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The objective of this study was to establish the cardioprotective effect of sulfaphenazole (SPZ), a selective inhibitor of cytochrome P450 2C9 enzyme, in an in vivo rat model of acute myocardial infarction (MI). MI was induced by 30min ligation of left anterior descending coronary artery, followed by 24h reperfusion (I/R). The study used 6 groups: I/R (control); SPZ; L-NAME; L-NAME+SPZ; 1400W (an inhibitor of iNOS); 1400W+SPZ. The agents were administered orally through drinking water for 3 days prior to induction of I/R. Myocardial oxygenation (pO2) at the I/R site was measured using EPR oximetry. The preischemic pO2 value was 18±2mm Hg in all groups. At 1h of reperfusion, the SPZ group showed a significantly higher hyperoxygenation when compared to control (45±1 vs. 34±2mm Hg). The SPZ group showed a significant improvement in the contractile functions and reduction in infarct size. Histochemical staining of SPZ-treated hearts exhibited significantly lower levels of superoxide and peroxynitrite, and markedly increased levels of iNOS activity and nitric oxide. Western blot analysis indicated upregulation of Akt and attenuation of p38MAPK activities in the reperfused myocardium. The study established that SPZ attenuated myocardial I/R injury through overexpression of iNOS, leading to enhancement of nitric oxide bioavailability and tissue oxygenation. Antioxid. Redox Signal. 11, 725–738.
Acute myocardial infarction (MI) occurs when the blood supply to a region of the heart is interrupted, most commonly due to rupture of a coronary plaque. The resulting ischemia or oxygen deprivation, if left untreated, can cause damage and death of heart muscle. Unfortunately, reintroduction of blood flow to the ischemic tissue can also cause tissue damage, termed as reperfusion injury. The pathogenesis of myocardial ischemia–reperfusion (I/R) injury is known to involve interplay of multiple mechanisms. Several studies have implicated reactive oxygen species (ROS), including superoxide radical (O2−.), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and peroxynitrite (ONOO−) that are generated upon reperfusion, in the I/R-mediated oxidative damage to the myocardium (25). The involvement of ROS in mediating I/R injury has been established based on the efficacy of antioxidants and free radical scavengers, such as superoxide dismutase (SOD), catalase, melatonin, and vitamin E, in minimizing I/R injury (12). Overexpression of manganese SOD (MnSOD), copper–zinc SOD (CuZnSOD), or glutathione peroxidase has been reported to protect the heart from I/R injury, further supporting the involvement of ROS in the reperfusion injury (8, 9, 65).
Unlike ROS, the involvement of nitric oxide (NO) in I/R injury has been controversial. NO, which is produced by a variety of mammalian cells, is an important mediator of both physiological and pathological vascular functions (37, 43). NO production is catalyzed by nitric oxide synthase (NOS). Enhanced NO generation was observed in the heart during I/R (13, 69). Decrease of NO production using NOS-inhibitors showed a decrease in the I/R-mediated functional impairment of the heart (44, 64). However, NO has also been shown to play a cardioprotective role in myocardial I/R injury (28, 36, 48, 54, 57, 63). Supplementation with L-arginine (substrate for NO production by NOS) and NO donors during reperfusion has been shown to be cardioprotective in regional, as well as in global ischemic models (36, 57, 63).
The cytochrome P450 (CYP) family of enzymes plays a significant role in normal cardiovascular homeostasis, as well as in cardiovascular pathogenesis (26). Particularly, CYP 2C9 has been implicated in myocardial I/R injury (23, 45, 55). CYP 2C9 has been identified as a potent source of superoxide radicals in the reperfused heart (18, 23, 45, 55). A role for CYP 2C9 in myocardial I/R injury was first demonstrated by Granville et al. (23) in an isolated rat heart model using CYP 2C6/9 inhibitors such as chloramphenicol, sulfaphenazole (SPZ), and cimetidine. The CYP 2C6/9 inhibitors were found to markedly attenuate infarct size and creatine kinase release. Superoxide was also found to be significantly reduced, while postischemic coronary flow was increased in the CYP inhibitor-treated hearts, indicating that NO scavenging and oxidative damage are likely to play a role in the protection against I/R-induced injury (23). These results were also reproduced in a rabbit coronary artery ligation model of focal I/R injury (23). A recent clinical study showed that SPZ could improve endothelium-dependent, NO-mediated vasodilation in patients with coronary artery disease (CAD) as assessed by increased acetylcholine-induced forearm blood-flow compared with control patients (17). The beneficial effect of SPZ administration was attributed to an increase in the bioavailability of NO in tissue and circulation during reperfusion. Similarly, a more recent study using SPZ administration in diabetic mice exhibited restoration of endothelium-dependent vasodilation, possibly by decreasing superoxide levels (14). However, the exact mechanism by which SPZ attenuates myocardial injury is not well understood.
