(Circulation. 2000;102:III-332.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Glutathione Reverses Endothelial Damage From Peroxynitrite, the Byproduct of Nitric Oxide Degradation, in Crystalloid Cardioplegia
From the Emory University School of Medicine, Atlanta, Ga and the Division of Cardiothoracic Surgery, Carlyle Fraser Heart Center of Emory University, Cardiothoracic Research Laboratory, Atlanta, Ga.
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Abstract |
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Background—NO has been advocated as an adjunct to cardioplegia solutions. However, NO undergoes a rapid biradical reaction with superoxide anions to produce peroxynitrite (ONOO-). ONOO- in crystalloid cardioplegia solution induces injury to coronary endothelium and to systolic function after cardioplegia and reperfusion. However, ONOO- may be degraded to less lethal or cardioprotective intermediates with glutathione (GSH) in reactions separate from its well known antioxidant effects. We hypothesized that GSH detoxifies ONOO- and reverses defects in endothelial function and systolic function when present in crystalloid cardioplegia.
Methods and Results—In anesthetized dogs on cardiopulmonary bypass, a 45-minute period of global normothermic ischemia was followed by 60 minutes of intermittent cold crystalloid cardioplegia (Plegisol) and 2 hours of reperfusion. The cardioplegia solution contained 5 µmol/L authentic ONOO-; catalase was included to attenuate the potential antioxidant effects of GSH and to unmask the effects on ONOO-. In 1 group (CP+GSH, n=5), the cardioplegia contained 500 µmol/L GSH, whereas 1 group received crystalloid cardioplegia without GSH (CCP, n=6). There were no group differences in postcardioplegia left ventricular systolic function (end-systolic pressure-volume relation, impedance catheter: CCP 10.0±2.4 versus CP+GSH 10.6±1.3 mm Hg/mL) or diastolic chamber stiffness (ß-coefficient: CCP 0.35±0.2 versus CP+GSH 0.31±0.18). Myocardial neutrophil accumulation (myeloperoxidase activity) was attenuated in CP+GSH versus CCP (2.2±0.7 versus 5.4±1.2, P<0.05). In postexperimental coronary arteries, maximal endothelium-dependent relaxation was greater in CP+GSH than in CCP (118±6% versus 92±5%, P<0.05), with a smaller EC50 value (-7.10±0.05 versus -6.98±0.03, respectively, P<0.05). Smooth muscle relaxation was complete in both groups. The adherence of neutrophils to postexperimental coronary arteries as a measure of endothelial function was less in CP+GSH than in CCP (98±18 versus 234±36 neutrophils/mm2, P<0.05). Nitrosoglutathione, a byproduct of the reaction between ONOO- and GSH, was greater in CP+GSH than in CCP (4.1±2.3 versus 0.4±0.2 µg/mL, P<0.05).
Conclusions—GSH in crystalloid cardioplegia detoxifies ONOO- and forms cardioprotective nitrosoglutathione, resulting in attenuated neutrophil adherence and selective endothelial protection through the inhibition of neutrophil-mediated damage.
