baseline level; *p<0.05, **p<0.01 vs. Sham; #p<0.05 vs. I/R; $p<0.05, $$ p<0.01 vs. corresponding group at the 7th min of reperfusion. NS nonsignificant. n=6 for each group Pflugers Arch - Eur J Physiol (2011) 462:219–233 100 50 §*$ 50 0 0 Sham PostC AB I/R b # 100 Activity of SOD at 7th min d Activity of SOD at 120th min p = NS p = NS p = NS 200 200 §§** $$ 100 50 0 (p<0.01 vs. baseline, Sham and the 7th min). That is, in I/R, the variations of post-ischemic enzyme activities observed at the 7th min are intensified at the 120th min. Yet, in the PostC samples, the CAT activity was 88±9% (p = NS vs. baseline, Sham and the 7th min; p<0.05 vs. I/R) and SOD activity was 123±5% (p<0.05 vs. baseline, Sham, I/R and the 7th min); SOD and CAT activities were similarly affected by AB infusion (Fig. 4c, d). That is, both PostC and AB prevent the decrease of CAT activity and attenuate the increase in SOD activity otherwise induced by I/R in late reperfusion. Levels of CAT, Cu/Zn-SOD, and Mn-SOD were not affected by either I/R, PostC, or AB in early reperfusion An enzyme level analysis was performed before and after (i.e., at the 7th and 120th min of reperfusion) the index ischemia in I/R, PostC, and AB groups, as well as in corresponding time-points in Sham group (Fig. 5). Notably, Western blotting analysis of tissues revealed that, after the 7th min of reperfusion, there were no significant changes in protein levels in all groups (Fig. 5a–c). Levels of Cu/Zn-SOD were reduced only in I/R, but not in PostC or AB, in late reperfusion However, after 120 min of reperfusion, a significant reduction (p<0.01 vs. Sham) of Cu/Zn-SOD was detected # §*# % of basal % of basal §* 150 150 §*# $ *# 100 50 0 only in I/R; this was reversed by PostC and AB (p<0.01 vs. I/R for both) (Fig. 5e). That is, in PostC, the levels of the two SODs were similar to those of Sham and AB hearts. The catalase levels were stable in all of the experimental conditions tested. Data suggest that there is a leakage of the cytoplasmatic Cu/Zn-SOD in late reperfusion, which was prevented by PostC and AB. Other enzymes are confined in the organelles and their levels do not change in any relevant way. Alternatively, the initial variations in enzyme transcription activated by stress may be responsible of the observed differences. PostC reversed the reduction in S-nitrosylated protein levels induced by I/R in a SOD-dependent manner in early reperfusion Nitrosylated protein levels were also studied before ischemia, at the 7th and 120th min of reperfusion in I/R, PostC, and PostC+SOD groups and at corresponding timepoints in Sham group (Fig. 6). At the 7th min, the post-ischemic levels of S-nitrosylated proteins were different from the baseline levels in both I/R and PostC. As can be seen in Fig. 6a, in a broad range (5– 70 kDa), a lower amount of S-nitrosylated proteins was present in I/R, whereas in PostC a higher grade of Snitrosylated proteins was observed. Their quantification is shown in Fig. 6b: the amount of S-nitrosylated proteins was Pflugers Arch - Eur J Physiol (2011) 462:219–233 227 Fig. 5 Antioxidant enzyme protein levels analyzed by Western blot in Sham, ischemia/reperfusion (I/R), postconditioning (PostC), and acid buffer (AB) hearts. a–c Mn/SOD, Cu–Zn/ SOD and catalase, respectively, at 7th min of reperfusion. d–f Mn/SOD, Cu–Zn/SOD and Catalase, respectively, at 120th min of reperfusion. Data in bar graph are mean (± SE) and are presented as percent variation of baseline level. **p<0.01 vs. Sham, ##p<0.01 vs. I/R. n=6 for each group (for further explanation, see text) low in I/R, markedly increased in PostC, and reduced by the addition of SOD (PostC+SOD). Enhanced Snitrosylation of the intracellular proteins (mitochondrial ETFAαβ and endothelial vWF) as a consequence of PostC and the reduction of their levels by SOD addition are shown in Fig. 7. PostC limited the reduction in S-nitrosylated protein levels induced by I/R in a SOD-dependent manner in late reperfusion At the end of reperfusion (Fig. 6c, d), the amount of protein S-nitrosylated was reduced significantly (p<0.01) in the I/R, PostC, and PostC+SOD with respect to the Sham group. Although the levels of S-nitrosylated proteins were significantly (p<0.05) lower than those observed at the 7th min, the grade of nitrosylation observed in the PostC group was still significantly (p<0.05) higher than those observed in I/R and PostC+SOD groups (p = NS, vs. each other). PostC reversed the increase in 3-nitrotyrosine levels induced by I/R in a SOD-independent manner in early reperfusion only In Fig. 8, 3-NT data are represented as percent variation with respect to baseline level (1,653±107 nM). In I/R group, 3-NT levels showed a significant (p<0.05) increase at the 7th min of reperfusion with respect to corresponding time-point in Sham (Fig. 8a). As observed by Iliodromitis et al. [28], intramyocardial 3-NT levels were significantly (p<0.05) decreased by PostC, being similar to Sham hearts in our experiments, regardless of the presence of exogenous SOD (Fig. 8a). 