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Am J Physiol Heart Circ Physiol 292: H109-H116, 2007. First published October 6, 2006; doi:10.1152/ajpheart.00925.2006
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Oxygen Sensing: Life and Death of a Cell

O2 delivery and redox state are determinants of compartment-specific reactive O2 species in myocardial reperfusion

Jason D. Stoner,1 Thomas L. Clanton,2,3 Sverre E. Aune,1,3 and Mark G. Angelos1,3

1Department of Emergency Medicine; 2Division of Pulmonary, Critical Care and Sleep Medicine, and 3Dorothy M. Davis Heart and Lung Research Institute, Ohio State University, Columbus, Ohio

Submitted 25 August 2006 ; accepted in final form 2 October 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reperfusion of the ischemic myocardium leads to a burst of reactive O2 species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O2 delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O2-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O2 delivery during the first 5 min of reperfusion was 84.7 ± 4.2 ml O2/min with 20% O2-saturated buffer and 354.4 ± 22.8 ml O2/min with 95% O2 (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O2 compared with 20% O2. However, the resorufin signal (extracellular ROS) was significantly increased with 20% O2 compared with 95% O2 during reperfusion. Perfusion of hearts with 20 mM glucose reduced the ·NADH during ischemia (P < 0.001) and the ·ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O2 delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O2 delivery promotes intracellular ROS and low O2 delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.

ischemia; reoxygenation; oxygen radicals

REINTRODUCTION OF O2 to the globally ischemic heart triggers a cascade of reperfusion events, including an initial burst of reactive O2 species (ROS). Studies of the isolated heart suggest that ROS production peaks within the first few minutes of reperfusion (37). Although ROS at low levels appear to have signaling capabilities that are cardioprotective, particularly during ischemia (13), it is thought that the much larger ROS burst at the onset of reperfusion impairs recovery of ventricular function (8). Cell injury from ROS may be wide ranging and include direct injury to membrane lipids, oxidative modification of protein and deoxyribonucleic acid, and cellular signaling for mitochondrial apoptotic transition (32). Known sources for ROS during reperfusion include endothelial xanthine oxidase (30), NAD(P)H oxidase (12), and the mitochondrial electron transport chain (complexes 1 and 3) (6), although other sources are also possible.

The role of ROS, including the ROS burst at the onset of reperfusion, in activating cardioprotective versus cardiodestructive mechanisms seems to primarily depend on the severity of ischemia preceding reperfusion. Ischemic preconditioning is known to be a powerful means of cardioprotection, maximizing contractile recovery after a later, prolonged ischemic insult. ROS are believed to be involved in this preconditioning signal and are likely to involve mechanisms such as activation of transcription factors (20), inhibition of inflammatory responses (5), and attenuation of apoptotic signaling (11). In contrast to the ROS preconditioning signaling response, with more prolonged ischemia, the deleterious effects of ROS appear to predominate, resulting in more aggressive injury to membranes and nucleotides, and activation of cell death cascades (6, 15). Targeting of the ROS burst after prolonged periods of global ischemia, such as those that occur following cardiac arrest, represents an unexplored strategy for therapeutic interventions to improve early cardiac arrest resuscitation and survival.

There is some evidence that controlling the initial reperfusion conditions, particularly the reintroduction of O2 and substrates, may alter the initial burst of ROS at reperfusion. Earlier studies utilizing the isolated perfused heart have noted improved reperfusion left venticular (LV) function and bioenergetics with alterations of glucose (29), glucose and insulin (3, 10), and reduced O2 delivery (17) at the time of reperfusion. However, none of these studies has examined the initial reperfusion ROS burst under these controlled reperfusion conditions.

