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Right arrow Cardiac - pharmacology

Ann Thorac Surg 2006;81:928-934
© 2006 The Society of Thoracic Surgeons

Original article: Cardiovascular

Pyruvate Mitigates Oxidative Stress During Reperfusion of Cardioplegia-Arrested Myocardium

E. Marty Knott a , b , Jie Sun, BS a , Yu Lei a , Myoung-Gwi Ryou, MS a , Albert H. Olivencia-Yurvati, DO b , Robert T. Mallet, PhD a , *

a Department of Integrative Physiology, University of North Texas Health Science Center, Fort Worth, Texas
b Department of Surgery, University of North Texas Health Science Center, Fort Worth, Texas

Accepted for publication August 25, 2005.

* Address correspondence to Dr Mallet, Department of Integrative Physiology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107-2699 (Email: malletr{at}hsc.unt.edu).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Cardioplegic arrest and reperfusion of the myocardium imposes oxidative stress that could potentially inactivate metabolic enzymes and compromise energy production. This study determined the impact of cardioplegic arrest and reperfusion on activities of several oxidant-sensitive enzymes, and tested whether pyruvate, a natural metabolic fuel and antioxidant, mitigates oxidant stress, protects enzymes, and bolsters myocardial energy state after reperfusion.

METHODS: In situ swine hearts were arrested for 60 minutes with 4:1 blood:crystalloid cardioplegia, and then reperfused for 3 minutes with cardioplegia-free blood with or without approximately 12 mM pyruvate. Tissue metabolites and enzyme activities were measured in left ventricular myocardium snap frozen at 45 minutes of arrest and 3 minutes of reperfusion.

RESULTS: The 8-isoprostane content, a measure of lipid peroxidation, sharply increased upon reperfusion, coincident with a 70% decline in redox state of the intracellular antioxidant glutathione. Aconitase and glucose 6-phosphate dehydrogenase activities fell during arrest; creatine kinase and phosphofructokinase were inactivated upon reperfusion. Pyruvate suppressed 8-isoprostane formation, maintained glutathione redox state, and enhanced phosphocreatine phosphorylation potential, a measure of myocardial energy state, during reperfusion. Pyruvate reactivated creatine kinase and aconitase, which are at least partially mitochondrial enzymes, but did not protect the cytosolic enzymes glucose 6-phosphate dehydrogenase and phosphofructokinase.

CONCLUSIONS: Administration of pyruvate upon reperfusion after cardioplegic arrest mitigates oxidative stress, protects mitochondrial enzymes and increases myocardial energy state. These results support therapeutic application of pyruvate-enhanced reperfusion to prevent cardiac injury after cardioplegic arrest.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Cardioplegic arrest is essential for a variety of surgical procedures including coronary revascularization, valvular repair, and correction of congenital heart defects. Unfortunately, cardioplegic arrest and subsequent reperfusion generate cytotoxic oxygen and nitrogen metabolites, including superoxide [1, 2], hydrogen peroxide [1], hydroxyl radical [3, 4], and peroxynitrite [5]. These reactive compounds could potentially cause myocardial dysfunction by chemically modifying cellular protein, lipid, and nucleic acid components. Several enzymes of intermediary and energy metabolism are among the principal biomolecular targets of reactive oxygen and nitrogen species. Inactivation of these enzymes could impair adenosine triphosphate (ATP) synthesis and delivery to the contractile machinery.

A natural carbohydrate and metabolic intermediate, pyruvate functions as an antioxidant by virtue of its {alpha}-keto-carboxylate chemical structure and its mitochondrial metabolism [6]. The latter favors production of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), the source of reducing power to maintain intracellular antioxidant defenses. Exogenous pyruvate increased the reducing power of the glutathione antioxidant system in postischemic stunned [7] and hydrogen peroxide–challenged [8] guinea pig myocardium. Pyruvate-fortified cardioplegia increased glutathione redox state during cardioplegic arrest but not during the first few minutes of reperfusion, when oxyradical formation is likely the most intense [9]. On the other hand, whether pyruvate administration during reperfusion after cardioplegic arrest can protect myocardial antioxidant defenses and prevent inactivation of enzymes has not been tested.