228 Pflugers Arch - Eur J Physiol (2011) 462:219–233 Fig. 6 S-nitrosylated proteins in Sham, ischemia/reperfusion (I/R), postconditioning (PostC), and PostC+SOD hearts. a–c Representative blots of S-nitrosylated proteins in rat heart homogenates at 7th and 120th min of reperfusion, respectively. To control the specificity of the biotin switch assay, ascorbate has been omitted in Sham samples (−Asc). b, d The bar graphs are means (± SE) of band concentrations at 7th and 120th min of reperfusion, respectively. Data are presented as percent variation of baseline level. *p<0.05, **p<0.01 vs. Sham; #p<0.05 vs. I/R; §p<0.05 vs. PostC; $p<0.05 vs. corresponding group at the 7th min of reperfusion. NS nonsignificant. n=6 for each group At the end of reperfusion (Fig. 8b), the levels of 3-NT in the I/R, PostC, and PostC+SOD groups were similar to those observed in Sham hearts. Discussion Here we show that postconditioning decreases protein nitration and favors protein SNO in the early phase of Fig. 7 S-nitrosylation of ETFAα,β and vWF in Sham, ischemia/ reperfusion (I/R), postconditioning (PostC), and PostC+SOD hearts. The protein bands show an increase in S-nitrosylation during PostC and a reduction during PostC+SOD at 7th min of reperfusion reperfusion; this is associated with a significant decrease in total SOD activity with respect to I/R. Intriguingly, cardioprotection was abrogated by SOD addition during PostC, supporting the idea that PostC triggering may be involved in a redox mechanism, via an increase in O2−• flux. This occurs in a moment in which PostC is prolonging acidosis and favoring NO production [8, 9, 25, 29, 62, 65]. We suggest that, in acidosis, the simultaneous increase of the flux of NO and O2−• favors protein S-nitrosylation and reduces nitration, via a secondary reaction between ONOO− and NO [60, 66] (see Fig. 1). Yet, since acidosis downregulates SOD, we also suggest a pivotal role for the limited denitrosylation by SOD-downregulation in PostC (Fig. 1) [41, 42, 56]. In the late reperfusion phase, SOD activity is increased and catalase activity decreased in I/R, effects that are reversed by PostC. Our results suggest that enzymes were covalently modified in situ either by PostC maneuvers or AB; in fact, the variation in activity started during early reperfusion and persisted in late reperfusion and could be observed also in tissue homogenate when pH was recovered. Hence, acidosis and reintroduction of O2 in early reperfusion permitted ROS/RNS signaling (during PostC Pflugers Arch - Eur J Physiol (2011) 462:219–233 Fig. 8 Levels of 3-nitrotyrosine in Sham, ischemia/reperfusion (I/R), postconditioning (PostC), and PostC+SOD hearts. a, b Mean (± SE) of 3-NT levels in the ventricular tissue after 7 and 120 min of reperfusion, respectively. Data are percent variations of baseline level. §§p<0.01 vs. baseline level; **p<0.01 vs. Sham; #p<0.05 vs. I/R. NS non significant. n=6 for each group maneuvers or AB infusion) which could covalently target enzymes, which in turn modify the redox environment (i.e., nitration/nitrosylation at 7th and 120th min). Our hypotheses and analyses were focused on the PostC triggering phase at the 7th min of reperfusion (early reperfusion) [4, 39, 62]. At this time-point, we studied, after I/R and PostC, both the levels and activities of CAT and SOD and measured the levels of 3-NT and SNO proteins. We also studied these parameters at 120-min reperfusion (late reperfusion) to have information on the persistence of effects and/or on variations of the compartmentalization of the redox environment in late reperfusion, when post-ischemic pH variations should be recovered [8, 9, 17, 29, 30, 55]. The main effects observed in the early and late reperfusion are summarized in Table 2. Early reperfusion Importantly, in early reperfusion, the enzyme levels are similar in I/R and PostC. However, the activity of SOD increases in I/R but is reduced immediately after PostC maneuvers or acidosis. Hence, whenever we add in early reperfusion an active exogenous SOD to the perfusate, PostC is no longer protective. It is likely that the implementation of this enzyme alters the scenario induced 229 by protective PostC, which per se reduces SOD activity. On the contrary, 3 min of an