In earlier work, we have observed myocardial [NADH] changes in response to changes in O2 delivery and glucose concentration (28). Recently, we noted a paradoxical ROS response at the onset of reperfusion when O2 concentration was varied (2). The burst of ROS at the onset of reperfusion was significantly increased under hypoxic reperfusion conditions in the isolated perfused heart, compared with reperfusion with 95% O2 saturated buffer. Since these measurements were made using a predominantly extracellular spin trap, 5,5-dimethyl-L-pyrroline N-oxide (DMPO), it is unclear whether the intracellular ROS signal varies with O2 reperfusion conditions.

The present study examines the hypothesis that ROS formation is a function of the O2 delivery during reperfusion and the cellular redox state in the ischemia period immediately before reperfusion. Second, it evaluates the compartmentalization of the intra- and extracellular ROS signals during reperfusion and how these signals are differentially affected by the conditions of reperfusion. The results demonstrate that both NADH accumulation during ischemia and O2 delivery during reperfusion are important determinants of ROS formation during initial reperfusion, and the effects of these variables on ROS formation differ in the intra- and extracellular compartments. Interventions to control the redox state and the O2 conditions during ischemia and reperfusion may direct future therapeutic advances, since currently no effective clinical strategies exist to effectively modify ROS production during the initial reperfusion of the heart.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolated heart preparation. Male Sprague-Dawley rats (350–450 g) supplied by Harlan (Indianapolis, IN) were used in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996), and the approval of the Ohio State University Laboratory Animal Resources Committee. Hearts were isolated and perfused in the Langendorff mode as we have previously described (21). Briefly, rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg). The right superficial jugular vein was isolated, and heparin (1,000 U/kg) was administered. The trachea was cannulated with a 16-gauge catheter attached to a rodent ventilator (Harvard Apparatus, South Natick, MA) set to provide adequate ventilation with room air. A midsternal thoracotomy was performed to expose the heart and to cannulate the aorta. Following rapid cannulation of the aorta, retrograde coronary perfusion with Krebs-Henseleit buffer (1.25 mM CaCl2, 5.5 mM glucose, 112 mM NaCl, 25 mM NaHCO3, 5 mM KCl, 1.2 mM MgSO4, 1 mM K2PO4, and 0.2 mM octanoic acid, bubbled with 95% O2-5% CO2, pH of 7.4) was initiated in situ. Hearts were quickly excised from the chest and transferred to the Langendorff apparatus with warmed (37°C) Krebs-Henseleit buffer perfusion at a constant perfusion pressure of 85 mmHg. A saline-filled latex balloon attached to a pressure transducer was inserted into the left ventricle for measurement of LV contractile function. The hearts were positioned in a temperature-controlled (37.4°C) glass chamber. Coronary flow rates were measured.

The LV balloon volume was inflated at the beginning of the experiment to a LV end-diastolic pressure of 5 mmHg. LV pressure was continuously sampled at a frequency response of 45 Hz and digitally processed by a heart performance analyzer (Digi-Med, Micro-Med, Louisville, KY). Continuous measures of LV function were derived by computer algorithm: LV systolic pressure, LV end-diastolic pressure, heart rate, and maximum rate of pressure change over time (dP/dtmax and –dP/dtmax). Developed pressure (systolic – diastolic pressure) and rate-pressure product (developed pressure x heart rate) were calculated.

Measurement of NADH, FAD, and ROS. NADH and FAD are intrinsic fluorophores, which fluoresce when excited by ultraviolet and visible light, respectively (1). The oxidized cofactor NAD+ and the reduced cofactor FADH2 do not fluoresce. With the use of the autofluorescent properties of NADH and FAD, the relative change in NADH and FAD can be continuously determined as a real-time measure of the cellular redox state.