Our recent study using isolated rat hearts showed that SPZ protected the hearts from I/R injury by scavenging ROS and increasing NO levels (31). However, it was not clear whether the increased NO levels in the SPZ-treated I/R-hearts were due to SPZ-mediated superoxide depletion or activation of endogenous pathways of NO production. This led us to hypothesize that SPZ may, in addition to inhibition and/or scavenging of superoxide generation, induce NO generation by activating endogenous inducible NOS (iNOS) in the reperfused heart. The elevated levels of NO bioavailability in the reperfused heart could have profound effects on the oxidative/nitrative stress, tissue oxygenation, infarction, and functional recovery. Therefore, the overall aim of this study was to investigate the cardioprotective effect of SPZ and to delineate the involvement of NO, superoxide, oxygenation and also to establish the signaling mechanism involved in cardioprotection in an in vivo rat model of acute myocardial infarction. The results from the present study showed that pretreatment of rats with SPZ significantly attenuated superoxide levels and induced iNOS expression in the reperfused heart with a concomitant enhancement of NO bioavailability and tissue oxygenation, leading to the improved recovery of cardiac function in vivo.
Sulfaphenazole [SPZ, 4-amino-N-(1-phenyl-1H-pyrazol-5-yl)], Nω-nitro-L-arginine methyl ester (L-NAME), dihydroethidium (DHE), 2,3,5-triphenyltetrazolium chloride (TTC), and monoclonal anti-β-actin antibody were obtained from Sigma (St. Louis, MO). 4-Amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate was obtained from Invitrogen (Carlsbad, CA). Polyclonal anti-iNOS antibody was procured from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit immuno-affinity-purified anti-nitrotyrosine antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Phospho-specific antibodies for Akt, ERK1/2, and p38 MAPK were obtained from Cell Signaling (Beverly, MA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Amersham Biosciences (Piscataway, NJ). Peroxynitrite was from Upstate Chemicals (Pickens, SC). Lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO) oximetry probe was synthesized as reported (47).
Male Sprague–Dawley rats (250–300g) were used in this study. Rats were randomly divided into six groups: I/R (control); SPZ (300μM); L-NAME (100μM); SPZ+L-NAME (300/100μM); 1400W (1mg/kg b.w.); 1400W+SPZ. The animals were treated with the drugs in drinking water for 3 days prior to experimentation. The rats consumed a daily average of 30ml water containing 300μM SPZ and/or 100μM L-NAME, which is equivalent to 8.1mg/kg b.w. of SPZ and/or 2.3mg/kg b.w. of L-NAME 1400W was administered i.p. 30min prior to the induction of I/R. Ischemia was induced by temporarily ligating the left anterior descending coronary artery (LAD) for 30min, followed by reperfusion for 1 or 24h by releasing the ligation. Myocardial tissue pO2 at the ischemic site was continuously monitored during the ischemia–reperfusion period up to 1h, and then at 24h of reperfusion. Left ventricular contractile functions were measured at 24h of reperfusion. Superoxide and nitric oxide levels in the excised heart tissue at 10min of reperfusion were determined by histochemical staining and fluorescence microscopy. Myocardial infarct size and immunohistochemical staining for iNOS and peroxynitrite were performed on excised heart tissues after 24h of reperfusion. All the procedures were performed with the approval of the Institutional Animal Care and Use Committee of the Ohio State University and conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23).
Rats were anesthetized with ketamine (50mg/kg, i.p.) and xylazine (5mg/kg, i.p.) followed by isoflurane (1–20%) with air. MI was induced by ligating the left anterior descending coronary artery (LAD), as described (5, 50). An oblique 12mm incision was made 8mm away from the left sternal border toward the left armpit. The chest cavity was opened with scissors by a small incision (10mm length) at the level of the third or fourth intercostal space, 3–4mm from the left sternal border. The LAD was visualized as a pulsating bright red spike, running through the midst of the heart wall from underneath the left atrium toward the apex. The LAD was ligated 2–3mm below the tip of the left auricle, using a tapered needle and a 6-0 polypropylene ligature. The ligature was passed underneath the LAD and a double knot was made to occlude the coronary artery. Occlusion was confirmed by the sudden change in color (pale) of the anterior wall of the left ventricle (LV). The ligature was released after 30min of ischemia, and the chest cavity was closed by tying together the 4th and 5th ribs with one 4-0 silk suture. The layers of muscle and skin were closed with a 4-0 polypropylene suture. After LAD ligation, successful infarction was confirmed by an ST elevation on electrocardiograms that were recognized in all rats. After 30min, the ligation was released, and the chest was closed. After resuming spontaneous respiration, animals were extubated and allowed to recover in a warm cage.
The MABP at baseline, ischemia (30min), and during reperfusion (1h) was measured by inserting a microtip transducer catheter (SPR-1000, ADInstruments, Colorado Springs, CO) into the right carotid artery. The catheter was connected to a computer-based data acquisition system (PowerLab, ML866, ADInstruments) for continuous monitoring of MABP and heart rate.