This study assessed the impacts of cardioplegic arrest and reperfusion on glycolytic, hexose monophosphate shunt, Krebs cycle, and energy shuttling enzymes, and determined whether reperfusion with pyruvate mitigates oxidative stress and protects or restores activities of these enzymes. In situ adult swine hearts were cardioplegically arrested for 60 minutes, then reperfused with cardioplegia-free whole blood with or without approximately 12 mM pyruvate. Left ventricular myocardial biopsies were taken during arrest and at 3 minutes of reperfusion for measurements of oxidative stress, antioxidant defenses and energy state, and enzyme activities. The 8-isoprostane, a product of lipid peroxidation, accumulated during reperfusion, and glutathione redox state concomitantly fell. Activities of aconitase and glucose 6-phosphate dehydrogenase fell during cardioplegic arrest, whereas creatine kinase and phosphofructokinase were inactivated upon reperfusion. Pyruvate prevented glutathione depletion and 8-isoprostane accumulation, and increased myocardial energy state. Pyruvate selectively reactivated creatine kinase and aconitase, enzymes located at least partially in the mitochondria, suggesting compartmentation of pyruvate metabolism [10, 11] may have concentrated its antioxidant actions on this organelle during the first minutes of reperfusion.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Animal experimentation was approved by the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center, and was conducted in accordance with the "Guide to the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1996). Twenty-seven adult domestic swine of either sex weighing 45 to 60 kg were randomly assigned to one of four experimental groups.

Surgical Procedures
The surgical preparation was described recently [9]. In brief, swine were sedated with ketamine plus xylazine, anesthetized with intravenous propofol, and mechanically ventilated with 1% to 3% isoflurane supplemented with oxygen. Cannulae were placed in femoral arteries to monitor arterial pressure and sample arterial blood. Plasma Lyte A (Baxter, Deerfield, Illinois) was administered through a femoral vein to maintain blood volume. The heart was exposed by median sternotomy and supported in a pericardial cradle. Coronary venous blood was sampled from a catheter placed in the coronary sinus through the right atrial appendage. The ventricles were drained with catheters inserted through the cardiac apex. A cannula was placed in the ascending aorta to deliver cardioplegia.

Cardioplegic Arrest and Reperfusion
Each pig was administered 300 U/kg of heparin intravenously. Arterial blood (1.2 L) was then withdrawn at a rate of 30 to 50 mL/min from a femoral artery using a roller pump (3M Health Care, Ann Arbor, Michigan). The blood was maintained at 37°C and used to prepare cardioplegia and to reperfuse the heart. Clamps were placed on the aorta distal to the cannula and on the superior and inferior vena cavae to effectively isolate the heart. Arrest was achieved with antegrade infusion, through the aortic cannula, of a minimum of 400 mL cold (4°C) blood cardioplegia (4 vol blood:1 vol crystalloid solution) as previously described [9]. Crystalloid cardioplegia solution (pH 7.6) contained 104 mM NaCl, 135 mM NaHCO3, 91 mM KCl, 6 mM CaCl2, 188 mM glucose, 68 U/L insulin, and 676 mg/L lidocaine. Arrest was maintained by infusing 100 to 150 mL blood cardioplegia into the aortic root at 20 and 40 minutes. After 60 minutes of arrest, the heart was reperfused by administering cardioplegia-free blood to the aorta at a rate of approximately 100 mL/min for 3 minutes. In 6 pigs, 1 M sodium pyruvate was infused into the aorta at a rate of 0.8 mL/min throughout reperfusion, resulting in a plasma pyruvate concentration of approximately 12 mM. The hearts did not fully recover mechanical function within 3 minutes, so after-arrest cardiac function was not monitored.

Plasma Pyruvate
Pyruvate in aortic and coronary sinus plasma extracts was assayed [12] in a Shimadzu Instruments UV-1601 spectrophotometer (Columbia, MD). Myocardial pyruvate uptake equaled arteriovenous pyruvate concentration difference multiplied by blood flow rate.

Myocardial Metabolites and Enzymes
Snap frozen biopsies (approximately 5 g) of the left ventricular apex were taken in situ by compressing tissue in Wollenberger tongs precooled in liquid N2 [13]. Myocardium was sampled at 45 minutes of arrest (n = 8), or at 3 minutes of reperfusion in absence (n = 7) or presence (n = 6) of pyruvate infusion. In sham controls (n = 6) myocardium was sampled 1 hour and 45 minutes after sternotomy but without cardiac instrumentation or arrest. Experiments were terminated after biopsy. Aliquots of frozen myocardium were weighed, dessicated to constant mass, and reweighed to determine tissue water content (mL/g), which equaled 1- (dry mass/wet mass).