Intracellular ROS were measured in the intact perfused heart utilizing the fluorescent probe dihydrofluorescein diacetate (dihydrofluorescein-DA, Molecular Probes). Earlier studies have demonstrated dihydrofluorescein-DA to have superior loading and sensitivity characteristics compared with other commonly used redox sensitive probes (23, 35). Dihydrofluorescein-DA passes across the cell membrane, where esterases cleave the acetate moiety, trapping the reduced probe in the intracellular compartment. In the presence of ROS, the dihydrofluorescein is oxidized to fluorescein. The probe is most sensitive to H2O2 (35) and hydroxyl radicals and relatively insensitive to superoxide (23), cytochrome c, and nitric oxide (35, 36). Dihydrofluorescein-DA was dissolved in a 10 mg/100 µl DMSO stock solution for a final dihydrofluorescein-DA concentration of 20 µM. We have noted this concentration of DMSO to have no measurable effect on heart contractile function. Hearts were loaded with 20 µM dihydrofluorescein-DA at a temperature of 37°C for 15 min.

In separate experiments, extracellular ROS were measured by using the extracellular fluorescent probe 10-acetyl-3,7-dihydroxyphenoxazine (Amplex red), which is an extremely sensitive extracellular marker for the presence of H2O2. When combined with horseradish peroxidase, it reacts stoichiometrically with H2O2 to produce resorufin, which is restricted to the extracellular space (27). Ten minutes before ischemia, Amplex red (25 µM) and horseradish peroxidase (0.1 U/ml) were added to the perfusate and continued through the first 10 min of reperfusion (27).

NADH, FAD, and fluorophore fluorescence were measured in isolated hearts within a black box that excluded extraneous light. A 6-mm-diameter fiberoptic bundle containing excitation and emission fibers was carefully positioned against the anterior LV wall. A securing screen was adjusted on the contralateral side of the heart to ensure continuous contact of the fiberoptic cable against the LV anterior wall. Positioning of the heart was done to prevent inhibition of LV function as measured by the heart performance analyzer. The proximal end of the fiberoptic cable was connected to a Radnoti Ratiometric Fluorometer (Radnoti Glass, Monrovia, CA). Excitation light arose from a 150-W xenon arc lamp filtered through one of four alternating excitation filters on a filter wheel. The bandpass filters used for excitation (Ex) and emission (Em) responses were for NADH [Ex 330 nm, bandwidth (BW) 80 nm; and Em 470 nm, BW 10], FAD (Ex 455 nm, BW 70 nm; and Em 630 nm, BW 50 nm), fluorescein (Ex 490 nm, BW 20 nm; and Em 535 nm, BW 35 nm) and resorufin (Ex 535 nm, BW 35 nm; and Em 595 nm, BW 45 nm). To minimize any photobleaching effect, the tissue was exposed to light for 8.5 ms of every 40-ms turn of the filter wheel and the lamp shutter was left open for only 7 s out of every 30 s. A photomultiplier tube within the spectrophotometer measured emission intensity. To compensate for movement, fluorescence emission signals were digitized, signal averaged over the sampling periods, and recorded in arbitrary fluorescence units as a fraction of baseline fluorescence. At the end of the reperfusion period, a 30-ml volume of perfusate containing 50 µM H2O2 was given over 2 min. This signal was used to standardize the fluorescein signal at reperfusion.

Metabolic and chemical analyses. In some experiments, coronary perfusate and coronary effluent were collected and analyzed at baseline and at scheduled intervals throughout the protocol. A gas analyzer (Synthesis 45, Instrumentation Laboratory, Lexington, MA) was used to measure PO2 and glucose. In this buffer-perfused model, myocardial O2 delivery (MDO2) was calculated utilizing coronary perfusate flow and measured O2 content: MDO2 = coronary perfusate flow (in ml/min) x [perfusate PO2 (in mmHg) x 24 µl O2·ml–1·mmHg–1]. In some experiments, tissue lactate and pyruvate concentrations were measured in hearts that were immediately freeze clamped at baseline before ischemia or at the end of ischemia before reperfusion. Metabolites were extracted under liquid nitrogen in perchloric acid. Lactate and pyruvate were measured with the use of standard spectrophotometric methods (7).