Cardiac contractile functions were measured in rats anaesthetized using pentobarbital sodium (50mg/kg) at 24h of reperfusion. A Millar catheter (SPR-1000) was advanced through the right carotid artery into the LV. The LV systolic pressure (LVSP), and maximum rate of increase (dP/dtmax) and decrease (dP/dtmin) of LV pressure were recorded and analyzed using PowerLab.
After 24h reperfusion, rats were reintubated and ventilated as mentioned above, the LAD was reoccluded, and 0.2ml of 2.0% Evans blue was injected from the inferior vena cava to delineate the nonischemic myocardial tissue. The animals were sacrificed immediately and their hearts were then cut into four transverse slices. The tissue slices were incubated at 37°C for 10min with a 1.5% solution of TTC in PBS to determine the infarct area and the area at risk (AAR). Images were captured digitally under a dissecting microscope. LV area, AAR, and infarct area were determined by computerized planimetry using MetaVue image analysis software (Molecular Devices, Downingtown, PA). The areas of myocardial tissue showing white and red colorations were defined as the regions of infarct and AAR, respectively, and the infarct size was determined. Infarct size was expressed as percentage of AAR.
Myocardial pO2 was measured using our established EPR oximetry method (47). The principle of EPR oximetry is based on molecular oxygen-induced line-width changes in the EPR spectrum of a paramagnetic probe. The probe usually is a microcrystal of stable nontoxic paramagnetic material that can be permanently implanted in the tissue region of interest using a 25-gauge surgical needle. Immediately following implantation, the probe responds to pO2 in the immediate microenvironment, mostly at the crystal–tissue interface, enabling magnetic resonance-based noninvasive and repeated measurements of pO2 for a prolonged period, months to years, from the same site. The technology has been well established and validated for measurements of pO2 from single cells to whole organs (30, 35, 47). To our knowledge, this is the first report on the use of EPR oximetry for pO2 measurements in the beating rat hearts, in vivo.
The myocardial pO2 measurements in the present study were performed using an in vivo EPR spectrometer (Magnettech, Berlin, Germany) equipped with automatic coupling and tuning controls for measurements in beating hearts. Microcrystals of LiNc-BuO were used as a probe for EPR oximetry. Rats, under inhalation anesthesia (air mixed with 1.5–2% isoflurane), were implanted with the oxygen-sensing probe in the left ventricular mid-myocardium. The animal was placed in a right lateral position with the chest open to the loop of a surface-coil resonator. EPR spectra were acquired as single 30-sec duration scans. The instrument settings were: microwave frequency, 1.2GHz (L-band), incident microwave power, 4mW; modulation amplitude, 180mG, modulation frequency 100kHz; receiver time constant, 0.2sec. The peak-to-peak width of the EPR spectrum was used to calculate pO2 using a standard calibration curve (47). Similarly, myocardial pO2 was measured after 24h of reperfusion.
Plasma nitrite/nitrate (NOx) levels in the reperfused hearts were determined by Griess assay. The assay is based on the enzymatic conversion of nitrate to nitrite by nitrate reductase, followed by spectrophotometric quantitation (at 550nm) of nitrite levels using the Griess reagent kit (Cayman Chemical, Ann Arbor, MI) and a Beckman AD 340 ELISA plate reader (Beckman Coulter, Fullerton, CA). Blood samples (0.5ml) were collected at 1h of reperfusion, centrifuged, and the plasma was stored at −80°C until analysis. The concentration of nitrite (indicative of NOx in the original samples) was calculated from a standard curve (1–35μM) and used for determination of total nitrite/nitrate concentrations. The NOx levels were expressed as concentration (μM).
Rats pretreated with vehicle-only (I/R), SPZ, L-NAME, SPZ+L-NAME were subjected to 30min of LAD ligation, followed by 10min reperfusion. Rats were sacrificed at 10min of reperfusion and hearts were placed immediately in cold PBS and then embedded in optimal cutting temperature (OCT) compound for cryosectioning. Superoxide generation in the heart tissue was determined using hydroxyethidine (HE) fluorescence (42). The cell-permeable DHE is oxidized to fluorescent HE by superoxide, which is then intercalated into DNA. It has been reported (42) that the superoxide generation in hearts subjected to I/R occurs during the first 10min of reperfusion. Hence, we measured the HE fluorescence at 10min of reperfusion. The frozen segments from the heart tissue were cut into 6μm thick sections that were then placed on glass slides. DHE (10μM) was topically applied to each tissue section. The tissue sections on slides were incubated in a dark chamber at 37°C for 30min and then washed three times with PBS and fixed with aqueous mounting medium (Gel Mount, Sigma Chemicals). The images of the tissue sections were obtained using a fluorescence microscope (Nikon TE 300, Tokyo, Japan) with a rhodamine filter set (excitation=550nm; emission=573nm). Fluorescence intensity, which positively correlated with the levels of superoxide generation, was determined in the myocardial tissue using MetaMorph image analysis software (Molecular Devices).