Metabolites were extracted from frozen myocardium [12, 13] and spectrophotometrically assayed [14]. Phosphocreatine phosphorylation potential provided a measure of myocardial energy state [12, 13]. Glutathione (GSH) and glutathione disulfide (GSSG) were measured in a Shimadzu Instruments LC-10AT high performance liquid chromatography system equipped with a fluorescence detector [9, 15]. The GSH/GSSG ratio provided a measure of myocardial antioxidant redox state [16]. Myocardial enzymes were extracted [8, 17] and activities measured by spectrophotometry [14]. Extract protein concentration was determined colorimetrically with the Coomassie Plus Kit (Pierce, Rockford, Illinois), and enzyme activities expressed as U/mg protein.

Myocardial 8-Isoprostane
To assess oxidative stress, total (esterified and non-esterified) 8-isoprostane, a product of lipid peroxidation, was measured in myocardium using a competitive immunoassay kit (Cayman Chemical, Ann Arbor, Michigan). The analyte was extracted from powdered tissue (250 mg) according to the kit instructions, then measured at 405 nm wavelength in a 96-well plate reader (BioTek KCjunior, Winooski, Vermont).

Statistical Analyses
Data are reported as means ± SEM. Factorial analysis of variance (ANOVA) was applied to determine differences in all measured variables. When ANOVA detected statistical significance, post hoc between-group comparisons were performed with Student-Newman-Kuels multiple comparison test. Statistical analyses were performed with SigmaStat version 3.1 software. All p values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Aortic infusion of pyruvate during reperfusion resulted in a plasma pyruvate concentration of 11.6 ± 0.5 mM being delivered to the heart. Net myocardial uptake of pyruvate was 0.7 ± 0.1 mmol/min at 3 minutes of reperfusion.

Myocardial 8-Isoprostane and Glutathione Redox State
Left ventricular myocardial content of 8-isoprostane, a product of lipid peroxidation and indicator of oxidative stress, did not change during arrest but doubled within 3 minutes of reperfusion (Fig 1). Pyruvate administration during reperfusion prevented the increase in 8-isoprostane. Thus, reperfusion engendered oxidative stress in myocardium, but pyruvate prevented lipid peroxidation due to this oxidant burst.


Figure 1
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  		Fig 1. Antioxidant state and oxidative stress. Glutathione redox state (GSH/GSSG, solid bars) and 8-isoprostane (hatched bars) content were measured in left ventricular myocardium sampled at 45 minutes' arrest (n = 8), at 3 minutes' reperfusion with (n = 6) and without (n = 7) pyruvate infusion, and at 105 minutes after sternotomy in nonarrested sham hearts (n = 6). Values are means ± SEM. *p < 0.05 versus sham; {dagger}p < 0.05 versus reperfusion.

 
The ratio of glutathione/glutathione disulfide content, GSH/GSSG, provides a global measure of the redox state of the myocardium's antioxidant defenses [16]. The GSH/GSSG increased during arrest, but fell sharply upon reperfusion, coincident with oxidative stress and lipid peroxidation (Fig 1). Pyruvate administration during reperfusion prevented the decline in GSH/GSSG.

Myocardial Water Content
Myocardial water content was measured to assess edema formation. Water content did not change during cardioplegic arrest, but noticeably increased during reperfusion, from 78.9 ± 0.2 to 81.1 ± 0.1 mL per 100 g of myocardium (Fig 2). Pyruvate treatment prevented the increase in myocardial water content during reperfusion.


Figure 2
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  		Fig 2. Myocardial water content. Tissue water content was determined from masses of fresh and dessicated tissue. Values are means ± SEM. *p < 0.05 versus all other groups.