Key Words: cardioplegia • nitric oxide • endothelium
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Introduction |
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NO was recently advocated as an adjunct to cardioplegia. NO is a potent cardioprotective agent that attenuates neutrophil-mediated damage.1 2 Accordingly, NO has been shown to attenuate neutrophil function and neutrophil–endothelial cell interactions2 3 4 which initiate the inflammatory cascade and sequelae of contractile dysfunction and infarction. Accordingly, NO therapy in cardioplegia solutions during experimental cardiac surgery has been associated with a reduction in infarct size, improved coronary artery endothelial function, attenuated neutrophil accumulation, and better contractile function.5 6
NO reacts with superoxide anions at a diffusion-limited rate to produce peroxynitrite (ONOO-). Superoxide anions are abundantly produced during ischemia and reperfusion, whereas NO is produced by the coronary artery endothelium and neutrophils. Accordingly, ONOO- has been reported to be generated by both vascular endothelium and neutrophils.7 8 Hence, the close proximity of NO and superoxide anions in the vascular space may promote ONOO- generation during and after the periods of ischemia encountered during cardiac arrest as induced with chemical cardioplegia solutions that contain therapeutic concentrations of NO donor agents. NO therapy has had varying results, with some investigators reporting cardioprotective effects,5 6 whereas others have reported cardiotoxic effects.9 10 This variability may be related to the generation of ONOO- and its subsequent physiological effects on coronary vascular endothelium and myocardium. Ronson et al11 reported that ONOO- demonstrated deleterious effects in crystalloid cardioplegia in a canine model of normothermic ischemia followed by cardioplegic arrest. In contrast, ONOO- in a blood environment (blood cardioplegia solution) demonstrated a beneficial effect,11 consistent with the observations of Lopez et al10 and Nossuli et al.12
The cardioprotective effects of ONOO- may be related to the presence of endogenous thiol-containing substances such as glutathione (GSH), albumin, and cysteine. These thiol-containing molecules potentially convert ONOO- to less harmful or even cardioprotective byproducts. In addition to its potent antioxidant effects through the conversion of hydrogen peroxide to molecular oxygen and water via GSH peroxidase, GSH converts ONOO- into potential NO donors such as S-nitrosoglutathione (GSNO) and S-nitroglutathione,13 14 which exhibit physiological and cardioprotective effects similar to those of NO.12
Because ischemically injured myocardium is significantly depleted of tissue GSH compared with normal myocardium, ONOO- generated by NO-enhanced cardioplegia solutions delivered to ischemic myocardium in which superoxide anions may be coincidentally generated may cause damage to myocardium and vascular endothelium, rather than the cardioprotection intended by the inclusion of NO. In the present study, we tested the hypotheses that GSH, added to crystalloid cardioplegia that contains authentic ONOO-, attenuates the deleterious effects of ONOO- on postcardioplegic endothelial dysfunction and cardiodynamic dysfunction.
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Methods |
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All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH publication No. 80-23, revised 1985).
Neutrophil Isolation
After anesthesia was induced and
arterial access was gained, 200 mL of
peripheral arterial blood was harvested for
postexperimental analysis of coronary artery
endothelial function with neutrophil (neutrophil)
adherence properties. The blood was mixed with 45 mL of anticoagulation
agents, which included 1.6% citric acid and 2.5% sodium citrate (pH
5.4) and 100 mL of 6% dextran solution in buffered Hanks’ balanced
salt solution (HBSS). Neutrophils were isolated with the use of the
Ficoll-Paque (Sigma Chemical Co) technique described in detail
elsewhere.2 The cells were placed in
Ca2+ and Mg2+–free
solution, counted, and adjusted to 
9x107
cells/mL. Final suspensions contained 94±1% neutrophils, and cell
viability averaged 99±0.5% as determined by trypan blue exclusion.
Heartworm-free dogs of either sex that weighed 22.6 to 35.4 kg (mean weight 28.4±1.1 kg) were premedicated with subcutaneous morphine sulfate (4 mg/kg) followed by initial anesthesia with intravenous 2.5% thiopental (20 mg/kg). After endotracheal intubation, mechanical ventilation was begun, and the respiratory rate was adjusted to maintain pH 7.35 to 7.45, PaO2 of >100 mm Hg, and PaCO2 of 35 to 45 mm Hg. Deep anesthesia was maintained with the continuous infusion of fentanyl citrate (0.4 µg · kg-1 · min-1) and diazepam (0.003 mg · kg-1 · min-1). The right femoral artery and vein were cannulated for arterial blood sampling and for fluid administration, respectively. After median sternotomy, the azygos vein was ligated, and the pericardium was incised and tented to form a cradle. Millar MPC-500 solid-state catheters (Millar Instruments) were placed in the proximal aorta through the right internal mammary artery to measure aortic pressure and into the left ventricle (LV) through an apical stab incision to measure instantaneous LV pressure. The left atrium was cannulated for the infusion of hypertonic saline (9%) to measure parallel conductance. After heparinization (300 U/kg), a 7F octapolar impedance catheter (Webster) was inserted into the LV through the right internal carotid artery to measure instantaneous LV conductance and blood volume for analysis of systolic and diastolic properties with pressure-volume analysis as previously described.11
The left subclavian artery was cannulated for arterial inflow, and the superior and inferior vena cavae were transatrially cannulated to harvest venous return. Cardiopulmonary bypass was instituted with a Cobe Optima membrane oxygenator (Cobe Cardiovascular, Inc) primed with 1.5 L hetastarch (Hespan; DuPont Pharmaceutical). Total cardiopulmonary bypass was initiated, and the left and right ventricles were vented through direct cannulation and gravity drainage. Finally, a pressure-monitoring aortic root cannula (DLP, Inc) was inserted into the proximal aorta for the delivery of cardioplegia. Thermistor probes were placed in the anterior and posterior wall to continuously monitor intramyocardial temperature.