The NO produced in I/R hearts was measured by fluorescence microscopy using DAF-FM diacetate. DAF-FM diacetate is cell permeable and passively diffuses across cellular membranes. Inside cells, DAF-FM diacetate is deacetylated by intracellular esterases to DAF-FM that reacts with NO to form fluorescent benzotriazole. Hearts, after 30min of ischemia, and 10min of reperfusion, were placed in an ice-cold PBS buffer and embedded in OCT compound for cryosectioning. The frozen tissues were cut into 6μm thick sections and incubated with 10μM DAF-FM diacetate for 30min at 37°C. Images of the tissue sections were obtained using a fluorescence microscope (Nikon TE 300) with a fluorescein isothiocyanate (FITC) filter set (excitation=495nm; emission=510nm). The fluorescence intensity, which positively correlated with the amount of NO generation, was quantitatively determined using MetaMorph image analysis software (Molecular Devices).
Immunostaining for iNOS was performed using formalin-fixed and paraffin-embedded heart tissue sections that were serially rehydrated in 100%, 95%, and 80% ethanol after deparaffinization with xylene. Slides were kept in a pre-heated steamer for 30min for antigen retrieval, and then washed with PBS three times for 5min each. Tissue sections were incubated with 2% goat serum and 5% bovine serum albumin in PBS to reduce nonspecific binding, followed by incubation with NOS2 (N-20) affinity-purified rabbit polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology) for 1h at room temperature in the humidity chamber. The sections were then incubated with secondary antibody (1:1,000 dilution) conjugated to green-fluorescent Alexa Fluor-488 dye. Separate tissue slides were stained without primary antibodies to examine nonspecific binding. The tissue sections were visualized using a Nikon fluorescence microscope equipped with an FITC filter set (excitation=495nm; emission=510nm) and the average intensity was calculated using MetaMorph image analysis software.
Paraformaldehyde-fixed and paraffin-embedded heart tissue sections were incubated with rabbit immuno-affinity-purified anti-nitrotyrosine antibody (at 1:100 dilution) for 1h at room temperature in a humidity chamber. The sections were then incubated with biotinylated secondary antibody (biotinylated goat anti-rabbit antibody, ready-to-use, LabVision Corporation, Fremont, CA) for 1h in the humidity chamber and streptavidin-horseradish peroxidase-conjugate solution for 30min. Color was developed by using peroxidase diaminobenzidine substrate and counterstained with H&E stain. Negative controls were prepared by incubating the primary antibody with 10mM nitrotyrosine in PBS for 1h at room temperature, and using this solution instead of the primary antibody. For positive controls, rat hearts were infused with peroxynitrite (1mM, 0.2ml) into the LV chamber, and the hearts were removed and fixed in formalin/paraformaldehyde after 15min of infusion. The slides were then processed and visualized using the above protocol.
Determination of iNOS protein was performed in samples taken from the area-at-risk of LV from I/R (control) and SPZ groups (3 rats in each group). Rats were anesthetized and sacrificed after 24h of reperfusion. The hearts were rapidly explanted, rinsed in cold PBS (pH 7.4), containing 0.16mg/ml heparin to remove red blood cells and clots, frozen in liquid nitrogen, and stored at −80°C. Tissue slices were taken from the area-at-risk (infarct region) of LV and were homogenized in buffer A (25mM Tris HCl, pH 7.4, 0.5mM EDTA, 0.5mM EGTA, 1mM phenylmethylsulfonyl fluoride, 1mM dithiothreitol, 25mM NaF, 1mM Na3VO4, and 1% protease inhibitor (Sigma), and centrifuged at 10,000g for 10min at 4°C. The pellets were then incubated on ice in buffer B (buffer A plus 1% Triton X-100) for 2h and centrifuged for 12min at 4°C. The resulting supernatants were collected as membranous fractions. The expressions of NOS isoforms were assessed by standard SDS–PAGE Western immunoblotting techniques using polyclonal anti-iNOS (1:500 dilution) and monoclonal anti-β-actin antibodies. Similarly, phospho-specific antibodies (1:500 dilution) for Akt, ERK1/2, and p38 MAPK, followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ) were used to determine the phosphorylation status of these proteins. The PVDF (polyvinylidene fluoride) membranes with transferred proteins were then developed by enhanced chemiluminescence. The same PVDF membranes were probed with antibodies for total Akt, ERK1/2, p38 MAPK, and β-actin. The protein intensities were quantified by an image-scanning densitometer (Scion Corporation, Frederick, MD). To quantify the phospho-specific signal in activated proteins, we first subtracted and then normalized the signal to the amount of actin or total target protein in the lysate (32). Data were expressed as percent of the expression in the control group.
