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Am J Physiol Heart Circ Physiol 285: H1385-H1395, 2003. First published June 19, 2003; doi:10.1152/ajpheart.00808.2002
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Influence of peroxynitrite on energy metabolism and cardiac function in a rat ischemia-reperfusion model

Warren H. Lee,1,3 John S. Gounarides,2 Eric S. Roos,2 and Michael S. Wolin3

1Metabolic Cardiovascular Diseases and 2Central Technologies, Novartis Institute for Biomedical Research, Summit, New Jersey 07901; and 3Department of Physiology, New York Medical College, Valhalla, New York 10595

Submitted 13 September 2002 ; accepted in final form 27 May 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Ischemia-reperfusion generates peroxynitrite (ONOO–), which interacts with many of the systems altered by ischemia-reperfusion. This study examines the influence of endogenously produced ONOO– on cardiac metabolism and function. Nitro-L-arginine (an inhibitor of ONOO– biosynthesis) and urate (a scavenger of ONOO–) were utilized to investigate potential pathophysiological roles for ONOO– in a rat Langendorff heart model perfused with glucose-containing saline at constant pressure and exposed to 30 min of ischemia followed by 60 min of reperfusion. In this model, ischemia-reperfusion decreased contractile function (e.g., left ventricular developed pressure), cardiac work (rate-pressure product), efficiency of O2 utilization, membrane-bound creatine kinase activity, and NMR-detectable ATP and creatine phosphate without significantly altering the recovery of coronary flow, heart rate, lactate release, and muscle pH. Treatment with urate and nitro-L-arginine produced a substantial recovery of left ventricular developed pressure, rate-pressure product, efficiency of O2 utilization, creatine kinase activity, and NMR-detectable creatine phosphate and a partial recovery of ATP. The pattern of effects observed in this study and in previously published work with similar models suggests that ONOO– may alter key steps in the efficiency of mitochondrial high-energy phosphate generation.

urate; nitric oxide; oxygen consumption; reactive oxygen species; heart perfusion

THE PRODUCTION OF REACTIVE oxygen species (ROS) from various cellular metabolic processes caused by the restoration of coronary flow after cardiac ischemia is thought to contribute to the observed myocardial damage (4, 14, 43). Antioxidants such as SOD provide partial cardioprotection most effectively when present during the initial reperfusion, suggesting that critical free radical-mediated injury occurs immediately on restoration of flow. ROS, such as superoxide anion (), have been shown to reach their highest concentrations in the ischemic heart within the first few minutes of reperfusion (5, 13, 43). Ischemia-reperfusion injury in isolated perfused hearts is also associated with evidence for an increased generation of peroxynitrite (ONOO–) from the essentially diffusion-limited reaction of with nitric oxide (NO), and this reactive species is thought to contribute to the observed dysfunction (10, 12, 30, 40–42).

Alterations in generation and/or use of energy are thought to be important contributing factors to the observed dysfunction caused by ischemia-reperfusion (34). Rapid resumption of oxidative phosphorylation is critical for the restoration of adequate energy metabolism (ATP and creatine phosphate production) and cellular survival. The abrupt rise in ROS as a result of the reoxygenation of ischemic or hypoxic cardiac muscle has been associated with a partial irreversible inhibition of mitochondrial respiration (38) through processes that appear to involve the generation of ONOO– from the reaction of with NO (39). It has been observed that ONOO– causes an irreversible inhibition of mitochondrial respiration through modification of iron-sulfur centers in respiratory enzymes (25). In addition, endogenous ONOO–, produced by hypoxia-reoxygenation, and exogenously generated ONOO– have been shown to depress developed tension in isolated rat papillary muscle (37). For example, the reduced contractility, as well as respiration, during posthypoxic reoxygenation was markedly attenuated by the NO synthase (NOS) inhibitor nitro-L-arginine (L-NA), the scavenger SOD, and the ONOO– scavenger urate (37). Similar studies in rat heart papillary muscles also showed that ONOO– attenuated developed force and resting tension (10), suggesting systolic and diastolic injury. Exogenous ONOO– has been reported to cause cardiac contractile dysfunction without reducing energy substrate utilization and O2 consumption (30). A decrease in cardiac contractile function may be due to the inhibition of creatine kinase, which is thought to impair the efficient coupling of the mitochondrial production of ATP and its utilization by myofibrils (27). Interestingly, the combination of NO and also leads to the inactivation of creatine kinase (19), presumably by modification and/or oxidation of the cysteine and tyrosine residues. Thus several systems associated with the generation and utilization of high-energy phosphates may be important targets for the ROS generated as a result of ischemia-reperfusion.