Experimental Protocol
After the initiation of cardiopulmonary bypass and
acquisition of baseline measurements, the aorta was cross-clamped for
45 minutes of normothermic global ischemia.
Subsequently, hypothermic (4°C), multidose (every 20 minutes)
crystalloid cardioplegia (CCP) solution (Plegisol; Abbott Laboratories)
was infused into the aortic root at 50 mm Hg with the Myocardial
Protection System delivery device (Quest Medical, Inc). Then, 600 mL
was administered for both induction and terminal cardioplegia, and 400
mL was delivered during intermittent infusions.11 Iced
saline slush was topically applied to maintain myocardial temperature
during cardiac arrest. During the delivery of crystalloid cardioplegia,
the coronary sinus effluent was harvested from the right
ventricle and discarded to avoid systemic effects of
ONOO- or its decomposition products and
direct effects of cumulative concentrations of
ONOO- exerted during reperfusion. Systemic mean
arterial pressure was kept at 70 mm Hg during
cardiopulmonary bypass.
The animals were randomized to receive either 5 mmol/L authentic ONOO- (CCP, n=6) or ONOO- (5 mmol/L) supplemented with 500 mmol/L GSH (CP+GSH, n=5). Catalase (100 U/L) was included in the cardioplegia of both groups to inhibit the antioxidant effect of GSH via GSH peroxidase through the rapid depletion of any buildup of hydrogen peroxide. Because catalase removes hydrogen peroxide at a 10 times faster rate than GSH peroxidase, the removal of substrate hydrogen peroxide would suppress the antioxidant effects of GSH peroxidase, thereby unmasking the effects of GSH on ONOO-. The concentration of GSH in cardioplegia was determined from in vitro pilot studies (data not shown). Because ONOO- is readily decomposed at relatively neutral pH, 0.1 N NaOH was added to the ONOO- stock solution to enhance stability, and the appropriate concentration of ONOO- was mixed with Plegisol immediately before the delivery of cardioplegia. The concentration of ONOO- was measured immediately before the first delivery and again after the final delivery of cardioplegia solution, and the percent decomposition was calculated as 1-(final absorbance/base absorbance)x100. The percent decomposition at the final delivery of cardioplegia was comparable between the groups (CCP 7.7±2.2%, CP+GSH 5.8±2.2%). The delivery of ONOO- was also confirmed through an analysis of tissue nitrotyrosine, the footprint of ONOO- (described later).
After the final delivery of cardioplegia and systemic rewarming to 37°C, systemic pressure was reduced to 50 mm Hg and the aortic cross-clamp was removed. After electromechanical resuscitation, the mean arterial pressure was gradually increased from 50 to 70 mm Hg. Ventricular fibrillation was counteracted with direct-current countershocks of 10 to 20 W-s. The heart was maintained in the total vented bypass state for the initial 30 minutes of reperfusion, after which cardiopulmonary bypass was discontinued, and functional data were collected every 30 minutes for the next 90 minutes off bypass. After the final data collection, euthanasia was accomplished with the administration of 100 mg/kg pentobarbital, and the hearts were excised and immediately immersed in cold Krebs-Henseleit (K-H) buffer for excision of the coronary arteries to determine vascular function (see later).
Experimental End Points
LV Performance and Chamber Stiffness
LV performance was described with the load-independent
end-systolic pressure-volume relationship (ESPVR) from
gradually decreasing pressure-volume loops acquired during transient
bicaval occlusion. ESPVR values were calculated with the equation
Pes=Ees(Ves-V0),
where Pes is the end-systolic pressure,
Ees is the slope of the linear ESPVR (elastance),
Ves is the end-systolic volume, and
V0 is the volume axis intercept when
Pes=0 mm Hg. LV chamber stiffness was
determined by fitting the gradually declining end-diastolic
pressure-volume points to the exponential relation
Ped=
(eßVed),
where Ped and Ved are the
end-diastolic pressure and volume points, respectively;
is the Ped intercept at V=0 mL; and ß is the
unitless modulus of chamber stiffness used to describe the degree of
curvature of the Ped-Ved
relation.
