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Am J Physiol Heart Circ Physiol 284: H698-H703, 2003. First published October 10, 2002; doi:10.1152/ajpheart.00693.2002
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Vol. 284, Issue 2, H698-H703, February 2003

Attenuation of ischemic preconditioning in pigs by scavenging of free oxyradicals with ascorbic acid

Andreas Skyschally1, Rainer Schulz1, Petra Gres1, Hans-Gert Korth2, and Gerd Heusch1

1 Institute of Pathophysiology, Center of Internal Medicine, University of Essen Medical School, 45122 Essen; and 2 Institute of Organic Chemistry, University of Essen, 45117 Essen, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Free oxyradicals are involved in the signal transduction of ischemic preconditioning in rats and rabbits. Data from larger mammals in which the infarct development is closer to that in humans are lacking. We have therefore investigated the impact of the radical scavenger ascorbic acid on ischemic preconditioning in pigs. In 33 anesthetized pigs, the left anterior descending coronary artery was perfused from an extracorporeal circuit. Infarct size (measured as percent area at risk) was determined by triphenyltetrazolium chloride staining. In placebo-treated animals undergoing 90 min of severe ischemia and 120 min of reperfusion, infarct size averaged 26.9 ± 3.9% (mean ± SE; n = 9). Ischemic preconditioning by 10 min of ischemia and 15 min of reperfusion reduced infarct size to 6.4 ± 2.4% (P < 0.05 vs. placebo; n = 9). Intravenous infusion of ascorbic acid (30 min before ischemic preconditioning or ischemia; 2-g bolus followed by 25 mg/min until the end of ischemia) had no effect on infarct size per se (22.6 ± 6.5%; n = 6), but largely abolished the infarct size reduction by ischemic preconditioning (19.1 ± 5.4%; n = 9). Scavenging of free oxyradicals with ascorbic acid largely attenuates the beneficial effect of ischemic preconditioning in pigs.

myocardial ischemia; infarct size


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC PRECONDITIONING IS an endogenous cardioprotective mechanism by which short episodes of ischemia-reperfusion protect the myocardium from the damage induced by a subsequent sustained period of ischemia. Since the initial description of ischemic preconditioning in dogs (20), infarct size reduction by ischemic preconditioning has been confirmed in all animal species tested so far (for review, see Ref. 29) and is possibly also operative in humans (15, 39). Free radicals are among the various triggers and mediators of ischemic preconditioning that have been identified (for review, see Ref. 29). Accordingly, the infusion of the radical scavengers superoxide dismutase or N-2-mercaptopropionyl glycine blocked the infarct size reduction via ischemic preconditioning in rabbits (33) and rats (24). Conversely, generation of free oxyradicals with purine and xanthine oxidase preconditioned the myocardium and reduced infarct size in isolated rabbit hearts. This protection by exogenous free oxyradicals was again blocked by superoxide dismutase and catalase (34). Although the involvement of free oxyradicals in ischemic preconditioning in rats and rabbits is established, data from larger mammals are lacking.

We have now tested the role of free oxyradicals in the signal transduction of ischemic preconditioning in pigs, a large mammal species in which coronary anatomy (37) and infarct development are more similar to those in man (27). The natural radical scavenger ascorbic acid was used to scavenge free oxyradicals; ascorbic acid transfers a hydrogen atom to an oxidizing radical thus producing the ascorbyl radical (7). The ascorbyl radical concentration can be determined by electron spin resonance spectroscopy, and the amplitude of the obtained spectrum has been used as an indicator of oxidative stress in vitro and in vivo (31, 36).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The experimental protocols used in this study were approved by the Bioethical Committee of the district of Düsseldorf and adhere to the guiding principles of the American Physiological Society.