The objectives of this study were to investigate whether the generation of ONOO– by ischemia-reperfusion contributes to cardiac dysfunction through alterations in systems that control high-energy phosphates by employing urate as a potential intracellular scavenger of ONOO–. Urate is an antioxidant that has a distinct profile, in that it scavenges intracellular ONOO– and reactive oxidants derived from H2O2 without scavenging NO or (3, 39). In an ischemia-reperfusion model of global myocardial function, the capacity of the heart to perform pressure-volume work under standardized conditions of preload and afterload was significantly greater when perfused with urate than SOD or catalase (3). The present study, as a consequence, sought to employ urate as a scavenger of ROS, such as ONOO–, for the purpose of preventing its potential pathophysiological actions on cardiac energy metabolism and contractile function. Because urate also scavenges other ROS and reactive free radicals (3), the actions of probes that prevent the generation of ONOO– were used in most experiments to help support interpretations of the role of this NO-derived species in alterations caused by ischemia-reperfusion. Attenuation of NO generation with the NOS inhibitor L-NA and scavenging of with Cu,Zn-SOD were used to prevent the formation of ONOO–. Overall, the actions of probes employed in this study of a saline-perfused rat heart model of ischemia-reperfusion are focused on defining the potential role of ONOO– in altering systems that control the levels of high-energy phosphates potentially associated with the observed loss of cardiac function.


   METHODS AND MATERIALS
TOP
 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Langendorff heart perfusion protocol for hemodynamic parameters. All procedures involving animals were performed in accordance with the standards of the US Department of Health and Human Services and were approved by the Novartis Animal Care and Use Committee. Hearts were rapidly removed from heparinized (5 mg ip) and anesthetized (pentobarbital sodium, 60 mg/kg ip) adult male Sprague-Dawley rats (270–300 g). The hearts were immediately placed in cold (0°C) Krebs-Henseleit buffer (KHB) to arrest contractions. The hearts were then mounted by the aorta on a glass cannula and arranged for retrograde perfusion on a nonrecirculating Langendorff perfusion apparatus. Two perfusate reservoirs were used for rapid alternations in control and drug treatments. The hearts were perfused at 37°C with a constant pressure of 60 mmHg with filtered (0.22 µm) KHB containing (in mM) 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2, 0.5 EDTA, 1.2 MgSO4, 25 NaHCO3, and 5.0 glucose. The pH of the buffer was maintained at 7.4 by saturation with a gas mixture of 95% O2-5% CO2. The hydrostatic pressure was ~60 cmH2O, and an apical stab was used to vent the left ventricle (LV). The difference between LV systolic and diastolic pressure or LV developed pressure (LVDP), heart rate, LV end-diastolic pressure, and the first derivative of LVDP (±dP/dt) were measured by a small fluid-filled latex balloon inserted through the left atrium into the LV cavity. A small part of the balloon was tied into the outer surface of the apex of the heart, and the catheter was tied in place at the atrial appendage (21). The balloon volume was adjusted with a micrometer syringe to an end-diastolic pressure of ~4–8 mmHg. The balloon was connected to a pressure transducer via a hydraulic line. All hearts were preperfused for 30 min to stabilize hemodynamic parameters before the experiment was started, and all determinations of ventricular performance were obtained on-line on a computer using an analysis system for the calculation of data (Buxco Electronic). Throughout the experimental period, coronary effluent was accumulated before ischemia (baseline) by timed collection of coronary perfusate dripping from the heart and continuously during reperfusion for the estimation substances (e.g., lactate) that are released. For treatment of hearts with urate, it was added in the desired amounts directly to the perfusion buffer.

Two different protocols were employed in the studies: one for endogenous generation of ROS, including ONOO– by ischemia-reperfusion, and the other for exogenous generation of ONOO– derived from the reaction of and NO generated by pyrogallol and S-nitroso-N-acetyl penicillamine (SNAP), respectively.

Protocol 1: global ischemia. Before the onset of ischemia, all hearts were stabilized for 30 min. Baseline values for functional parameters were obtained after 10 min of perfusion. SOD or L-NA was perfused 5 min before ischemia. Thereafter, the hearts were subjected to 30 min of global no-flow normothermic ischemia. During ischemia, hearts were immersed in a limited volume of perfusion buffer at 37°C to avoid hypothermia. Reperfusion was then initiated and continued for 60 min in the presence of urate, L-NA, SOD, or plain buffer (control).

Protocol 2: perfusion only. After 30 min of initial stabilization, stock solutions of SNAP, pyrogallol, and SNAP plus pyrogallol were infused into the aortic cannula of Langendorff preparations for the following 30 min at rates calculated to yield the desired concentration in the coronary perfusate. For experiments using urate, perfusion was started 25 min after initial stabilization and continued for the following 35 min alone or in conjunction with SNAP plus pyrogallol infusion. For the final 60 min of perfusion, all hearts were perfused with KHB.