 
Myocardial Pyruvate and Derivatives
Cytosolic lactate dehydrogenase converts pyruvate to its reduced congener, lactate. In mitochondria, pyruvate carboxylation generates Krebs cycle intermediates, leading to increased citrate content [18]. Pyruvate and its derivatives were measured in left ventricular myocardium of arrested, reperfused, and sham control hearts (Fig 3). Pyruvate content did not increase in the control group during arrest or reperfusion, but sharply increased as expected during pyruvate administration. Lactate content increased twofold during arrest and another threefold during pyruvate-free reperfusion. Pyruvate treatment prevented lactate accumulation during reperfusion. Citrate content increased during arrest and remained elevated during reperfusion in the control group. Pyruvate administration further increased citrate content.


Figure 3
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  		Fig 3. Myocardial pyruvate and its metabolic derivatives. Pyruvate (solid bars), lactate (hatched bars), and citrate (open bars) were measured in left ventricular myocardium. Values are means ± SEM. *p < 0.05 versus sham; {dagger}p < 0.05 versus reperfusion.

 
Myocardial Enzymes
Activities of glycolytic (phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase), hexose monophosphate shunt (glucose 6-phosphate dehydrogenase), Krebs cycle (aconitase), and energy shuttling (creatine kinase) enzymes were measured in arrested, reperfused, and sham control myocardium. Creatine kinase activity (Fig 4A) did not change during arrest but fell 43% within 3 minutes of reperfusion. Exogenous pyruvate attenuated creatine kinase inactivation. Like creatine kinase, phosphofructokinase was unaltered during arrest but fell upon reperfusion (Fig 4B). However, pyruvate did not protect phosphofructokinase activity during reperfusion. Aconitase activity fell 40% during arrest and, unlike the other enzymes, partially recovered during reperfusion (Fig 4C). Pyruvate markedly increased aconitase activity, to a level even 30% higher than in sham myocardium. Glucose 6-phosphate dehydrogenase was inactivated by 26% during arrest, but was unaffected by reperfusion with or without pyruvate (Fig 4D). Activities of glyceraldehyde 3-phosphate dehydrogenase (Fig 4E) and lactate dehydrogenase (Fig 4F) were unaltered by arrest, reperfusion, or pyruvate.


Figure 4
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  		Fig 4. Myocardial enzymes. Activities (U/mg protein) of (A) creatine kinase (CK), (B) phosphofructokinase (PFK), (C) aconitase, (D) glucose-6-phosphate dehydrogenase (G6PDH), (E) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and (F) lactate dehydrogenase (LDH) were measured in left ventricular myocardial extracts. Values are means ± SEM. *p < 0.05 versus sham; {dagger}p < 0.05 versus reperfusion.

 
Myocardial Energy State
Left ventricular myocardial ATP content tended to fall during arrest and declined even further upon reperfusion (Fig 5). Pyruvate treatment during reperfusion restored ATP content to the level observed in nonarrested sham controls. To assess the effects of cardioplegic arrest and reperfusion and the impact of pyruvate on myocardial energy state, phosphocreatine phosphorylation potential was computed from intracellular concentrations of phosphocreatine, creatine and inorganic phosphate. Phosphorylation potential nearly tripled during arrest but fell to near sham values within 3 minutes of reperfusion (Fig 5). Pyruvate prevented the fall in phosphorylation potential during reperfusion.


Figure 5
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  		Fig 5. Myocardial phosphorylation potential and adenosine triphosphate (ATP, hatched bars) content. Phosphocreatine phosphorylation potential ([PCr]/([Cr][Pi]), solid bars) was computed from intracellular concentrations of phosphocreatine (PCr), creatine (Cr), and inorganic phosphate (Pi) measured in left ventricular myocardium. The ATP content was measured in the same samples. Values are means ± SEM. *p < 0.05 versus sham; {dagger}p < 0.05 versus reperfusion.

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
This investigation aimed to determine if the natural antioxidant pyruvate could prevent inactivation of essential metabolic enzymes during the intense oxidative stress produced by reperfusion of cardioplegically arrested myocardium. Enzyme activities, oxidative stress and tissue edema, antioxidant redox state, and phosphorylation potential were measured in left ventricular myocardium sampled during arrest and at 3 minutes of reperfusion with and without pyruvate infusion. Recently [9], pyruvate-fortified cardioplegia was found to increase GSH/GSSG during arrest, but this antioxidant action quickly subsided during pyruvate-free reperfusion and failed to prevent GSH depletion and myocardial edema. Accordingly, this study investigated whether pyruvate administration exclusively during reperfusion after arrest with pyruvate-free cardioplegia could prevent lipid peroxidation, tissue edema, and GSH depletion, and protect enzymes known to be targets of oxidants.