Plasma Creatine Kinase Activity
Arterial blood samples (3 mL) were withdrawn at
baseline, at the end of normothermic ischemia, at
the end of cardioplegic arrest, and at 2 hours of reperfusion. The
samples were centrifuged at 2500g and 4°C for 10
minutes. The plasma was analyzed spectrophotometrically for
creatine kinase (CK) activity (Sigma Diagnostics) and for
protein concentration as described previously.15
Plasma CK activity was expressed as international units per gram of
protein.
Tissue Water Content
Tissue water content was determined through desiccation of
subepicardial and subendocardial samples of the LV free wall at 85°C
for 48 hours. Tissue water content was calculated with the formula
[(wet weight-dry weight)/wet weight]x100.
Myeloperoxidase Activity for Neutrophil Accumulation in
Cardiac Tissue
Myeloperoxidase (MPO), an enzyme that is specific for
neutrophils, was analyzed spectrophotometrically at 460 nm in
postexperimental LV tissue samples as described
elsewhere.15
Postexperimental Coronary Artery Endothelial
Function
Isolated Coronary Artery Rings
Agonist-stimulated endothelial relaxation responses to agonist stimulators of NO synthase were determined in postexperimental coronary artery rings as a bioassay of endothelial function as described in detail previously.16 17 Indomethacin (10 µmol/L) was added to organ chambers to inhibit prostaglandin effects. The coronary rings were subsequently preconstricted with the thromboxane A2 mimetic U46619 (5 nmol/L) and dilated with cumulative concentrations of the endothelium-dependent vasodilators acetylcholine (receptor dependent) and A23187 (receptor independent) and the endothelium-independent vasodilator nitroprusside.
Neutrophil Adherence Assay
Basal endothelial function related to the inhibition of neutrophil adherence by endogenously released NO was assessed according to the adherence of fluorescently labeled (PKH26 vital fluorescent dye; Sigma Chemical Co) neutrophils to postexperimental coronary artery endothelium with the use of epifluorescence microscopy as described in detail previously.2 6
Nitrosoglutathione Concentration of Cardioplegic Solution
GSH reacts with ONOO- to form
GSNO.18 The concentration of GSNO was analyzed
spectrophotometrically in samples of cardioplegic solution withdrawn
from the aortic root and the coronary sinus during the delivery
of cardioplegia.
Quantification of Tissue Nitrotyrosine in LV Myocardium
The quantification of LV free wall nitrotyrosine levels as an
estimate of ONOO- delivery was performed with
ELISA with a mouse IgG monoclonal anti-nitrotyrosine primary antibody
(Upstate Biotechnology) as previously published.11 Tissue
levels of nitrotyrosine were compared with nitrated protein solution
(0.04% BSA) prepared as a standard. The amount of nitrotyrosine
content in tissue samples was calculated with use of the standard curve
generated from nitrated BSA that contains known amounts of
nitrotyrosine and expressed as nanogram of nitrotyrosine per milligram
of protein.
NO Levels in Cardioplegia Solution Estimated by
Nitrate/Nitrite Concentration
The major decomposition products of NO (potentially
regenerated from S-GSNO) are nitrate and nitrite. The
concentration of nitrate/nitrite in effluent cardioplegia was measured
according to the vanadium reduction technique reported
previously19 and is expressed in micromoles per liter
per liter.
Statistical Analysis
Time-related differences and group-time interactions were
analyzed by 2-way ANOVA for repeated measures. Single-event,
nonrepeated variables were compared between groups by standard
t test or nonparametric tests based on the
normality of the distribution of data. Relaxation responses in
postexperimental rings were expressed as the percent change in tension
from the precontracted levels, and these data were compared at each
concentration between groups by standard t test or
nonparametric test if data were not normally distributed.
EC50, the dose of the drug required to effect
50% of maximum relaxation, was calculated and expressed as the
negative log of the drug concentration. A value of P<0.05
was considered statistically significant. All data are
presented as mean±SEM.
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Results |
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One animal in each of the CP+GSH and CCP groups was excluded due to technical intraoperative complications. In addition, 1 animal in the CCP group was unable to be weaned from cardiopulmonary bypass due to severe LV dysfunction. Five animals in each group were included in the final analysis.