Measurement of myocardial O2 uptake. A section of polyethylene tubing (PE-100, Intramedic) was inserted into the pulmonary artery, with the tip of the tubing positioned in the right ventricular apex. The O2 content in the coronary effluent was determined with an O2 meter (model 5300, Yellow Springs Instrument). PO2 was monitored and recorded continuously with an on-line Clark-type O2 electrode (model 5357, Instech) in temperature-jacketed chambers, located to measure the inflow and outflow perfusate O2 concentration. The O2 concentration was calibrated against air- and 95% O2-saturated KHB solution. At each flow rate, the perfusate O2 content was recorded. Coronary flow was determined by measuring the volume of effluent as a function of time. O2 consumption (µmol O2 · g dry wt–1 · min–1) was obtained using the following equation


Cardiac efficiency was calculated as the ratio of the rate-pressure product to O2 consumption.

NMR spectroscopy. The time course of ATP, phosphocreatine (PCr), and pH levels in the isolated hearts was followed using 31P NMR. The procedures for preparation and perfusion were similar to those described above. Isolated hearts were contained in a 20-mm NMR tube that was modified to allow the inflow and aspiration of perfusate. Perfusate was aspirated by means of a suction line positioned just above the level of the aortic cannula, such that the heart was totally immersed in a bath of perfusate. This, in conjunction with the variable-temperature control unit of the spectrometer, ensured that the temperature throughout the experiment was maintained at 37 ± 0.2°C. Three groups of hearts (n = 6 in each group) were studied using 31P NMR, with spectra obtained during baseline, ischemia, and reperfusion. 31P NMR spectra were acquired using a wide-bore spectrometer (model DMX-400, Bruker). Shimming was performed using the free induction decay of the 1H2O resonance. Tuning and shimming procedures were completed within the first 9 min after completion of the attachment of the isolated heart to the cannula and its subsequent placement into the magnet.

31P NMR spectra were acquired with 128 transients, a spectral width of 5841.12 Hz, 8,192 complex points, and a recycle time of 3 s. The total acquisition time (which defines temporal resolution) was 4 min 45 s. 1H decoupling was not performed to minimize the potential for sample heating during spectral acquisition. A sealed capillary tube containing 250 mM trisodium trimetaphosphate solution was included in the NMR tube as a chemical shift and integration reference. 31P chemical shifts were referenced relative to the trisodium trimetaphosphate signal, which was set at –18.1 ppm. Myocardial intracellular pH (pHi) was calculated from the chemical shift of the intracellular inorganic phosphate (Pi-intra) resonance relative to PCr resonance using the following equation: pHi = 6.75 + log (Pi – 3.25)/(5.69 – Pi) (2).

Creatine kinase activity measurements. The method for measuring membrane-bound creatine kinase was modified according to the procedure by De Sousa et al. (9). Frozen tissue samples were weighed and homogenized in ice-cold buffer (50 mg/ml) containing 5 mM HEPES, pH 8.7, 1 mM EGTA, 1 mM DTT, 5 mM MgCl2, and 0.1% Triton X-100. The mixture was incubated for 60 min at 0°C to ensure complete enzyme extraction. The total activities of creatine kinase were assayed at 30°C and pH 7.5 using a coupled enzyme spectrophotometric assay kit measuring the reduction of NAD to NADH by the increase in absorbance at 340 nm. The rate of change in absorbance is directly proportional to creatine kinase activity. Values are expressed as moles per milligram of protein per minute.

Measurement of actin nitration. Fifty milligrams of frozen cardiac muscle were homogenized with a Polytron (Brinkman) in 500 µl of 50 mM Tris buffer (pH 7.8) and 500 µl of sample buffer (2% Triton X-100, 300 mM NaCl, 20 mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA, pH 8.0, 0.4 mM sodium orthovanadate, 0.4 mM PMSF, and 1.0% NP-40). The proteins for all these samples were extracted on ice for 30 min and cleared by centrifugation for 5 min at 10,000 g. For detection of nitrotyrosine, {alpha}-sarcomeric actin antibody was used for immunoprecipitation and later probed with antinitrotyrosine. To perform immunoprecipitations, {alpha}-sarcomeric actin antibody was added and rocked for 1 h. Protein A-agarose was added, and the suspension was placed on a rocking platform for 1 h. The agarose beads were spun down and washed three times with 50 mM Tris buffer, pH 7.8. The antigens were released and denatured by addition of SDS sample buffer. Samples were matched for protein (80 µg) and subjected to 14% SDS-PAGE. The proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The blotted PVDF membrane was blocked in freshly prepared phosphate-buffered saline (PBS) containing 3% nonfat dry milk for 60 min at 20–25°C with constant agitation. The PVDF membranes were probed with antinitrotyrosine antibody diluted in freshly prepared PBS containing 3% nonfat dry milk overnight with agitation at 4°C. The membranes were washed twice with PBS containing 0.05% Tween 20. Peroxidase-conjugated goat anti-rabbit secondary antibodies were incubated with PVDF membranes for 3 h at room temperature with agitation. The membranes were again washed twice with PBS-Tween 20 buffer. The reactive bands were visualized using an enhanced chemiluminescence system (Renaissance, NEN). Autoradiographs of Western blots were analyzed by densitometry and quantified.