Pyruvate Metabolism
Pyruvate infusion into the aorta delivered a plasma pyruvate concentration of 11.6 mM to the heart. This treatment increased pyruvate and citrate contents in the myocardium at 3 minutes of reperfusion, indicating rapid uptake and mitochondrial metabolism of pyruvate. On the other hand, pyruvate actually prevented lactate accumulation, even though pyruvate is the direct precursor of lactate through lactate dehydrogenase. Radioisotope and stable isotope studies in isolated hearts demonstrated substantial subcellular compartmentation of pyruvate metabolism. Thus, exogenous pyruvate was mainly metabolized in the mitochondria, and only gradually converted to lactate [11, 19]; indeed, intracellular lactate was not in isotopic equilibrium with exogenous pyruvate [10, 19]. Moreover, pyruvate carboxylation in the mitochondria generates the Krebs cycle intermediates malate and oxaloacetate, leading to increased citrate content [18]. An allosteric inhibitor of phosphofructokinase [20], citrate suppresses glycolysis, the principal metabolic source of lactate, and thereby may prevent lactate accumulation. Moreover, pyruvate carboxylation increases the capacity of the Krebs cycle to oxidize acetyl CoA, which would favor pyruvate oxidation over lactate formation.

Antioxidant Effects of Pyruvate
Reactive oxygen and nitrogen species formed during reperfusion may contribute to myocardial injury after cardioplegic arrest [1–5]. The metabolic antioxidant pyruvate [9] and a pharmacological antioxidant, N -acetylcysteine [21], suppressed myocardial 8-isoprostane release during cardioplegic arrest. In this study, oxidative stress was minimal during arrest, but substantial stress accompanied reperfusion with whole blood. Reperfusion with pyruvate-enhanced blood prevented myocardial lipid peroxidation and rapid decline of GSH/GSSG. Thus, pyruvate mitigated reperfusion-induced oxidative stress. Pyruvate functions as an antioxidant by directly detoxifying peroxynitrite [22] and hydrogen peroxide [23] or, through its metabolism to citrate, increasing the supply of NADPH to maintain GSH/GSSG [6, 7].

Enzyme Activities
Mitochondrial and cytosolic creatine kinase isoenzymes exchange phosphate groups between ATP and phosphocreatine to efficiently transfer high energy phosphate from mitochondria to extramitochondrial ATP-consuming processes. During periods of oxidative stress, creatine kinase may be inactivated by S -thiolation or S -nitrosation of its catalytic cysteine residue through reaction with glutathione disulfide [24], peroxynitrite [25], hydrogen peroxide [26, 27], or superoxide [28], or by nitration of tyrosine residues by peroxynitrite [29]. This investigation for the first time determined the impact of reperfusion on this important enzyme after cardioplegic arrest. Creatine kinase was unaltered during arrest but inactivated upon reperfusion. Pyruvate may protect creatine kinase by maintaining a high glutathione redox state during reperfusion. Indeed, creatine kinase, inactivated by S -thiolation of its catalytic cysteine, can be directly dethiolated and reactivated by GSH [24, 30].

Aconitase, a Krebs cycle enzyme that converts citrate to isocitrate, is another target of oxidants, including nitric oxide [31], hydrogen peroxide [8], superoxide [32], and peroxynitrite [32]. Indeed, loss of aconitase activity is commonly used as a biomarker of oxidative damage [33]. Aconitase activity depends on the redox state of its cubane [4Fe-4S]2+ cluster [34]. Oxidants disassemble the cubane cluster and inactivate the enzyme. Inactivation is reversible if oxidative stress is quickly resolved but may become irreversible when oxidative stress is prolonged [35]. This investigation demonstrated inactivation of aconitase during cardioplegic arrest, partial recovery after reperfusion, and robust reactivation of the enzyme by pyruvate. Current evidence suggests that citrate is required for reinsertion of iron into the cubane cluster reversing the posttranslational modifications responsible for aconitase inactivation [35]. Thus, pyruvate enhancement of myocardial citrate content may have produced a salutary effect on aconitase.