Reperfusion protocols. In the first series, hearts were loaded with either the molecular probe dihydrofluorescein-DA or Amplex red. Hearts underwent 20 min of global ischemia and then reperfusion with either high (95%) or low (20%) O2-saturated perfusate for the first 5 min to determine the initial intra- and extracellular ROS response to low and high O2 at reperfusion. After the initial 5 min of reperfusion, perfusion was changed back to 95% O2-saturated perfusate for the remaining 25 min of reperfusion. In a second series of experiments, hearts were perfused with either 5.5 or 20 mM glucose perfusate and then underwent 20 min of global ischemia, followed by 30 min of reperfusion to evaluate the influence of this substrate change on redox state and ROS formation during early reperfusion. In a third series, hearts were perfused with either 5.5 or 20 mM glucose in buffer equilibrated with either 95% or 20% O2 for the first 5 min of reperfusion, as in the first protocol, to determine the crossed effects of glucose and O2 on the recovery of contractile function and other variables. In this series, the experimental design yielded four reperfusion groups: 1) normal glucose, high O2; 2) normal glucose, low O2; 3) high glucose, high O2; and 4) high glucose, low O2.

Data analysis. Changes in the fluorescence of NADH, FAD, fluorescein, and Amplex red signals during ischemia were normalized to baseline levels (F/F0) where F0 is the baseline fluorescence immediately following fluorophore loading. We infused a standardized bolus of H2O2 at the end of the experiment to standardize the fluorescein response (50 µM H2O2) and the resorufin response (500 nM H2O2). In reasonable time periods (e.g., short bursts following reperfusion), one can usually establish two baseline points, before and after the burst of ROS, where the fluorescence is stable. We can then extrapolate the burst from a line connecting these reference points. Group differences were assessed by using the Kruskal-Wallis nonparametric test. LV function measurements of rate-pressure product, developed pressure, and dP/dtmax in each heart were expressed as a ratio of reperfusion to baseline (preischemic) function and analyzed using ANOVA and a Tukey's post hoc test. All group values were expressed as means ± SE. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
An immediate increase and plateau in the NADH signal was noted with the onset of global ischemia, and a rapid recovery was noted with reperfusion in all experiments. In hearts loaded with dihyrofluorescein or Amplex red, following 20 min of global ischemia, an increase in the fluorescein (Fig. 1) and the resorufin (Fig. 2) signals were noted within the first minutes of reperfusion, indicating intracellular and extracellular ROS production, respectively. In nonischemic 60-min-perfused control hearts, no changes in the NADH or fluorescein signal were seen (data not shown). To determine the contribution of H2O2 to the fluorescein signal at reperfusion, in a separate group of experiments, hearts were loaded with dihydrofluorescein-DA and then pretreated with 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) (22 µM), a glutathione peroxidase mimetic that scavenges H2O2 production through reactions with endogenous glutathione (26). In ebselen-treated hearts, no increase in the fluorescein signal was seen at reperfusion (data not shown).


Figure 1
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  		Fig. 1. Fluorescein and NADH fluorescence during ischemia and reperfusion (95% O2). Fluorescein signal (top trace) and NADH autofluorescence (bottom trace) during global ischemia of 20 min followed by reperfusion from a representative experiment. An increase in fluorescein signal is seen at the onset of ischemia and the onset of reperfusion corresponding to increased reactive O2 species (ROS) formation. A controlled infusion of 50 µM H2O2 is utilized to create a standardized response by which to normalize the reperfusion ROS burst. NADH rises to a plateau during global ischemia and recovers to near baseline with return to baseline O2 delivery conditions.