Statistical analysis. Values are means ± SE, with n equal to the number of animal preparations studied. Statistical analysis of the time-dependent changes in hemodynamic and high-energy phosphate data was performed by two-way ANOVA. Statistical analysis of all other data was evaluated with a one-way ANOVA followed, if appropriate, by Tukey's test for multiple comparisons. P < 0.05 was considered statistically significant.

Materials. Creatine kinase and lactate assay kits, pyrogallol, urate, L-NA, SNAP, and Cu,Zn-SOD from bovine blood were purchased from Sigma-Aldrich; all other reagents were of the highest purity commercially available.


   RESULTS
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of urate on recovery of cardiac function after ischemia-reperfusion in isolated rat hearts. Preliminary experiments indicated that 10–50 µM urate appeared to cause cardioprotection in the ischemia-reperfusion model employed in this study. Changes in LVDP of hearts subjected to ischemia-reperfusion treated with 20 µM urate are shown in Fig. 1. Ischemia induced a rapid decline in LVDP (Fig. 1). The LVDP dropped to zero within 5 min after the onset of ischemia; thereafter, it was not generated during ischemia. LVDP of the untreated control heart recovered to ~38% of the preischemic value by the end of the reperfusion period. Perfusion of hearts with KHB buffer only or KHB with 20 µM urate for 2 h without ischemia-reperfusion indicated that the presence of urate did not significantly alter LVDP or other functional parameters (not shown), demonstrating that urate did not cause detectable secondary effects during the time course of the experimental protocol used with ischemia-reperfusion. Western blot analysis of nitrotyrosine on {alpha}-sarcomeric actin in cardiac muscle of hearts exposed to the ischemia-reperfusion protocol employed in the present study showed that ischemia-reperfusion markedly increased tyrosine nitration and that 20 µM urate and 100 µM L-NA attenuated nitrotyrosine detection (Fig. 2).



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  		Fig. 1. Time course of changes in left ventricular developed pressure (LVDP, A), heart rate (HR, B), and flow rate (FR, C) in isolated rat hearts exposed to 30 min of global ischemia and 60 min of reperfusion (control, {bullet}), ischemia-reperfusion with preischemic administration of 100 µM nitro-L-arginine (L-NA) ({blacktriangledown}) and 200 U/ml SOD ({triangledown}), and ischemia-reperfusion with postischemic administration of 20 µM urate ({circ}). There were no significant differences in heart rate and flow rate between any treatment group and the control animals. Values are means ± SE (n = 6). *P < 0.05 vs. control.

 


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  		Fig. 2. Western blot analysis of nitrotyrosine in {alpha}-sarcomeric actin on 30 min of ischemia and 60 min of reperfusion (I/R) of isolated rat hearts. Nitration of {alpha}-sarcomeric actin is shown relative to control, which is presented as 100 (n = 4). Values are means ± SE. *P < 0.05 vs. control (with ischemia-reperfusion).

 

Effects of urate, L-NA, and SOD on alterations in cardiac function caused by exposure of isolated rat hearts to ischemia and reperfusion. Table 1 shows heart rate, ±dP/dt, and LVDP for isolated rat hearts from control and urate-, L-NA-, and SOD-treated groups during the initial 30 min of equilibration, 30 min of global no-flow ischemia, and 60 min of reperfusion. Myocardial hemodynamic parameters for each group of hearts during reperfusion were expressed as the percentage of the respective parameters just before ischemia. Ischemia induced a rapid decline in LVDP (Fig. 1A), heart rate (Fig. 1B), and ±dP/dt (Table 1). These parameters dropped to zero within 5 min after the onset of ischemia, and they did not change during ischemia. With reperfusion, there was an initial period of fibrillation followed by return to a more normal cardiac rhythm associated with a substantially decreased LVDP. LVDP remained significantly depressed throughout the reperfusion period, and after 60 min of reperfusion, it was 38% of the preischemic value. L-NA (100 µM), SOD (200 U/ml) added 5 min before ischemia, and urate (20 µM) added on reperfusion improved the recovery of LVDP. The presence of urate, L-NA, and SOD resulted in a significant enhancement of the recovery of LVDP (compared with 38% in the control) at 60 min of reperfusion to 73, 57, and 54% of the preischemic values, respectively (Fig. 1A). Data in Table 1 show that ±dP/dt recovered in a manner similar to the improvement in LVDP. The recovery of heart rate was similar in untreated control hearts and hearts treated with SOD, L-NA, and urate (Fig. 1B). Heart rates of these groups at 60 min of postischemic reperfusion were not significantly different from each other (Fig. 1B).


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  		Table 1. Effects of urate, L-NA, and SOD on alterations in flow rate, heart rate, LVDP, LVEDP, and ±dP/dt in ischemia and reperfusion of isolated rat hearts

 

There was a small increase in coronary flow in the SOD- and urate-treated groups compared with the control group (Fig. 1C). In hearts pretreated with L-NA, however, lower values of coronary flow were observed throughout the time course of reflow, presumably resulting from inhibition of endothelial NOS. Nevertheless, control and urate-, SOD-, and L-NA-treated groups did not show significant differences in coronary flow from each other during the reperfusion period. The data in Fig. 1 and Table 1 suggest that the presence of L-NA, SOD, and urate in ischemia-reperfusion contributes to cardioprotection under conditions that are not associated with alterations in coronary flow.