Blood gas data obtained before and after stabilization on bypass were not significantly different between groups at any time point. Anterior and posterior myocardial temperatures during antecedent global ischemia and cardioplegic arrest were comparable between groups throughout these periods. Aortic cross-clamp time (CCP 112±1.7 minutes versus CP+GSH 112±0.7 minutes), cardioplegic arrest time (CCP 65.8±0.9 minutes versus CP+GSH 66.6±0.4 minutes), and total cardiopulmonary bypass times (CCP 163±2.3 minutes versus CP+GSH 164±4.8 minutes) were comparable between groups. Hematocrit values before (CCP 35±1.2% versus CP+GSH 34±5.7%) and during CPB (CCP 19±1.4% versus CP+GSH 19.6±0.6%) were also comparable between the groups. Both groups required at least 1 direct-current countershock to convert ventricular fibrillation without differences in the number of cardioversions during the early period of reperfusion. PO2 and Pco2 of the cardioplegia solutions were comparable between groups.
Steady-State Hemodynamic Parameters
There were no group differences in steady-state mean
arterial blood pressure, heart rate, or LV
end-diastolic pressure at any time during the
experiment.
LV Systolic Performance and Chamber
Stiffness
Table 1
shows postbypass LV
performance and chamber stiffness. After the discontinuation of
cardiopulmonary bypass, Ees,
V0, and diastolic chamber stiffness
(ß-coefficient) were comparable between groups throughout the
reperfusion phase. In addition, there was no significant difference
between any preischemic and postischemic
cardiodynamic variable in either group.
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Plasma CK Activity
Baseline plasma CK activities were comparable between groups
(Figure 1). There was no significant
increase in plasma CK activity after global ischemia. At 90
minutes after the discontinuation of cardiopulmonary bypass,
plasma CK level tended (P=0.12) to be greater in the CCP
group than in the CP+GSH group. However, these differences did not
reach statistical significance. These data are consistent with
the LV performance data.
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Tissue Water Content
Postexperimental transmural LV tissue water contents were
comparable between the 2 groups (CCP 79.5±0.6% versus CP+GSH
79.6±0.3%, P=0.8). These data are consistent with
the lack of group differences for diastolic chamber
stiffness data.
MPO Activity in LV Tissue
Postexperimental LV MPO activity in the CP+GSH group (2.2±0.6
absorbance units/min) was significantly lower than that in the CCP
group (5.4±1.1absorbance units/min, P<0.05), suggesting
that neutrophil accumulation in the postcardioplegic
myocardium was attenuated by the inclusion of GSH in
ONOO--enhanced crystalloid cardioplegia.
Postexperimental Coronary Artery Endothelial
Function
Agonist-Stimulated Vascular Reactivity
There were significant differences in concentration-dependent
relaxation responses to the endothelium-dependent,
receptor-dependent vasodilator acetylcholine in postexperimental
coronary arteries (Figure 2).
Compared with the CP+GSH group, the concentration-response
curves to acetylcholine were shifted significantly to the right in the
CCP group, with a reduction in the maximum relaxation (92±5% versus
118±6%, P=0.02 overall, Figure 2) and an increase
in EC50 (-6.98±0.03 versus -7.10±0.05,
P=0.04). There was no overall decrease in relaxation with
the endothelium-dependent receptor independent
vasodilator A23187; there were no group differences in maximal
relaxation or EC50 (CCP -7.15±0.04 versus
CP+GSH -7.18±0.05). Responses to the
endothelium-independent vasodilator sodium
nitroprusside showed no group differences in maximum relaxation or
EC50 between CCP (-7.15±0.04) and CP+GSH
(-7.18±0.05). These data suggest that adjunct GSH protects the
receptor-mediated function of the postcardioplegic coronary
vascular endothelium from damage induced by
ONOO-.
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Basal Postexperimental Neutrophil Adherence
The adherence of unstimulated neutrophils to postexperimental
coronary artery endothelium was significantly
less in the CP+GSH group than in the CCP group (98±18 versus 234±36
neutrophils/mm2, P<0.05). These data
demonstrate that neutrophil adherence to postexperimental
coronary artery endothelium, related to basal
production of NO, in CCP was attenuated by GSH.