active exogenous CAT does not limit the cardioprotective effects of PostC, which, in fact, per se tends to increase the activity of endogenous CAT with respect to I/R. These data indicate that specific antioxidant enzymes (SOD or CAT) can or cannot abrogate PostC-triggering. However, large spectrum antioxidants (NAC or MPG) abolish both preconditioning and PostC protection in several settings [8–10, 13, 22, 38, 51, 54, 63]. Although the infused enzymes do not easily enter into the cells, enough SOD enters, at least into endothelial cells, to perturb the redox environment created by PostC (i.e., SOD down-regulation), thus blocking protection. Indeed PostC attenuates endothelial cell dysfunction by increasing eNOS activity and NO bioavailability in neighboring cells [20, 67]. The increased availability of NO may, in turn, contribute to SNO formation and PostC protection. It is likely that exogenous SOD during PostC maneuvers may also alter the necessary crosstalk between endothelium and myocardium. The increased S-nitrosylation of endothelial (vWF) and mitochondrial (ETFA α,β) proteins supports the idea that the mechanism is due, at least in part, to a NO signaling from endothelium to cardiomyocytes, which are the richest cells in mitochondria. A pivotal cardioprotective role for NO• from enzymatic and non-enzymatic origin has been shown for both pre- and postconditioning [13, 15, 25, 26, 46–50, 60, 66, 69]. In fact, NO• is a cardiovascular protective molecule via multiple effects, both in normoxia and particularly during reperfusion [15, 50, 66]. Accordingly, in preconditioning, a central role is played by protein SNO, which provides protection preventing further cellular oxidative and nitrosative stress in reperfusion [24, 40, 56]. Yet, the high reduction potential of NO• severely limits the formation of peroxynitrite (ONOO−) in this context [46, 60, 66]. Actually, ONOO− has also been proposed to be cardioprotective at very low concentrations [15, 32, 65, 69]. In particular, PostC in the presence of a peroxynitrite decomposition catalyst (FeTPPS, 5,10,15,20-tetrakis-[4sulfonatophenyl]-porphyrinato-iron [III]) failed to reduce infarct size in rat hearts [32]. This is an indirect observation supporting the fact that an early increase in peroxynitriteinduced nitrosative stress is involved in the triggering mechanism of cardioprotection by PostC. To the best of our knowledge, no studies have shown that peroxynitrite given at reperfusion may mimic the beneficial effects of PostC. On the basis of our study, we suggest that, in protected hearts, adequate levels of NO• can quench the transiently formed ONOO− via a secondary reaction, thus forming N2O3 and leading to protein SNO [40, 66] (Fig. 1). According to Wang et al. [65] and Iliodromitis et al. [28], who observed a marked decrease in 3-NT 10 min after the beginning of reperfusion, but in contrast with Kupai et al. [32], 230 Pflugers Arch - Eur J Physiol (2011) 462:219–233 Table 2 Variations of studied parameters in the early (7th min) and late stages (120th min) in I/ R and postC hearts in comparison with baseline levels The arrows indicate significant percent variations with respect to baseline levels. The number of arrows is related to the extent of percent variation = non-significant variation SOD activity CAT activity Enzyme levels Mn-SOD Cu/Zn-SOD CAT S-nitrosylation Tyrosine nitration Early reperfusion (7th min) Late reperfusion (120th min) I/R PostC I/R PostC ↑↑ = ↓↓ = ↑↑↑ ↓↓ ↑ = = = = ↓↓ ↑↑↑ = = = ↑↑ ↑ = ↓ = ↓↓↓ = = = = ↓ = who observed an increase 5 min after the beginning of reperfusion, we observed a decrease in 3-NT 7 min after the beginning of reperfusion in PostC hearts. To reconcile these apparent opposing results, we can speculate that, after an initial formation of ONOO−, as the reaction product of O2−• and NO•, a further increase in NO•—via enzymatic and nonenzymatic processes [54, 60, 69]—may allow a secondary reaction between NO• and ONOO− [59, 60, 66]. This secondary reaction may be responsible of both ONOO− and 3-NT level lowering and SNO augmentation (Fig. 1). The mechanism of NO-conferred prevention of peroxynitrite-dependent damage has recently been reported and is based on the formation of a nitrosating species in this process [11]. We can argue that the simultaneous presence of elevated levels of NO• and reduced activity of SOD may favor the appropriate amount of protein SNO (Fig. 6a). Actually, our data are in accordance with the reported denitrosylase activity of SOD [21, 42, 56]. In fact, besides an increased nitrosylating activity, an increased level of Snitrosylated protein in PostC hearts may be due to