The statistical significance of the results was evaluated using the one-way ANOVA and Student's t-test. The values were expressed as mean±SD. A p value of <0.05 was considered significant.
I/R is associated with drastic changes in tissue oxygenation, which may have important implications in the early events leading to myocardial injury and dysfunction. In order to study the dynamics of tissue oxygenation during the I/R episode and to examine the effect of SPZ, we used in vivo EPR oximetry to monitor myocardial pO2 in the infarct region continuously. We implanted an oxygen-sensing microcrystalline probe in the left ventricular mid-myocardium, in the region where ischemia would be expected to occur following LAD ligation, and performed the pO2 measurements under in vivo conditions (Fig. 1A–C). Figure 1D shows the changes in pO2 during the 30min ischemia followed by 60min reperfusion in rats pretreated with SPZ+L-NAME, 1400W, and 1400W+SPZ. The basal (preischemic) levels of myocardial pO2 were in the range of 18–20mm Hg, and there were no significant differences among the groups. Immediately after induction of ischemia, the pO2 levels dropped quickly and remained at ~2mm Hg during the 30min ischemic duration. Upon restoration of blood flow (reperfusion), a rapid increase in pO2 leading to marked hyperoxygenation was observed in all groups. Further, the hyperoxygenation persisted for 60min and beyond. Figure 1E shows the mean values of pO2 obtained at the end of 60min, as well as after 24h reperfusion. At 60min of reperfusion, the level of reoxygenation in untreated (I/R) hearts was significantly higher compared to non-I/R control (35±2 vs. 19±1mm Hg). A similar hyperoxygenation was observed in all the treated groups. The SPZ group showed the maximum hyperoxygenation (45±2mm Hg), a more than twofold increase from the preischemic level. The hyperoxygenation was observed to be persistent and significant. However, the SPZ group showed a significant decrease in pO2 at 24h compared to 1h, although the value was still substantially elevated in SPZ-treated hearts (38±2mm Hg) at 24h. The hyperoxygenation in the SPZ group at 24h was significantly attenuated compared to the I/R group (56±6mmHg). L-NAME or 1400W showed a similar increase in pO2 up on reperfusion, however, the magnitude of hyperoxygenation was significantly less when compared to I/R or SPZ group at 1h. The results further showed that co-administration of 1400W with SPZ significantly abrogated the hyperoxygenation observed in the SPZ group (30±3 vs. 45±2mm Hg). The pO2 data suggested that the hyperoxygenation observed in the SPZ group was possibly mediated through iNOS.
Heart rate and mean arterial blood pressure were measured in rats before ischemia (baseline) and after 30min ischemia and 1h reperfusion (Fig. 2). There were no significant differences in the heart rates among I/R, SPZ, L-NAME, and SPZ+L-NAME groups during ischemia and reperfusion (Fig. 2). However, the mean arterial blood pressure was significantly reduced in all groups at the end of 30min of ischemia. At 1h reperfusion, the SPZ group showed a further significant drop in blood pressure when compared to the other groups.
Left ventricular contractile functions were measured after 24h of reperfusion (Fig. 3). Rats pretreated with SPZ showed a significant improvement in LVSP, dP/dtmax, and dP/dtmin when compared to the I/R group. On the other hand, L-NAME or SPZ+L-NAME groups did not show any significant difference in the contractile functions when compared to the I/R group. Co-treatment of L-NAME and SPZ significantly blunted the recovery of contractility observed in the SPZ group.
Myocardial infarct size was measured in rats subjected to 30min of LAD ligation, followed by 24h reperfusion (Fig. 4). The infarct area, expressed as percent of area-at-risk (AAR), was significantly less in rats pretreated with SPZ compared to untreated (I/R) rats (19±2.5% vs. 34±3.3%). There was no significant difference in the infarct size in groups pretreated with L-NAME (32±3.1%), SPZ+L-NAME (30±3.6%), 1400W (33±4%) or SPZ+1400W (31±3.2%) when compared to the I/R group. Combined treatment of 1400W or L-NAME with SPZ significantly blunted the beneficial effect of SPZ.
To determine the effect of SPZ on the NO levels, the plasma levels of stable metabolites of NO, namely, nitrite and nitrate (NOx) were determined after 1-h reperfusion (Fig. 5). Control hearts subjected to I/R showed a significantly increased plasma NOx level compared to non-I/R hearts. On the other hand, SPZ-treated hearts showed a further significant increase in NOx levels when compared to the I/R group. L-NAME and SPZ+L-NAME treatments significantly attenuated the plasma NOx levels, suggesting that the increase in NOx levels in the SPZ-treated group was derived from an L-NAME inhibitable source.