GSNO Concentration
There was an increased concentration of GSNO in the CP+GSH
cardioplegia solution compared with the CCP group (P<0.05)
(Figure 3, left). In the coronary
sinus effluent, the concentration of GSNO was comparable in both
groups; there was a significant transcardiac difference in
the CP+GSH group, whereas there was no such difference in the CCP
group, suggesting an uptake of GSNO by the myocardium in
the CP+GSH group.
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Nitrate/Nitrite Concentration in Cardioplegia Effluent
Nitrate/nitrite concentrations in cardioplegia effluent and plasma
during early reperfusion are summarized in Table 2. There was a trend toward a greater
concentration of nitrate/nitrite in coronary sinus effluent
during the delivery of the second cardioplegia in the CP+GSH group, but
this did not reach statistical significance. Nitrate/nitrite
concentrations at all other time points were comparable between the two
groups. These data suggest that GSNO was not converted to free NO.
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Tissue Nitrotyrosine
Figure 4 demonstrates that there was
a comparable level of nitrotyrosine in LV myocardium in
both CCP and CP+GSH groups. These data suggest that comparable amounts
of ONOO- were delivered to the
myocardium in both groups or that the protective mechanisms
of GSH did not attenuate the nitration of tyrosine in the CP+GSH
group.
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Discussion |
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ONOO- is a degradation product generated by the biradical reaction between NO and superoxide anions. Due to the 1:1 stoichiometry, the rate of ONOO- appearance increases in proportion to the concentration of its 2 substrates. Hence, ONOO- levels may increase when superoxide anion and NO generation increase during reperfusion20 21 or with stimulation of the endothelium7 in an environment in which NO is simultaneously produced.22 Although NO has been reported to be cardioprotective in many in vivo studies,1 23 it has been demonstrated to be deleterious in crystalloid (in vitro) environments.21 24 It has been suggested that these deleterious effects of NO in crystalloid environments may be mediated directly via ONOO-, via ground-state peroxynitrous acid,25 or via the hydroxyl radical–like metabolite of ONOO-.26 Accordingly, Ronson et al11 reported that 5 µmol/L ONOO- added to crystalloid cardioplegia markedly reduced postcardioplegia systolic functional recovery, increased neutrophil accumulation in LV myocardium, and increased tissue edema. In contrast, this same concentration of ONOO- in blood cardioplegia had cardioprotective effects. In the present study with a model similar to that of Ronson et al,11 500 µmol/L GSH was added to crystalloid cardioplegia that contained 5 µmol/L authentic ONOO-. In support of a reaction between ONOO- and GSH, the reaction product GSNO was generated in the GSH-enhanced cardioplegia group, consistent with other reports13 27 but not in the group without GSH. However, we could not demonstrate that GSNO was further broken down to authentic NO, because there was no group difference in nitrate/nitrite levels in effluent cardioplegia solution. GSH-enhanced crystalloid cardioplegia had no effect on postcardioplegia systolic or diastolic function or tissue water content. However, GSH did reduce neutrophil accumulation in postcardioplegia LV myocardium and was associated with significantly better coronary artery endothelial function (agonist-stimulated relaxation and basal neutrophil adherence properties). Hence, GSH added to crystalloid cardioplegia that contained ONOO- reversed the deleterious effects of this anion, possibly by generating GSNO. These data suggest that GSH may be useful in preventing the deleterious effects of ONOO- derived from NO if NO donors are used as adjuncts to crystalloid cardioplegia solutions in which detoxifying thiol agents are absent.
In the present study, 45 minutes of ischemia was imposed to
sensitize the myocardium to injury. In this model of severe
LV injury, GSH could act either to reduce the oxidant species hydrogen
peroxide to water and molecular oxygen via GSH peroxidase activity or
to react with ONOO-. To separate the antioxidant
actions from the reactions with ONOO-, catalase
was added to the cardioplegia solution in each experimental group in a
concentration that favored a rapid reaction with oxidant species. The
rate constant for the reaction of catalase with hydrogen peroxide is 10
times faster than the corresponding value for GSH.28
Therefore, catalase should outcompete GSH for hydrogen peroxide and,
hence, its antioxidant activity. The general antioxidant effect of
catalase in both groups may be responsible for the excellent recovery
of postcardioplegia systolic and diastolic function
in both groups, consistent with the observations of
others,29 30 compared with the marked dysfunction observed
by Ronson et al11 in an identical model in which catalase
was not added to cardioplegia solution. Hence, a protective effect on
functional recovery could not be exerted in the absence of injury.