a decreased rate of denitrosylation by SOD downregulation. Indeed preconditioning also delays the denitrosylation that is favored by the high oxygen availability [56, 57], typically of reperfusion. Accordingly, preconditioning also delays the normalization of tissue pH and requires ROS signaling in early reperfusion after the index ischemia [22, 38]. Of note, persistence of acidosis is important for both nonenzymatic NO• production [60, 69] and PostC cardioprotection [8, 9, 17, 29, 34, 55]. In fact, a common mechanism by which ischemic PostC protects the heart is by delaying the normalization of tissue pH, and, in fact, early reperfusion with an acidotic buffer reduces infarct size to the same extent as PostC [8, 9, 17, 29, 34, 55] (and unpublished observations of the authors). It is likely that low pH buffers act primarily on the endothelial cells, supporting the importance of crosstalk between endothelium and cardiomyocytes. Full reperfusion quickly restores the intracellular pH and initiates several adverse effects, which are collectively known as “pH paradox”. PostC delays re-alkalinization of the heart during early reperfusion, i.e., tissue pH remains acidic longer after PostC compared with an abruptly reperfused heart [8, 9, 17, 29, 34, 55]. For instance, intracellular acidosis during early reperfusion inhibits calpain activity and contributes to PostC protection [29, 30]. Since the enzyme activities are strongly influenced by the intracellular pH, we suggest that the acidic intracellular environment maintained by PostC in the early reperfusion plays an important role in the observed variation of SOD (optimal pH 7.8) and CAT (optimal pH 7.0) activities. In fact, the enzyme activities are similarly influenced by AB infusion and PostC (Fig. 4). Notably, protection by early reperfusion with an acidotic buffer is also redox dependent [8, 9] (and unpublished observations) and keeps a lower post-ischemic SOD activity for at least 2 h (present study; Fig. 4). However, it is not easy to study how the pH varies in different organelles during I/R and/or PostC. For this reason, in a perspective of future investigations on the role of pH, here we have opted for a study of the total activity of SOD rather than a study of individual isoforms confined in organelles. Nevertheless, in the early reperfusion, there are no significant changes in the iso-enzyme levels in I/ R and PostC (Fig. 5). We suggest that the observed changes in the activities of antioxidant enzymes contribute to PostC triggering. Overall, in early reperfusion, the observed changes in enzyme activities, nitration, and nitrosylation may represent a modification of the redox environment in a delicate moment for the triggering of protection. This redox mechanism includes an increase in NO• and derivative production [9, 15, 20, 34, 49, 56] that increases the amount of protein SNO, which is also maintained by reduced nitrosylation breakdown by SOD downregulation (as hypothesized; Fig. 1). The high protein S-nitrosylation may provide “protection preventing further cellular oxidative and nitrosative stress” as well as channel opening [56]. Since the main sources of ROS are mitochondria, we can argue that mitochondrial proteins can be among the main targets for either SNO or 3-NT. Actually, several mitochondrial SNO proteins have been seen to be protective in Pflugers Arch - Eur J Physiol (2011) 462:219–233 preconditioning and reperfusion [11, 36, 56, 57]. In fact, N2O3 has an increased stability in the hydrophobic milieu of the mitochondria, where the high levels of reactive cysteines would favor SNO formation [56]. Whether these occur in PostC deserves future studies. Late reperfusion In late reperfusion, the activity of SOD is still reduced and that of CAT increased by both PostC and AB, if compared with their activities in I/R group (different covalent modifications (?), see below). The increased activity of CAT by PostC or AB in this phase may be important to prevent the further reduction of H2O2 to hydroxyl radical (OH•), which represents a dangerous step because an increase in toxicity can occur. Yet, protection with PostC and AB prevents the reduction of the levels of the cytosolic Cu/Zn SOD, which are otherwise reduced after I/R. The dichotomy on activities and levels between cytosolic and organelle-confined enzymes [68] supports a role for compartmentalization of the redox environment in late reperfusion [52]. Importantly, SNO proteins are reduced by I/R, but this reduction is limited by PostC and not by PostC +SOD. Therefore, also in late reperfusion, SNO proteins are still higher in PostC hearts. Whether compartmentalization