 

Figure 2
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  		Fig. 2. Resorufin (Amplex red) and NADH fluorescence during ischemia and reperfusion (20% O2). Resorufin fluorescent signal (top trace) and NADH autofluorescence (bottom trace) during global ischemia of 20 min followed by reperfusion with 20% O2 from a representative experiment. Amplex red and horseradish peroxidase are loaded just before ischemia and then again during reperfusion, since the probe remains extracellular and washes out. An increase in signal is seen at reperfusion, indicating a burst of ROS that interacts with the extracellular probe. A control bolus of 50 µM H2O2 solution is utilized to create a standardized response by which to normalize the reperfusion ROS burst.

 
Hearts reperfused with 95% O2-saturated buffer were noted to have an increased fluorescein signal at reperfusion, corresponding to increased intracellular ROS production, compared with hearts undergoing an identical period of ischemia and reperfused with 20% O2-saturated buffer (Fig. 3). This is in contrast to hearts loaded with Amplex red, undergoing identical ischemia and reperfusion conditions. In these hearts, the resorufin signal, corresponding to extracellular ROS production, was significantly higher in hearts reperfused with 20% O2-saturated buffer compared with hearts reperfused with 95% O2-saturated buffer (Fig. 3).


Figure 3
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  		Fig. 3. Intracellular and extracellular ROS burst at reperfusion with 95% and 20% O2. Left: hearts are loaded with dihydrofluorescein and reperfused with 95% or 20% O2-saturated perfusate after 20 min of global ischemia (n = 8/group). The ROS burst seen in the first minutes of reperfusion is significantly higher (*P < 0.001) when reperfused with 95% O2 compared with 20% O2. This signal represents intracellular ROS generation and was normalized to the signal generated by a known bolus of H2O2 injected into the heart at the end of each experiment (F1/FH2O2). FH2O2, fluorescent signal from known concentration of H2O2 injected into the heart at the end of the experiment. Right: hearts loaded with Amplex red and horseradish peroxidase and reperfused with 95% or 20% O2-saturated perfusate after 20 min of global ischemia (n = 4/group). This signal is expressed as F1/F0, with F0 defined as the fluorescent signal at baseline. This is an extracellular ROS signal.

 
In series 2, changes in redox state, as measured by tissue NADH and FAD autofluorescence, were evaluated in hearts perfused with either 5.5 or 20 mM glucose perfusate (n = 4/group). In hearts perfused with 20 mM glucose, an increase in NADH was noted under nonischemic perfusion conditions (Fig. 4). However, the relative change in NADH during ischemia and reperfusion was significantly less in 20 mM glucose-perfused hearts compared with 5.5 mM glucose-perfused hearts (Fig. 4). In contrast to NADH changes during ischemia and reperfusion, FAD changes were similar between groups (Fig. 4). To test whether hyperglycemic perfusion of the heart altered ischemic NADH concentrations through changes in glycolytic flux, two additional groups of hearts were studied. Hearts perfused with standard oxygenated perfusate and 5.5 or 20 mM glucose were freeze clamped at baseline or at the end of 20 min of ischemia (n = 4/group). The tissue lactate-to-pyruvate ratio at preischemia and at the end of ischemia was similar between groups (Fig. 5).


Figure 4
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  		Fig. 4. FAD and NADH fluorescence in 5.5 and 20 mM glucose-perfused hearts. With the change from 5.5 to 20 mM glucose in one group (F0), there was a significant increase in NADH autofluorescence (*P < 0.01) to a new baseline (F1). The relative ·NADH during ischemia (F2 – F1) and reperfusion (F3 – F2) was significantly less in the 20 mM glucose hearts compared with 5.5 mM glucose hearts (**P < 0.01). The ·FAD during baseline, ischemia, and reperfusion were similar between groups (n = 4/group).

 

Figure 5
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  		Fig. 5. Lactate-to-pyruvate ratio at baseline (BL) and end of ischemia (Isch). The lactate-to-pyruvate ratio significantly increased (*P < 0.001) in both groups during ischemia; however, neither the ratio at baseline nor that at ischemia was significantly different between the 5 and 20 mM glucose (Gluc)-perfused hearts.