Superoxide, NO, peroxynitrite, and iNOS levels in the infarct tissue sections of hearts subjected to 30min LAD occlusion, followed by 10min or 24h reperfusion, were measured by histochemical staining and fluorescence microscopy (Fig. 6). The level of superoxide at 10min reperfusion was significantly elevated in the I/R group. The SPZ-treated group showed a significant reduction in superoxide generation when compared to the I/R group. Administration of L-NAME, either alone or in combination with SPZ, did not have any significant effect on superoxide generation upon reperfusion. The results clearly indicated that SPZ attenuated the formation of superoxide radicals during the early phase of reperfusion. The NO level in the hearts at 10min of reperfusion was significantly higher in SPZ-treated hearts, when compared to the I/R group (Fig. 6). However, in L-NAME and SPZ+L-NAME groups, there was a significant decrease of NO as compared to the SPZ group. The results suggested that SPZ enhanced myocardial tissue NO bioavailability during the early phase of reperfusion.
Peroxynitrite (OONO−), a potent oxidant formed by the reaction of superoxide with NO, has been implicated in I/R injury (52). In order to determine whether SPZ could attenuate peroxynitrite generation in the reperfused heart, we used nitrotyrosine-staining to quantify the myocardial tissue levels of peroxynitrite. Untreated rats subjected to 24h reperfusion exhibited a substantially high level of peroxynitrite (Fig. 6). Pretreatment of rats with SPZ showed a significant reduction of peroxynitrite formation. On the other hand, rats pretreated with L-NAME alone or L-NAME+SPZ did not show any significant difference in the peroxynitrite levels when compared to the I/R group.
SPZ has been shown to increase the bioavailability of NO by decreasing superoxide generation in the reperfused heart (23, 31). However, its effect on the induction of NO-generating enzymes such as iNOS in the heart upon reperfusion is not known. We measured the level of iNOS protein in the tissue sections of rat hearts subjected to 24h reperfusion (Fig. 6). The iNOS level was significantly increased (threefold) in the SPZ-treated hearts when compared to the untreated (I/R) group. Rats pretreated with L-NAME alone or L-NAME with SPZ did not show any significant difference in the iNOS levels when compared to the untreated I/R group. The upregulation of iNOS by SPZ was further confirmed by Western blot analysis. There was a significant increase of iNOS expression in the SPZ group when compared to I/R control (Fig. 7).
To further understand the underlying mechanism of signaling pathways leading to the attenuation of post-ischemic reperfusion injury in the hearts treated with SPZ, we performed SDS-PAGE and Western blot assays for phosphorylated Akt, ERK1/2, and p38 MAPK in the rat heart homogenates. The SPZ group showed a significant increase in the phosphorylation of Akt when compared to the I/R group (Fig. 8). However, there was no significant change in the phosphorylation of ERK1/2 in the SPZ and I/R groups. In contrast, there was a significant increase in p38 MAPK in the I/R group when compared to the SPZ group. SPZ treatment markedly ameliorated I/R-induced activation of p38 MAPK compared to the I/R group. Overall, the Western blot analyses indicated that SPZ treatment enhanced the activation of Akt and attenuated the phosphorylation of p38 MAPK, thereby conferring cardioprotection. However, the results do not confirm a direct effect of SPZ on these proteins.
The present study showed that pretreatment of rats with SPZ significantly diminished the myocardial damage and dysfunction caused by reperfusion. The beneficial effects correlated with a significant elevation of tissue oxygenation and bioavailability of NO. Reperfusion of ischemic myocardium is known to compromise the bioavailability of NO, which is mainly attributed to impairment of key enzymes and substrates responsible for its production (41, 68). In addition, the loss of NO bioavailability could also occur as a result of inactivation of NO by superoxide radicals that are known to be abundantly generated in the reperfused myocardium (69). The present results suggest that the augmentation of NO bioavailability by SPZ is mainly due to overexpression of iNOS enzyme. The results further suggest that the superoxide-scavenging ability of SPZ may also be responsible for the enhanced bioavailability of NO and decreased levels of peroxynitrite in the reperfused myocardium. The involvement of NO in cardioprotection is obvious from the observation that addition of L-NAME or 1400W attenuated both NO levels and the cardioprotective capacity of SPZ. Most importantly, the large increase in myocardial tissue oxygenation observed in the SPZ group could be due to NO bioavailability, leading to increased myocardial blood flow at reperfusion.
The superoxide, peroxynitrite, and NO levels at 10min of reperfusion (Fig. 6) suggest that the cardioprotective effect of SPZ may be due to any one, or a combination of the following factors: (a) inhibition/scavenging of superoxide radicals; (b) increased bioavailability of NO upon reperfusion; (c) decreased systemic vascular resistance and/or aortic input impedance (most importantly, coronary vascular) resistance; and (d) improved myocardial energetics. The myocardial energetics refer to the balance between myocardial oxygen delivery and consumption.