plays a role on both enzyme activity and protein nitrosylation needs to be studied further. Preliminary data obtained with two hearts for each group suggest that both the prolonged SOD upregulation by I/R and the downregulation by PostC are likely due to different covalent oxidations; in fact, a large spectrum scavenger (MPG) prevents these modifications in activity. Preliminary data obtained with three hearts for each group also show that the activity of the cytoplasmatic enzyme GPx does not change either after I/R, PostC, or AB (activity ranging between 90% and 110% of the baseline level in all hearts). Yet, GPx levels tend to decrease (about −20% vs. the baseline level) in late reperfusion after I/R, but not after PostC or AB (n=3 for each group). Also, the dichotomy of effect (activity/level) on GPx supports a role for compartmentalization of the redox environment [52], which deserves future studies. In fact, we argue that in different compartments, where pH, enzyme activities, and/or levels may be differently influenced, the redox environment may be subtly/patchy varied. Nevertheless, overall reduced activity of SOD by PostC is evident. Methodological problems We did not measure radicals but assessed SOD and CAT activities and the levels of 3-NT and SNO proteins in vitro. These may give information about the results of changing ROS/RNS production and help to overcome the difficulties 231 in measuring the levels of changing radicals in situ. In fact, the various radicals react reciprocally with an unpredictable outcome in their levels. For example, we could measure O2− and NO• but cannot easily predict whether ONOO− or N2O3 formation will prevail (Fig. 1). Thus, by studying 3NT and SNO proteins, which are more stable products, we could simultaneously have reliable indices of redox environment and of reaction direction. However, enzyme activities were studied at Vmax; thus, we cannot have information on the efficacy of changed activities in situ. Moreover, we have studied these redox effects in the isolated hearts and could not predict whether the effects can be replicated in vivo. Nevertheless, we have shown a redox signaling in PostC in isolated heart [54] and this observation has been confirmed in vivo in different laboratories [8, 9, 63]. Also, these new results need to be confirmed in vivo. Moreover, due to the variable amount of necrosis and protein leakage from different compartments, the comprehensive interpretation of results requires future systems biology studies, which should clarify the role of compartmentalization in enzyme/proteins, their specific nitration/nitrosylation, and the consequent effects on their structure, activity, and/or response to oxidative/nitrosative stress. Finally, 3 min of CAT and/or SOD in our I/R setting is not sufficient to limit per se infarct size and the recovery of cardiac function, as more prolonged infusions can do in some experimental settings [41]. Here, we specifically tested the possibility to alter PostC-triggering with these two different antioxidants and did not test longer infusions which have already been largely studied. In summary, in early reperfusion, PostC induces SOD downregulation, which together with the persistence of acidosis [8, 9, 17, 29, 30, 34] and the NO• augmentation (enzymatic and non-enzymatic production [49, 60, 65, 69]) may favor nitrosylation and/or may limit protein denitrosylation; these together with limited nitrosative stress and preserved CAT activity accompany PostC triggering. Intriguingly, exogenous SOD prevents PostC triggering, whereas exogenous CAT does not interfere with PostC protection. That is, the addition of exogenous SOD does not allow the early reduction in SOD activity, normally induced by PostC or AB. In late reperfusion, SOD activity is still reduced and that of CAT increased by PostC and AB, with respect to I/R. However, the levels of cytoplasmatic enzymes (e.g., Cu/Zn-SOD) are reduced by I/R and preserved by PostC, whereas the levels of enzymes confined in the organelles (e.g., CAT, MnSOD) are not significantly affected by either I/R or PostC. Yet, the levels of nitrosylated proteins are still higher in PostC. In conclusion, variations in the activity of redox enzymes, reduced levels of 3-NT, and increased levels of SNO proteins may contribute to cardioprotection. 232 Acknowledgments This work was funded by: National Institutes of Cardiovascular Research (INRC; FM, PP), Regione Piemonte, PRIN, ex60%, and Compagnia di San Paolo. We thank Prof. Donatella Gattullo for her invaluable support and Jennifer Lee for language revision. 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