 
In series 3, hearts loaded with dihydrofluorescein and perfused with high glucose concentrations (20 mM) had a significantly lower ·NADH during ischemia and a depressed ROS burst at reperfusion compared with 5.5 mM glucose-perfused hearts (Fig. 6). In relating the cellular redox state at the end of ischemia to the initial ROS burst at the onset of reperfusion, we noted a significant correlation between the NADH signal at the end of ischemia and the magnitude of the fluorescein signal at reperfusion (Fig. 7). Baseline LV function, including developed pressure, rate-pressure product, and dP/dtmax, was similar between all groups in series 3 (n = 8/group) (Table 1). During the 5-min-controlled reperfusion period, MDO2 during the first 5 min of reperfusion was 84.7 ± 4.2 ml O2/min in hearts perfused with 20% O2 and 354.4 ± 22.8 ml O2/min in hearts perfused with 95% O2 (n = 8/group, P < 0.001). Recovery of LV function after 20 min of ischemia was similar in both glucose groups reperfused with 95% O2 and the 20 mM glucose group reperfused with 20% O2. However, in the 5.5 mM glucose hearts initially reperfused with 20% O2, LV function during reperfusion remained significantly depressed relative to the other three groups (Fig. 8). Reperfusion flow was similar between all groups (n = 8/group).


Figure 6
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  		Fig. 6. Tissue NADH levels at the end of ischemia and ROS reperfusion burst in hearts perfused with 5.5 and 20 mM glucose. Tissue NADH levels at the end of global ischemia in hearts perfused with 5.5 glucose or 20 mM glucose buffer (top). NADH levels were significantly higher in 5.5 mM glucose-perfused hearts, which correlated with a larger increase in ROS production at the onset of reperfusion (bottom). The ROS burst (fluorescein signal), following 20 min of global ischemia, was significantly decreased in hearts perfused with 20 mM glucose perfusate compared with 5.5 mM glucose-perfused hearts (*P < 0.001, n = 8/group). The fluorescein signal was normalized to a known quantity of H2O2. Both groups were reperfused with 95% O2-saturated buffer after 20 min of ischemia.

 

Figure 7
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  		Fig. 7. The ROS burst at reperfusion as a function of the reductive pressure during ischemia as measured by NADH. A strong correlation was noted in all groups between the change from baseline of NADH measured at the end of global ischemia and the fluorescein signal increase noted at the onset of reperfusion (linear regression, r = 0.693, P < 0.001). Hearts were loaded with dihydrofluorescein. Fluorescein signal was determined during reperfusion and compared with the NADH signal at the end of global ischemia.

 

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  		Table 1. Baseline LV function

 