Glucose 6-phosphate dehydrogenase, a cytosolic enzyme that catalyzes the initial, rate-controlling reaction of the hexose monophosphate shunt, is inactivated by oxidative modification of a lysine residue in its catalytic core [36]. Activity of this enzyme fell 26% during arrest but no further upon reperfusion, and was unresponsive to pyruvate. The allosterically regulated glycolytic enzyme phosphofructokinase was inactivated only upon reperfusion, yet it, too was unprotected by pyruvate. Neither glyceraldehyde 3-phosphate dehydrogenase, a known oxidant target [37, 38], nor lactate dehydrogenase were affected by arrest, reperfusion, or pyruvate treatment.

The two enzymes reactivated by pyruvate are at least partially mitochondrial (aconitase, creatine kinase mitochondrial isoenzyme), while cytosolic enzymes phosphofructokinase and glucose 6-phosphate dehydrogenase did not respond to pyruvate treatment. It is plausible that pyruvate exerts its antioxidant effects initially in the mitochondria owing to its metabolic compartmentation. Citrate accumulation indicates that pyruvate was taken up and metabolized by the mitochondria, yet its conversion to lactate, a cytosolic process, did not occur within 3-minute reperfusion.

Myocardial Energy State
Oxidant- and pyruvate-induced changes in enzyme activities may affect myocardial energy production. Phosphocreatine phosphorylation potential, a measure of myocardial energy state [39], increased during arrest, but fell at 3 minutes of reperfusion [9]. Pyruvate administration during early reperfusion maintained phosphorylation potential and ATP content. Enhancement of phosphorylation potential by pyruvate [40] increases Gibbs free energy of ATP hydrolysis, which defines the amount of energy available for ATP-dependent cardiac performance. In addition to providing readily oxidized fuel for the myocardium [40], pyruvate may have enhanced phosphorylation potential by preventing creatine kinase inactivation during reperfusion. However, it must be noted that despite inactivation of creatine kinase, phosphorylation potential during pyruvate-free reperfusion was similar to that of nonarrested sham hearts.

Limitations
This in situ heart preparation permits direct examination of the effects of cardioplegia and reperfusion on the organ. However, only 1.2 l of arterial blood could be safely withdrawn from the pig without provoking circulatory collapse despite replacement with Plasma Lyte. The volume of whole blood available after blood cardioplegia administration was only sufficient for 3 minutes of reperfusion, which did not permit complete recovery of mechanical function. Blood from donor animals was not administered to avoid the proinflammatory and pro-oxidant effects of allogenic blood. Therefore, it was not possible to determine if pyruvate protection of creatine kinase and aconitase and enhancement of energy state would have improved cardiac mechanical recovery, or prevented arrhythmias or infarction. The optimal pyruvate concentration and duration of treatment remain to be determined.

Summary and Conclusions
This is the first investigation of alterations in myocardial enzyme activities during and immediately after cardioplegic arrest. Creatine kinase activity was maintained during arrest but fell by 3 minutes of reperfusion, whereas aconitase activity fell during arrest and partially recovered upon reperfusion, despite intense oxidative stress. Pyruvate administration during reperfusion reduced oxidative stress and increased antioxidant state. Pyruvate protected the mitochondrial enzymes creatine kinase and aconitase from inactivation but had no salutary effect on cytosolic enzymes. Pyruvate also increased myocardial energy state.