Effects of urate on alterations in cardiac function caused by exposure of isolated rat hearts to an exogenous source of ONOO– generation. ONOO– was generated endogenously in the perfused heart preparation as a result of the reaction of NO derived from the NO donor SNAP with generated by the autoxidation of pyrogallol. The effects of SNAP and pyrogallol and their combination on heart rate and LVDP, rate-pressure product, and dP/dt are shown in Table 2. After 30 min of equilibration, a 30-min infusion of SNAP plus pyrogallol caused a rapid and significant depression of the rate-pressure product of isolated hearts. During the subsequent 60 min of perfusion, there was a partial recovery in the rate-pressure product; however, this remained significantly lower than that of control hearts. When hearts were treated with SNAP or pyrogallol alone, there was no significant difference in mechanical function throughout the 60 min of perfusion compared with the control group. Although SNAP plus pyrogallol did not significantly alter heart rate compared with control hearts, there was an attenuation of cardiac LVDP relative to controls (42% vs. 12%). This loss of contractile function was significantly attenuated when 20 µM urate was infused together with SNAP and pyrogallol.


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  		Table 2. Effects of urate on impairment of cardiac function by ONOO– generated by SNAP plus pyrogallol

 

Influence of endogenous NO and ONOO– on myocardial O2 consumption and cardiac efficiency during postischemic reperfusion of isolated rat hearts. After 20 min of reperfusion, the mean O2 consumption in the control group fell to 61.7% of the preischemic values compared with 89.1 and 78.1% in the urate- and L-NA-treated groups, respectively (Fig. 3A). The decline in O2 consumption of the control and urate- and L-NA-groups at 20 min was significantly less than the observed decrease in the rate-pressure product (Fig. 3B), indicating an increase in myocardial O2 consumption (MO2) per unit of rate-pressure product of cardiac work. The ratio of rate-pressure product to MO2 was calculated as an estimation of the aerobic metabolic efficiency. The rate-pressure product-to-MO2 ratio in the urate- and L-NA-treated hearts was persistently elevated throughout the reperfusion period compared with control. The ratio in urate- and L-NA-treated hearts vs. control was greatest in the first 20 min of reflow, but the efficiency continued to be greater than control throughout the reperfusion period (Fig. 3C).



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  		Fig. 3. Changes in myocardial O2 consumption (MO2, A), rate-pressure product (RPP, B), and cardiac efficiency (RPP/MO2, C) during 30 min of total ischemia and 60 min of reperfusion in control hearts ({bullet}) and hearts treated with 20 µM urate ({circ}) and 100 µM L-NA ({blacktriangledown}). Values are means ± SE (n = 4). Preischemic values for control group are as follows: MO2 = 28 ± 4 µmol · g dry wt–1 · min–1, RPP = 35 ± 1.7 mmHg/s (x103), and flow rate = 12 ± 1 ml/min; these parameters were not altered by urate and L-NA (not shown). *P < 0.05, urate or L-NA vs. control. {dagger}P < 0.05, urate vs. L-NA.

 

Effects of NO and ONOO– on energetics by NMR in ischemia and reperfusion of the isolated rat hearts. Examples of the NMR spectra obtained from a rat heart treated with 100 µM L-NA are shown in Fig. 4. Resonances corresponding to the {alpha}-, {beta}-, and {gamma}-phosphates of ATP and PCr are observed. In addition, signals arising from the intracellular and inorganic phosphate in the media are present. Figure 5, A and B, summarizes the changes in ATP and PCr during the initial equilibration, ischemia, and reperfusion intervals. During ischemia, ATP and PCr become almost undetectable. On reperfusion, the recovery of ATP lags behind the rapid recovery of PCr. In hearts subjected to global ischemia and reperfusion, there was a limited recovery of intracellular levels of high-energy phosphates during reperfusion. The beneficial effects of urate and L-NA on recovery of mechanical function were accompanied by a partial preservation of energy status during reperfusion. The recovery of PCr was significantly enhanced in the urate-treated hearts compared with the control hearts (93 ± 2.9 vs. 74 ± 5.7% of preischemic values, P < 0.01, n = 6). Similarly, the recovery of ATP was also significantly enhanced between the urate-treated and control hearts (42 ± 2.4 vs. 32 ± 3.7%, P < 0.04, n = 6). Urate significantly increased the recovery of PCr, and the beneficial effect on ATP levels became apparent after 10 min of reperfusion. Nevertheless, the NOS inhibitor L-NA (100 µM) restored ATP to levels similar to those in control hearts during the 60-min reperfusion. In addition to information on intracellular energetics, these spectra may also be used to measure pHi from the pH-dependent change in the chemical shift of the intracellular Pi resonance. There was no difference in pHi among the control and urate- and L-NA-treated groups (Fig. 5C).