Figure 8
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  		Fig. 8. Left ventricular contractile recovery after reperfusion. Recovery of maximum rate of left ventricular pressure change over time (dP/dtmax), developed pressure (Dev Press), and rate-pressure product (Rate Pres Prod) were significantly depressed in 5.5 mM hearts initially reperfused with 20% O2 (*P < 0.001). Reperfusion function was preserved in 20 mM hearts reperfused with 20% O2.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Reperfusion of the ischemic heart is accompanied by a burst of ROS, which peaks in the first minutes of reperfusion (37). In this study, we report in the whole heart that reperfusion conditions, particularly O2 concentrations and to a lesser extent redox state, modulate this initial burst of ROS and demonstrate a compartment-specific response. We note that O2 delivery at reperfusion is an important factor in the magnitude of the ROS burst and subsequent recovery of LV function. Reperfusion with hypoxic-saturated (20% O2) perfusate in the first minutes of reperfusion significantly attenuated the intracellular ROS burst but increased the extracellular ROS burst. In the acellular perfusate of the isolated perfused heart, O2 saturation of 20%, although equivalent to normal atmospheric FIO2 conditions, constitutes a significant reduction in O2 delivery such that O2 consumption is delivery dependent (28). The increase seen in initial extracellular ROS production with hypoxic reperfusion supports our earlier work in the whole heart, where we noted a similar increase in extracellular ROS generation with hypoxic reperfusion using electron paramagnetic resonance methodology (2). In the earlier study, ROS was measured with the spin trap DMPO, which forms an adduct with primarily extracellular O2bullet– and/or bulletOH. In the present study, both intracellular and extracellular oxidant-sensitive probes were used to measure ROS, which showed an early paradoxical compartment-specific response to O2 delivery at reperfusion. These important new findings suggest that different ROS-generating mechanisms in different compartments have different responses to environmental conditions. The exact mechanisms responsible for these compartment-specific responses are not specifically addressed in this study but likely vary with the predominant ROS-generating source in each compartment and the cell type. Both increased and decreased ROS formation has been reported in different cell lines exposed to hypoxia. For example, cultured porcine pulmonary artery endothelial cells exposed to hypoxia had a significant decline in ROS formation (33), whereas cardiomyocytes exposed to hypoxia showed an increase in ROS production (13). These experiments emphasize the importance of the initial reperfusion conditions, because hypoxic reperfusion was continued in both studies only for the first 5 min after which normal O2 delivery with 95% O2-saturated perfusate was reinstituted. Despite the relatively short hypoxic reperfusion period of 5 min, recovery of LV function remained significantly depressed at the end of the 30-min reperfusion period. The early O2 delivery to the ischemic myocardium appears to be an important reperfusion determinant of the magnitude of the initial intracellular ROS burst and subsequent recovery of LV function.

Previous studies have utilized autofluorescence spectroscopy methodology to measure NADH in isolated hearts and myocardial tissue (9, 19, 22). Within the total NAD and NADP pool, NADP makes up ~6% of the total pool (24). The majority of NADH measured is thought to originate from the mitochondria (4, 14). A reduction in O2 delivery (ischemia) leads to an accumulation of NADH (15, 31) from impaired electron transport. Previous experiments have demonstrated tissue NADH to be a near instantaneous indicator of cellular hypoxia (28). Nevertheless, surface fluorometry of the LV has limitations based on the positional dependence of the signal. With regard to fluorescent probes, variable probe uptake during myocyte loading can also represent a significant problem. These technical limitations are partially overcome by using each heart as its own control and referencing the fluorescent signal to the baseline signal (F0) or the signal generated by a standardized dilute H2O2 bolus. Additionally, other tissue fluorescence studies commonly cite hemoglobin interference as the most important confounding factor in assessing tissue NADH fluorescence (1, 18). In our non-blood perfused model, this major limitation does not impact the observed results. The predominant ROS signal measured with dihyrofluorescein appears to arise from intracellular hydrogen peroxide (35). Further evidence of the dihydrofluorescein sensitivity to H2O2 is provided in our study by replication of the ROS signal with infusion of a dilute H2O2 bolus and blocking the fluorescein signal rise at the time of reperfusion with ebselen, a glutathione peroxidase mimic (26).

We also noted a reduction of the ROS signal at reperfusion with modification of the cellular redox state as indicated by changing [NADH] levels during ischemia. Perfusion of hearts with a hyperglycemic perfusate (20 mM glucose) resulted in a gradual increase in NADH before ischemia, a decrease in NADH accumulation during ischemia, and a reduced level of initial ROS generation at reperfusion. In contrast to hyperglycemic hearts, hearts perfused with normal glucose levels (5.5 mM) had no change in NADH levels at preischemia, a greater accumulation of NADH during global ischemia, and a larger ROS burst at the onset of reperfusion. This is consistent with previous work suggesting an association of NADH levels with the burst of ROS production at reperfusion (16). These earlier investigators noted that oxidation of NADH catalyzed by xanthine oxidoreductase is coupled to the reduction of O2, suggesting a mechanism by which elevated NADH levels at the end of ischemia can enhance ROS production during reperfusion.