Although we cannot be sure that the antioxidant effects of GSH were
completely eliminated with catalase, the conversion of
ONOO- to GSNO was strongly supported by
our data (Figure 3
).
In the present study, 5 µmol/L ONOO- was added to the crystalloid cardioplegia solution to represent potential ONOO- derived from exogenously added NO donor agents. Under normal conditions, the generation of NO is relatively low (1 to 20 nmol/L). However, tissue NO concentrations can increase 1000-fold with activation of the inducible form of NO synthase or activation of neutrophils or with parenteral administration of NO donor agents. A coincident increase in superoxide anion would provide substrates for ONOO- production. A high concentration of ONOO- may be achieved during early reperfusion when NO is derived from both the vascular endothelium and activated neutrophils or when NO levels are augmented by exogenous NO donors. Carreras et al8 reported an increase in ONOO- generation coincident with the generation of superoxide anions and NO in activated neutrophils. Therefore, with the assumption of a 1:1 stoichiometry between NO and superoxide anions, the 5 µmol/L concentration of ONOO- used in the present study may be relevant to cases in which NO donors are used in cardioplegia solutions.6
The mechanisms of vasculoprotection by GSH and reduction in neutrophil accumulation are not clear. The improved coronary artery endothelial function may have been related to the attenuation of neutrophil adherence to endothelium and consequent endothelial injury, observed by Nossuli et al,12 which were mediated by GSNO. In the presence of ONOO-, GSH has been shown by others to (1) cause vasorelaxation,13 27 (2) stimulate the levels and activity of guanylate cyclase in endothelial cells,14 (3) attenuate ONOO--induced hemolysis,31 and (4) inhibit platelet aggregation.18 The effects of GSH in the presence of ONOO- can be inhibited by hemoglobin18 or inhibitors of guanylate cyclase.13 27 The physiological effects of GSH in the presence of ONOO- are consistent with the actions of an NO-like substance. Indeed, GSH reacts with ONOO- to form S-GSNO12 or S-nitroglutathione (GSNO2),27 both of which are purported NO donors,13 27 or further breakdown of these molecules may generate authentic NO itself.14 Recently, Balazy et al32 showed that the reaction of ONOO- with GSH produced GSNO2, which released NO and demonstrated potent vascular relaxant activity. Nossuli et al12 observed a concentration-dependent appearance of GSNO in an in vitro reaction between GSH and 10 to 100 µmol/L ONOO-, which is within the range of ONOO- used in the present study. In fact, GSNO was found in the present study to appear in the delivered cardioplegia solution of the CP+GSH group, and this GSNO was absent from the coronary venous effluent, which implies myocardial uptake of the nitrosothiol. The vascular protection and reduction in neutrophil accumulation in LV tissue when GSH was present in ONOO- cardioplegia may therefore be related to the generation GSNO or other products of thiol nitrosation.
In summary, with a clinically relevant model of cardioplegia myocardial protection in which 5 µmol/L ONOO- conferred significant contractile and endothelial dysfunction,11 the addition of GSH attenuated postcardioplegia vascular endothelial dysfunction and attenuated myocardial neutrophil accumulation. GSH did not, however, improve postcardioplegia dysfunction, potentially via a reduction in oxidant-mediated injury to the contractile process by catalase added to both groups. For surgeons who consider using NO-related therapy in a crystalloid cardioplegia environment to reduce ischemic-reperfusion injury during surgical revascularization, the addition of GSH to the cardioplegia solution may detoxify ONOO- derived from NO donors or NO precursors and thereby allow full advantage to be taken of the potent cardioprotection afforded by NO therapy.
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Acknowledgments |
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This work was supported by a grant-in-aid from the American Heart Association, National Center, to Dr Vinten-Johansen and by contributions from the Carlyle Fraser Heart Center of Emory University. We appreciate the support of Quest Medical, Inc (Allen, Tex) for cardioplegia delivery supplies. The authors are grateful for the assistance of Gail H. Nechtman in the preparation of the manuscript.
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Footnotes |
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Reprint requests to Dr Jakob Vinten-Johansen, Cardiothoracic Research Laboratory, 550 Peachtree St, NE, Atlanta, GA 30365-2225.
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