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  		Fig. 4. 31P NMR spectra of an isolated rat heart treated with 100 µM L-NA during reperfusion after 30 min of global ischemia. Bottom trace: spectrum obtained at baseline before ischemia; middle trace: spectrum obtained during 25–30 min of ischemia; top trace: spectrum obtained at the end of reperfusion. Peaks from left to right indicate media inorganic phosphate (Pi, at 5.3 ppm), cellular Pi (initially at 5 ppm), and {alpha}-, {beta}-, and {gamma}-phosphate of ATP (at –7.5, –16, and –2.5 ppm, respectively). Bottom trace demonstrates a well-oxygenated heart with excellent energetics and very little Pi. Trace obtained at the end of ischemia reveals an almost complete lack of high-energy phosphates, a very large Pi peak, and a change in the chemical shift of this Pi peak relative to trisodium trimetaphosphate (TTP), indicative of severe intracellular acidosis. Spectrum obtained during reperfusion demonstrates a partial recovery of energetics and a restoration of intracellular pH. PCr, phosphocreatine.

 


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  		Fig. 5. Changes in ATP (A) and PCr (B) levels and intracellular pH (C) determined by 31P NMR spectroscopy during 30 min of total ischemia and 60 min of reperfusion in control hearts ({bullet}) and hearts treated with 20 µM urate ({circ}) and 100 µM L-NA ({blacktriangledown}). Values are means ± SE (n = 6). *P < 0.05 vs. control.

 

Effects of urate and L-NA on loss of membrane-bound creatine kinase activity caused by ischemia-reperfusion. The activity of membrane-bound creatine kinase was attenuated 18% from the activity observed in control hearts (120 min of perfusion) as a result of exposure to ischemia-reperfusion (not shown). When cardiac tissue samples from urate- and L-NA-treated hearts exposed to ischemia-reperfusion were compared with control hearts subjected to ischemia-reperfusion, the loss of creatine kinase activity was attenuated by 50% (P < 0.04, n = 6) and 57% (P < 0.03, n = 6), respectively. Figure 6 summarizes the results of measurement of membrane-bound creatine kinase activity.



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  		Fig. 6. Attenuation of loss of creatine kinase (CK) activity by administration of 20 µM urate and 100 µM L-NA in the membrane-bound fraction of cardiac muscle. Heart tissues were obtained at the end of reperfusion. Values are means ± SE; n = 6 for each group. *P < 0.05 vs. control (with ischemia-reperfusion). **P < 0.05 vs. control (no ischemia-reperfusion).

 

Effects of urate on lactate release upon reperfusion. As shown in Fig. 7, there was an initial washout on reperfusion of the high levels of lactate accumulated during ischemia, and lactate release gradually returned to basal levels. Nevertheless, there was no statistically significant difference between values for control and urate-treated groups during the 60 min of reperfusion.



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  		Fig. 7. Effects of control ({bullet}) and administration of 20 µM urate ({circ}) on lactate efflux after 30 min of ischemia and 60 min of reperfusion in isolated perfused rat hearts. Values are means ± SE (n = 6).

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The protective actions of urate and L-NA are consistent with the concept of ONOO– generation potentially having an important role in the cardiac dysfunction after ischemia-reperfusion in the saline-perfused rat heart model used in the present study. The observed protective actions of the probes for ONOO– are consistent with several roles for ONOO– in causing alterations in mechanisms that control high-energy phosphate metabolism in the impairment of cardiac contractile function. Although MO2 is not markedly altered by the ischemia-reperfusion treatment employed, the efficiency of O2 utilization appears to be impaired. Data in the present study indicate that changes in heart rate, coronary flow, and pHi do not appear to be important contributors to the observed dysfunction. Because L-NA and urate did not alter the marked decrease in high-energy phosphates and pH that occurs during ischemia, these actions of ischemia also do not directly cause the observed marked impairment of contractile function and high-energy phosphate metabolism observed during reperfusion. On the basis of an evaluation of the relations between cardiac work, MO2, and high-energy phosphate metabolism observed in the present study and previous investigation of similar ischemia-reperfusion models, consideration is given to the possibility that the loss of efficiency may originate primarily from a deterioration in the coupling of O2 utilization to high-energy phosphate generation compared with the coupling of energy use to cardiac work.