We investigated whether the basis for a smaller rise in NADH concentration during global ischemia under hyperglycemic conditions is due to alterations of glycolytic flux. With ischemia, the ratio of NADH to NAD+ increases in the cytosol, due to both inhibition of pyruvate conversion to acetyl-CoA with subsequent entry into the mitochondria and inhibition of the malate-aspartate shuttle, which shuttles cytoplasmic NADH to the mitochondria. In the mitochondria during ischemia, NADH accumulates due to the cessation of NADH oxidation in the mitochondrial respiratory chain. A small level of oxidation of NADH occurs with the conversion of pyruvate to lactate under anaerobic conditions, but this too ceases after significant buildup and feedback of metabolic by-products. If during ischemia, under hyperglycemic conditions, more pyruvate is converted to lactate in the cytosol, more NADH is oxidized to NAD+, which could result in a smaller increase in the measured NADH concentrations during ischemia. However, in the absence of significant changes in ischemic tissue lactate and pyruvate levels between hyperglycemic and normoglycemic groups, as seen in this study, it does not appear that changes in glycolytic flux accounted for the differential accumulation of NADH during ischemia between groups. Instead it appears that the basis for changes in ischemic NADH accumulation was localized to the mitochondria. This is consistent with estimates of the mitochondrial NAD+ and NADH pool. Under normal physiological conditions, the NAD+ and NADH pool in the cytosol is ~10% of the total, compared with 90% in the mitochondria (25). Significant changes between groups were seen during ischemia in both FAD and NADH signals, albeit in opposite directions. Because of the autofluorescent nature of FAD, during ischemia a reduction in FAD to the nonautofluorescent FADH2 is seen, which mirrors the NADH signal. This redox reaction is primarily confined to the mitochondria. Further support of a mitochondrial source for NADH changes during ischemia is found in earlier work showing that the NADH fluorescence in the isolated perfused rat heart originates primarily from the mitochondria (19). In another study, a hyperglycemic-mediated increase in ROS production in nonischemic vascular endothelial cells was noted to be mediated by tricarboxylic acid cycle NADH but not cytosolic-derived NADH (34).

The greater increase in ischemic NADH in normoglycemic hearts reflects a more reduced redox state in the mitochondria, such that with the reintroduction of O2, there is a greater reductive force that could presumably increase the pressure to reduce O2 forming superoxide and thus could lead to an increased burst of ROS in these hearts. As such, it would appear that ischemic NADH and, to a lesser extent, FAD are prereperfusion markers of the magnitude of the initial ROS burst accompanying reperfusion and that interventions which attenuate the rise of ischemic NADH may also attenuate the reperfusion ROS burst.

In summary, tissue O2 levels appear to be a critical factor in the burst of ROS typically seen in the first minutes of reperfusion and are compartment specific. Similarly, the ischemic redox state preceding reperfusion may be an important determinant of ROS formation at reperfusion and appears to be mediated by NADH/NAD+ changes in the mitochondria before the onset of reperfusion. Thus, in the isolated whole heart, cellular redox state appears to be an important prereperfusion factor and O2 delivery a key post reperfusion factor modulating the magnitude of the ROS burst in the heart at reperfusion.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The research was supported by a training grant from the Emergency Medicine Foundation Research Training Award (to J. D. Stoner); National Heart, Lung, and Blood Institute Grant HL-53333 (to T. L. Clanton); and the American Heart Association Ohio Affiliate (to M. G. Angelos).


   ACKNOWLEDGMENTS
 
We thank Alan Blumberg and Valery Wright for technical assistance in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. G. Angelos, Dept. of Emergency Medicine, The Ohio State Univ., 146 Means Hall, 1654 Upham Dr., Columbus, OH 43210 (e-mail: angelos.1{at}osu.edu)

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


   REFERENCES
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 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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