Pyruvate may be a beneficial antioxidant and energy-generating intervention in the setting of cardioplegic arrest.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This study was supported by grants from the Osteopathic Heritage Foundation (02-18-522) and the National Heart, Lung and Blood Institute (HL071684). This work was completed in partial fulfillment of the requirements for the PhD degree for E. Marty Knott. The outstanding technical assistance of Arti B. Sharma, Linda Howard, and Abraham Heymann is gratefully acknowledged. Mirza Baig, PharmD, and Amar Joumma, Department of Pharmacy, Plaza Medical Center of Fort Worth, Fort Worth, Texas, prepared the cardioplegia solutions.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Prasad K, Chan WP, Bharadwaj B. Superoxide dismutase and catalase in protection of cardiopulmonary bypass-induced cardiac dysfunction and cellular injury Can J Cardiol 1996;12:1083-1091.[Medline]
  2. Kalawski R, Balinski M, Bugajski P, Wysocki H, Olszewski R, Siminiak T. Stimulation of neutrophil activation during coronary artery bypass graftingcomparison of crystalloid and blood cardioplegia. Ann Thorac Surg 2001;71:827-831.[Abstract/Free Full Text]
  3. Menasche P, Grousset C, Gauduel Y, Mouas C, Piwnica A. Prevention of hydroxyl radical formation: a critical concept for improving cardioplegia. Protective effects of deferoxamine Circulation 1987;76(Suppl V):V180-V185.
  4. Sellke FW, Shafique T, Ely DL, Weintraub RM. Coronary endothelial injury after cardiopulmonary bypass and ischemic cardioplegia is mediated by oxygen-derived free radicals Circulation 1993;88(Suppl II):II395-II400.
  5. Mehlhorn U, Krahwinkel A, Geissler HJ, et al. Nitrotyrosine and 8-isoprostane formation indicate free radical-mediated injury in hearts of patients subjected to cardioplegia J Thorac Cardiovasc Surg 2003;125:178-183.[Abstract/Free Full Text]
  6. Mallet RT, Sun J. Antioxidant properties of myocardial fuels Mol Cell Biochem 2003;253:103-111.[Medline]
  7. Tejero-Taldo MI, Caffrey JL, Sun J, Mallet RT. Antioxidant properties of pyruvate mediate its potentiation of beta-adrenergic inotropism in stunned myocardium J Mol Cell Cardiol 1999;31:1863-1872.[Medline]
  8. Mallet RT, Squires JE, Bhatia S, Sun J. Pyruvate restores contractile function and antioxidant defenses of hydrogen peroxide-challenged myocardium J Mol Cell Cardiol 2002;34:1173-1184.[Medline]
  9. Knott EM, Ryou M-G, Sun J, et al. Pyruvate-fortified cardioplegia suppresses oxidative stress and enhances phosphorylation potential of arrested myocardium Am J Physiol Heart Circ Physiol 2005;289:H1123-H1130.[Abstract/Free Full Text]
  10. Bünger R. Compartmented pyruvate in perfused working heart Am J Physiol Heart Circ Physiol 1985;249:H439-H449.[Abstract/Free Full Text]
  11. Lloyd S, Brocks C, Chatham JC. Differential modulation of glucose, lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart Am J Physiol Heart Circ Physiol 2003;285:H163-H172.[Abstract/Free Full Text]
  12. Yi KD, Downey HF, Bian X, Fu M, Mallet RT. Dobutamine enhances both contractile function and energy reserves in hypoperfused canine right ventricle Am J Physiol Heart Circ Physiol 2000;279:H2975-H2985.[Abstract/Free Full Text]
  13. Itoya M, Mallet RT, Gao ZP, Williams Jr AG, Downey HF. Stability of high-energy phosphates in right ventriclemyocardial energetics during right coronary hypotension. Am J Physiol Heart Circ Physiol 1996;271:H320-H328.[Abstract/Free Full Text]
  14. Bergmeyer HU. Methods of enzymatic analysis. New York: Academic Press; 1983.
  15. Sharma AB, Knott EM, Bi J, Martinez RR, Sun J, Mallet RT. Pyruvate improves electromechanical and metabolic recovery from cardiopulmonary arrest and resuscitation Resuscitation 2005;66:71-81.[Medline]
  16. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple Free Radic Biol Med 2001;30:1191-1212.[Medline]
  17. Stuewe SR, Gwirtz PA, Agarwal N, Mallet RT. Exercise training enhances glycolytic and oxidative enzymes in canine ventricular myocardium J Mol Cell Cardiol 2000;32:903-913.[Medline]