In the present study, the baseline (preischemic) myocardial tissue oxygenation did not change among the four groups. However, there was a substantial hyperoxygenation in all groups upon reperfusion. The hyperoxygenation in the SPZ group at 1h reperfusion was significantly higher when compared to the untreated, L-NAME, or 1400W group. The increase in pO2 at 1h reperfusion in the SPZ group could be attributed to the enhanced NO levels, which may cause increased blood flow upon reperfusion. The marked hyperoxygenation could also occur as a result of decreased oxygen consumption due to NO-mediated inhibition of mitochondrial respiration (67). On the other hand, the differences in the levels of myocardial oxygenation among the groups at 24h reperfusion could reflect the decrease in oxygen demand as a result of varying levels of infarction. It should be noted that only in the case of the SPZ group did we observe a decrease in the oxygenation at 24h compared to 1h into reperfusion, which is reflective of the substantial reduction in the infarct size found within the SPZ group. Overall, the magnitude and temporal changes in myocardial tissue oxygenation apparently reflects the physiological and metabolic alterations due to acute changes in the tissue levels of nitric oxide, oxidants, and oxygen consumption.
The three prime determinants of oxygen consumption are heart rate, contractility, and peak myocardial tension (afterload). Heart rate was unchanged by SPZ, but the increase in dP/dtmax and peak systolic pressure would be expected to increase oxygen demand and to decrease myocardial oxygen content. However, we observed an increase in the myocardial oxygen content upon reperfusion of SPZ-treated hearts. There are two possible explanations as to why myocardial oxygen content was increased by SPZ. One possibility is that the preload end-diastolic radius of the LV chamber could be reduced, the LV wall would thicken, and both decreased radius and increased wall-thickness would lead to a decrease in afterload despite elevation of systolic pressure. Second, the decrease in coronary vascular resistance (caused by NO) might have been out of proportion to the effect on the general systemic arterial resistance of vessels. Thus, the decrease in preload and afterload, along with the increased coronary flow, would result in the observed increase in myocardial oxygen content.
The increase in peak systolic pressure in the SPZ-treated hearts (Fig. 3B) may be attributed to increased stroke volume, stiffening of the aorta (increasing elasticity modulus), or increased systemic vascular resistance. However, the depressed systemic arterial blood pressure (Fig. 2B) might indicate a decrease in the systemic arterial diastolic pressure, which is the determinant of afterload and myocardial oxygen consumption. The decrease in systemic arterial pressure and the increase in stroke volume are consistent with the known pharmacology of NO (29).
Among the inhibitors of nitric oxide synthases, 1400W is by far the most selective for inhibiting the activity of iNOS. Its ratio of selectivity for iNOS versus eNOS is >4,000-fold, which is in sharp contrast to that of aminoguanidine (11-fold), N5-iminoethyl-L-ornithine (49-fold) (1) or isothiourea (2–6 fold) (21). In addition, the in vitro potency of 1400W in inhibiting iNOS is 135- and 19-times that of aminoguanidine and N5-iminoethyl-L-ornithine, respectively (1). Studies have shown that 1400W is effective in reversing vascular abnormalities known to be associated with the induction of iNOS (21, 53, 58). The widely accepted mechanism of action of 1400W is its inhibitory effect on iNOS activity.
Studies have shown that ROS produced in the myocardium at the onset of reperfusion can cause tissue and functional injury, which is preventable, at least in part, by antioxidants (2, 32–34, 51, 56). Peroxynitrite (ONOO−), a dominant reaction product of NO and superoxide (16), has been reported to increase apoptosis in a variety of cell types. The proapoptotic mechanisms of ONOO− include protein and DNA oxidation (10), lipid peroxidation (60), protein nitration (19, 22, 40), apoptosis-inducing factor release (66), and endoplasmic reticulum stress, with the subsequent release of caspase (46). Our results showed that pretreatment with SPZ markedly reduced cardiac nitrotyrosine content, indicating that SPZ blocks nitrative stress through inhibition of peroxynitrite formation. The decrease in peroxynitrite in the SPZ-treated rat hearts could be due to attenuation of superoxide radicals by SPZ, thereby limiting the reaction of superoxide with NO (31).
The iNOS-dependent biosynthesis of NO is a common pathway involved in the protection of heart against various forms of stress (6, 7). Ischemic preconditioning (IPC) has been shown to upregulate iNOS expression in cardiac myocytes (27, 62). A relatively modest upregulation of iNOS, such as that known to occur during late preconditioning, is cardioprotective, whereas a massive upregulation of iNOS that may occur during inflammation or septic shock could be detrimental (24, 62). Enhanced expression of iNOS and NO production has been implicated in the cardioprotection by several drugs including resveratrol (27, 62), sildenafil (53), and atorvastatin (3). The results of the present study clearly indicated an upregulation of iNOS expression by SPZ, suggesting that NO derived from iNOS was responsible for the cardioprotection in the SPZ-treated hearts.