The protective actions of L-NA and urate against cardiac dysfunction and increases in the nitration of tyrosine on {alpha}-sarcomeric actin caused by ischemia-reperfusion in the isolated heart model examined in the present study further support previously reported evidence for the importance of ONOO– formation on reperfusion (37, 42). These previous studies provided evidence that the endogenous production of ONOO– is enhanced during the first min of reperfusion, when there is a simultaneous increase in production of NO and , which contributes to the impaired recovery of myocardial function during reperfusion. The hypoxia-reoxygenation that occurs during ischemia-reperfusion is likely to be the major stimulus for dysfunction, because exposure of isolated cardiac muscle to hypoxia-reperfusion inhibits contractile function in isolated rat cardiac papillary muscle through mechanisms that appear to be ONOO– dependent (37). Respiration is inhibited by processes that also appear to be ONOO– dependent in isolated rat cardiac muscle under these conditions (37). Although it is well established that ischemia-reperfusion treatments similar to the protocol used in the present study inhibit sites in the mitochondrial electron transport chain, such as the Fe-S center (i.e., complex I), high-energy phosphate generation, and respiratory function (8, 15), evidence for a role for ONOO– in these processes has only recently been reported (1). Cell types normally present in hearts, such as endothelium and cardiomyocytes, are likely to contribute to the formation of ONOO– in cardiac muscle that is exposed to hypoxia-reoxygenation or ischemia-reperfusion (7, 36, 37). Treatment of hearts in the present study with urate on reperfusion or with L-NA and SOD 5 min before the onset of global ischemia resulted in a significant improvement in the loss of LVDP, the rate-pressure product, and ±dP/dt observed in control hearts exposed to the ischemia-reperfusion protocol. The mechanism these three probes have in common in reducing contractile dysfunction caused by ischemia-reperfusion in the rat hearts is likely to be dominated by their ability to scavenge or prevent the formation of ONOO–, because urate is not known to directly interact with or NO. ONOO– generated by pyrogallol plus SNAP decreased LVDP, the rate-pressure product, and ±dP/dt in a manner that was antagonized by 20 µM urate, consistent with the proposed role of ONOO– in ischemia-reperfusion injury. Because the results of previous studies (36–39) and the present experiments examining the effects of high levels of NO generation suggest that elevated levels of NO in the absence of an exogenous source of generation do not cause irreversible respiratory inhibition or impaired contractile function, reactive NO-derived species and ROS generated by the redox effects of elevated levels of NO do not appear to contribute to the dysfunction that is observed under the conditions examined in the present study. Thus, although urate can scavenge many reactive species, the comparable effects of inhibition of NOS and urate suggest that urate is functioning by scavenging an NO-derived species that is likely to be ONOO–. Although the reaction between urate and ONOO– has been only partially characterized (16), urate has been considered to be an efficient scavenger of ONOO– at physiological pH (26, 32). Overall, these observations suggest that ONOO– is a key mediator of the cardiac dysfunction caused by the ischemia-reperfusion protocol used in the present study.

Endogenously formed ONOO– potentially alters important processes involved in the generation of high-energy phosphates or their use in the dysfunction that results from ischemia-reperfusion. This interpretation is based on observations in the present study associated with urate and L-NA attenuating the loss of efficiency of O2 utilization for cardiac contractile function and decreased high-energy phosphates during ischemia-reperfusion. Studies in various experimental ischemia-reperfusion models have demonstrated that MO2 rapidly recovers to a greater extent than contractile function, resulting in a loss of efficiency (20). Other investigators have previously noted that infusion of NO (18) or ONOO– (11) into isolated heart preparations also causes a depression in cardiac function associated with a reduction in myocardial energy generation and/or efficiency of O2 utilization. It has not been clearly established whether this action potentially mediated by ONOO– is due to an uncoupling of MO2 from high-energy phosphate generation or the efficiency of high-energy phosphate utilization for contractile function. The observed increase in PCr as a result of the presence of probes that would lower ONOO– levels and the absence of elevated lactate production during reperfusion, which could result from the increased stimulation of glycolysis by elevated cytosolic ADP, are potentially consistent with minimal impairment of the efficiency of high-energy phosphate utilization for contractile function after reperfusion. Previous studies measuring the influence of ischemia-reperfusion on the relation between rates of conversion of PCr into ATP and cardiac work in several different isolated heart models similar to the model used in the present study have generally found that this relation appears to remain tightly coupled, suggesting that it does not contribute to the loss of efficiency (23, 28). However, a loss of efficiency of high-energy phosphate utilization appears to occur in ischemia-reperfusion models where the acidification observed during ischemia persists through the reperfusion phase (34). This is thought to occur as a result of processes such as fatty acid metabolism stimulating lactate and proton (H+) formation by anaerobic glycolysis. Because the acidification and increased lactate formation observed during ischemia did not continue during the reperfusion phase in the present study, an acidification-mediated loss in the efficiency of high-energy phosphate utilization should not have contributed to responses observed beyond the initial period of reperfusion. Thus data examining the actions of urate and L-NA are generally not consistent with detection of an important role for ONOO– in impairing the efficiency of high-energy phosphate utilization for contractile function in the isolated saline-perfused rat heart ischemia-reperfusion model employed in the present study.