  18. Panchal AR, Comte B, Huang H, et al. Partitioning of pyruvate between oxidation and anaplerosis in swine hearts Am J Physiol Heart Circ Physiol 2000;279:H2390-H2398.[Abstract/Free Full Text]
  19. Peuhkurinen KJ, Hassinen IE. Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart Biochem J 1982;202:67-76.[Medline]
  20. Garland PB, Randle PJ, Newsholme EA. Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes, and starvation Nature 1963;200:169-170.[Medline]
  21. Fischer UM, Cox Jr CS, Allen SJ, Stewart RH, Mehlhorn U, Laine GA. The antioxidant N-acetylcysteine preserves myocardial function and diminishes oxidative stress after cardioplegic arrest J Thorac Cardiovasc Surg 2003;126:1483-1488.[Abstract/Free Full Text]
  22. Vásquez-Vivar J, Denicola A, Radi R, Augusto O. Peroxynitrite-mediated decarboxylation of pyruvate to both carbon dioxide and carbon dioxide radical anion Chem Res Toxicol 1997;10:786-794.[Medline]
  23. Constantopoulos G, Barranger JA. Nonenzymatic decarboxylation of pyruvate Anal Biochem 1984;139:353-358.[Medline]
  24. Reddy S, Jones AD, Cross CE, Wong PS, Van Der Vliet A. Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue Biochem J 2000;347:821-827.
  25. Konorev EA, Hogg N, Kalyanaraman B. Rapid and irreversible inhibition of creatine kinase by peroxynitrite FEBS Lett 1998;427:171-174.[Medline]
  26. Mekhfi H, Veksler V, Mateo P, Maupoil V, Rochette L, Ventura-Clapier R. Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils Circ Res 1996;78:1016-1027.[Abstract/Free Full Text]
  27. Suzuki YJ, Edmondson JD, Ford GD. Inactivation of rabbit muscle creatine kinase by hydrogen peroxide Free Radic Res Commun 1992;16:131-136.[Medline]
  28. McCord JM, Russell WJ. Inactivation of creatine phosphokinase by superoxide during reperfusion injury Basic Life Sci 1988;49:869-873.[Medline]
  29. Mihm MJ, Bauer JA. Peroxynitrite-induced inhibition and nitration of cardiac myofibrillar creatine kinase Biochimie 2002;84:1013-1019.[Medline]
  30. Konorev EA, Kalyanaraman B, Hogg N. Modification of creatine kinase by S-nitrosothiolsS-nitrosation versus S-thiolation. Free Radic Biol Med 2000;28:1671-1678.[Medline]
  31. Pellat C, Henry Y, Drapier JC. IFN-gamma-activated macrophagesdetection by electron paramagnetic resonance of complexes between L-arginine-derived nitric oxide and non-heme iron proteins. Biochem Biophys Res Commun 1990;166:119-125.[Medline]
  32. Hausladen A, Fridovich I. Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not J Biol Chem 1994;269:29405-29408.[Abstract/Free Full Text]
  33. Bulteau AL, Lundberg KC, Ikeda-Saito M, Isaya G, Szweda LI. Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia/reperfusion Proc Natl Acad Sci USA 2005;102:5987-5991.[Abstract/Free Full Text]
  34. Ramsay RR, Dreyer JL, Schloss JV, et al. Relationship of the oxidation state of the iron-sulfur cluster of aconitase to activity and substrate binding Biochemistry 1981;20:7476-7482.[Medline]
  35. Bulteau AL, Ikeda-Saito M, Szweda LI. Redox-dependent modulation of aconitase activity in intact mitochondria Biochemistry 2003;42:14846-14855.[Medline]
  36. Friguet B, Szweda LI, Stadtman ER. Susceptibility of glucose-6-phosphate dehydrogenase modified by 4-hydroxy-2-nonenal and metal-catalyzed oxidation to proteolysis by the multicatalytic protease Arch Biochem Biophys 1994;311:168-173.[Medline]
  37. Janero DR, Hreniuk D, Sharif HM. Hydroperoxide-induced oxidative stress impairs heart muscle cell carbohydrate metabolism Am J Physiol Cell Physiol 1994;266:C179-C188.[Abstract/Free Full Text]
  38. Eaton P, Wright N, Hearse DJ, Shattock MJ. Glyceraldehyde phosphate dehydrogenase oxidation during cardiac ischemia and reperfusion J Mol Cell Cardiol 2002;34:1549-1560.[Medline]
  39. Veech RL, Lawson JW, Cornell NW, Krebs HA. Cytosolic phosphorylation potential J Biol Chem 1979;254:6538-6547.[Abstract/Free Full Text]
  40. Mallet RT. Pyruvatemetabolic protector of cardiac performance. Proc Soc Exp Biol Med 2000;223:136-148.[Abstract/Free Full Text]



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