Activation of Akt and ERK1/2 has been shown to be cardioprotective (38, 59). In the heart, the p38 MAPK pathway has been implicated in the regulation of cardiac gene expression, myocyte hypertrophy, inflammation, bioenergetics, contractility and proliferation, and apoptosis (4, 15, 49). Studies have also shown that activation of p38 MAPK enhances lethal injury and inhibition of p38 MAPK by SB203580 (MAPK inhibitor) reduces postischemic myocardial apoptosis (39). Several proapoptotic proteins have been identified as direct Akt substrates, including Bad, caspase-9, and apoptosis-signal regulating kinase (ASK1). Phosphorylation of these molecules by Akt may reduce apoptotic cell death by inhibiting caspase-9 activity, releasing antiapototic molecule Bcl-2, blocking proapoptotic molecules Fas ligand expression, and inhibiting proapoptotic p38 MAPK activation (11, 20). In the present study, we observed that SPZ activated Akt and decreased the activity of p38 MAPK, which might lead to a decrease in myocyte apoptosis and thereby protecting the heart against I/R injury. However, the exact mechanisms by which SPZ mediates enhancement of Akt and inhibition of p38 MAPK activities are yet to be elucidated.
Wang et al. (61) have demonstrated that Akt phosphorylation by diazoxide is upstream of NOS, and NOS and Akt phosphorylation are important in preventing cell death in the ischemic myocardium. Diazoxide was shown to be an important agonist of mitoKATP channel opener and exert its antiapoptotic effect through the PI3 kinase-Akt pathway. Further, the phosphorylation of eNOS with subsequent NO production is an important downstream effector that contributes significantly to the cardioprotective effect of diazoxide against I/R injury. Similarly, in our study we have observed that SPZ activated Akt expression and decreased the expression of p38 MAPK, which might lead to decrease in myocyte apoptosis and thereby protecting the heart against I/R injury. However, the exact mechanism by which SPZ mediates enhancement of Akt and inhibition of p38 MAPK activity is yet to be understood.
The pO2 measurements were performed by EPR oximetry using an oxygen-sensing paramagnetic microcrystalline probe (47). The probe is nontoxic and stable in tissues without undergoing phagocytosis by macrophages or clearance from the site of implant. The probe is also stable against biological oxido-reductants including superoxide, nitric oxide, hydrogen peroxide, ascorbate, or glutathione. Both the EPR-detection sensitivity (paramagnetism) and oxygen-sensitivity calibration (line-broadening by oxygen) are stable for long periods, thus, enabling repeated measurements. Finally, the probe reports absolute values of pO2 in the microenvironment at the site of implantation. Since multiple crystals are used, the measured value is an average of readings from the entire implant, which is usually a point deposit, as seen in Fig. 1B.
To our knowledge, this is the first report of in situ oxygenation measurements in the beating heart of a live rat. The EPR oximetry in the heart requires one-time surgical implantation of the oxygen-sensing microcrystals at the site of interest. Subsequent measurements can be performed noninvasively and precisely at the site of implantation for extended periods of time, possibly weeks or months, without having to open the chest or reintroduce the probe (30). At present, these measurements are only limited to the hearts of closed-chest rodents such as mice and small rats. In larger-sized rats, such as that used in the present study, it is necessary to make an incision in the chest to allow the resonator (pick-up coil) access to the region of interest to obtain a detectable EPR signal. Nevertheless, the measurements reported in this work were done on open-chest rats during the 90min period, and then on reopened chests after 24h. Despite the technical challenges and limitations, very precise and reliable oxygen measurements from the beating heart could be obtained using EPR oximetry.
In summary, pretreatment of rats with SPZ ameliorated I/R-induced myocardial damage and improved cardiac contractile functions by decreasing superoxide production, peroxynitrite formation, and by enhancing NO bioavailability, myocardial tissue oxygenation, and iNOS expression. SPZ treatment also activated pro-survival Akt, leading to the attenuation of proapoptotic p38 MAPK. Overall, this study established that SPZ induces iNOS expression and modulates important signaling pathways involved in cardioprotection.
This work was supported by National Institutes of Health Grant EB006153.
AAR, area at risk; CYP, cytochrome P450; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein; DHE, dihydroethidium; EPR, electron paramagnetic resonance; HE, hydroxyethidine; I/R, ischemia/reperfusion; iNOS, inducible nitric oxide synthase; IPC, ischemic preconditioning; LAD, left anterior descending coronary artery; LiNc-BuO, lithium octa-n-butoxynaphthalocyanine; L-NAME, Nω-nitro-L-arginine methylester; LVSP, left ventricular systolic pressure; MI, myocardial infarction; NO, nitric oxide; NOS, nitric oxide synthase; OONO−, peroxynitrite; ROS, reactive oxygen species; SPZ, sulfaphenazole; TTC, 2,3,5-triphenyltetrazolium chloride.
No competing financial interests exist.