The actions of urate and L-NA can be used to evaluate the function of certain other components of the systems that potentially influence efficiency through the coupling of MO2 to energy metabolism. Creatine kinase is altered by ischemia-reperfusion and ONOO– in a manner that potentially influences the efficiency relation between energy metabolism and cardiac function. According to the creatine kinase-PCr energy-shuttle hypothesis (35), PCr serves to transfer a high-energy phosphate from ATP produced by mitochondria to sites of ATP utilization by contractile proteins on myofibrils. During metabolic stress, this energy shuttle becomes essential for maintaining high-performance states, and ischemia-reperfusion has been previously shown to impair the function of components of this system (27). For example, if creatine kinase is inhibited, respiration requires high ADP diffusion flux back into the mitochondrion, which is one of the regulators of respiration (27). Creatine kinase is also extremely sensitive to inhibition by authentic ONOO– and the simultaneous generation of NO and (19). In the present study, ischemia-reperfusion inhibits membrane-bound creatine kinase in a manner that was prevented by urate and L-NA, which is consistent with a role for ONOO– in the observed inhibition. Because the membrane-bound creatine kinase activity is thought to be primarily dependent on the mitochondrial form of this enzyme (17), the modest loss of this activity could contribute to alterations in efficiency caused by ischemia-reperfusion. Mitochondrial creatine kinase is a prime target for modification and inactivation by ONOO– (33), and its inactivation by ONOO– has been shown to impair cardiac function and mitochondrial energy metabolism in a Langendorff perfusion model (31). Overall, the substantial recovery of PCr in the presence of L-NA and urate compared with only a modest recovery of ATP lost during ischemia further supports a role for ONOO– in inhibiting this enzyme during ischemia-reperfusion and the importance of this energy shuttle in maintaining high-performance states.

The potential impact of alterations in mitochondrial function on the origins of deviations in efficiency of energy metabolism used for cardiac work needs to be given additional consideration. This is because of the inconsistency of observations that ischemia-reperfusion inhibits mitochondrial function without markedly altering the recovery of O2 utilization associated with cardiac work in multiple ischemia-reperfusion models similar to the model used in the present study. Previous studies have detected an inhibition of O2 consumption in arrested hearts exposed to ischemia-reperfusion (29) and inhibition of electron transport and ADP-stimulated (state 3) respiration in mitochondria isolated from hearts exposed to ischemia-reperfusion protocols (6, 24) similar to that used in the present study. Our previous work with isolated rat and bovine cardiac muscle provided evidence that the hypoxia-reoxygenation component of ischemia-reperfusion inhibits respiration in an ONOO–-dependent manner, because the effects of hypoxia-reoxygenation are prevented by L-NA, SOD, and urate (39). A subsequent study has shown that an NO-dependent mechanism appears to mediate the alterations in mitochondrial respiratory function observed in hearts exposed to ischemia-reperfusion (1). Interestingly, in our previous studies, a mitochondrial uncoupler (dinitrophenol) was able to stimulate O2 consumption in cardiac muscle exposed to hypoxia-reoxygenation or ONOO– to levels that were higher than basal but lower than levels observed in the presence of the uncoupler without exposure to hypoxia-reoxygenation or ONOO– (39). On the basis of the present understanding of how mitochondrial respiration is controlled by multiple processes (22), it is possible that processes associated with cardiac work activated after exposure to ischemia-reperfusion can stimulate respiration back to the levels observed in the presence of an impairment of other components essential to mitochondrial function. Processes caused by ischemia-reperfusion, such as the uncoupling of electron transport as a result of proton leakage across the mitochondrial membrane (34) or electron transfer to molecular O2 and the formation of ROS, could be important factors that increase O2 consumption in the presence of an inhibition of electron transport, which potentially contribute to the loss of efficiency.

Although multiple signaling and metabolic systems are likely to be altered by ischemia-reperfusion, this study provides evidence that ONOO– is potentially a prominent factor in the contractile dysfunction observed in the saline-perfused rat heart. The dysfunction in this model appears to result from an ONOO–-mediated impairment of high-energy phosphate metabolism associated with a loss of efficiency of O2 utilization for cardiac work. Data in the present study and similar models of ischemia-reperfusion suggest that consideration should be given to processes altered by ONOO– that are involved in the coupling of mitochondrial respiration to high-energy phosphate generation as potentially important sites contributing to the loss of efficiency. Because the blood-perfused human coronary circulation contains inflammatory and thrombotic systems regulated by NO, other important nutrients such as fatty acids, and antioxidants including urate, mechanisms other than the processes potentially dependent on ONOO– examined in the present study may also be important factors in the observed responses to periods of ischemia followed by reperfusion. However, the mechanisms potentially dependent on ONOO– examined in the present study may be important factors in cardiac stunning and a more severe loss of cardiac function caused by ischemia-reperfusion.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-31069, HL-43023, and HL-66331.


   ACKNOWLEDGMENTS
 
Studies reported here were conducted by W. H. Lee in partial fulfillment of the requirements for a Ph.D. degree in physiology at New York Medical College.

Part of the information reported here was presented at the Experimental Biology 1999 Meeting, Anaheim, CA, and was published in abstract form (FASEB J 13: A754, 1999).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. S. Wolin, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (E-mail: mike_wolin{at}nymc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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