Experimental Preparation

Thirty-three Göttinger minipigs (body wt, 20-40 kg) of either sex were initially sedated using ketamine hydrochloride (1 g im) and then anesthetized with thiopental (Trapanal, 500 mg iv). Through a midline cervical incision, the trachea was intubated for connection to a respirator (Dräger; Lübeck, Germany). Anesthesia was then maintained using enflurane (1-1.5%) with a 40% oxygen-60% nitrous oxide mixture. Arterial blood gas measurements were monitored frequently in the initial stages of the preparation until levels were stable, and measurements were made periodically throughout the study (Radiometer; Copenhagen, Denmark). Rectal temperature was monitored and maintained between 37 and 38°C by use of a heated surgical table and drapes. The common carotid arteries were cannulated with two polyethylene (PE) catheters: one to measure arterial pressure, and the other to supply blood to the extracorporeal circuit. The jugular veins were cannulated for volume replacement using warmed 0.9% NaCl and for the return of blood to the animal from the coronary venous line.

A left lateral thoracotomy was performed in the fourth intercostal space, and the pericardium was opened. A micromanometer (P7; Konigsberg Instruments; Pasadena, CA) was placed in the left ventricle through the apex together with a saline-filled PE catheter (used to calibrate the micromanometer in situ). Ultrasonic dimension gauges were implanted in the left ventricular (LV) myocardium to measure the thickness of the anterior and posterior (control) walls. The left anterior descending (LAD) coronary artery was dissected over a distance of 1.5 cm, ligated, cannulated, and perfused from an extracorporeal circuit. Before coronary cannulation, the pigs were anticoagulated with 20,000 IU sodium heparin; additional doses of 10,000 IU were given at hourly intervals. The system included a roller pump, windkessel, and a side port for the injection of radiolabeled microspheres. Coronary arterial pressure was measured from the sidearm of a PE T-connector (Cole-Parmer; Chicago, IL) used as a catheter tip with an external transducer (pvb Medizintechnik; Kirchseon, Germany). Minimal coronary arterial pressure was held at >70 mmHg by adjusting the roller pump of the extracorporeal circuit to avoid hypoperfusion before ischemia. The large epicardial vein parallel to the LAD coronary artery was dissected and cannulated. Coronary venous blood was drained to an unpressurized reservoir and then returned to a jugular vein through the use of a second roller pump. Heart rate was controlled throughout the study by left atrial pacing (model 215/T; Hugo Sachs Elektronik; Hugstetten, Germany).

Regional Myocardial Blood Flow

Radiolabeled microspheres (141Ce, 114In, 51Cr, 103Ru, 95Nb, or 46Sc; 15 µm in diameter; NEN-DuPont; Boston, MA) were injected into the coronary perfusion circuit to determine the regional myocardial blood flow and its distribution throughout the LAD perfusion bed (model 5912; Gammaszint BF 5300; Packard, Germany).

Morphology

At the end of each study, the heart was removed and sectioned from base to apex into five transverse slices in a plane parallel to the atrioventricular groove. The slices were immersed in a sodium phosphate buffer (0.09 mol/l, pH 7.4) that contained 1.0% triphenyltetrazolium chloride (TTC; Sigma Aldrich; Munich, Germany) and 8% dextran (mol wt 77,800) for 20 min at 37°C to identify infarcted tissue. The amount of infarcted tissue is expressed as the percentage of the LV area at risk as defined by a reduction of regional myocardial blood flow during ischemia by >85% (25).

Ascorbyl Radicals

Ascorbyl radicals were determined by electron spin resonance spectroscopy. Two myocardial drill biopsies of ~25 mg each were taken at each time point from the area at risk and immediately frozen in liquid nitrogen to preserve the ascorbyl radical from degradation. Electron spin resonance spectroscopy was performed on frozen biopsies using a Bruker ESP 300E spectrometer (8-10 GHz, 15-in magnet; Bruker BioSpin; Billerica, MA). The spectral component of the ascorbyl radical (Fig. 1) was identified by the corresponding g-value (2.0054). The peak-to-peak amplitude of the spectral component corresponding to the ascorbyl radical concentration was determined and corrected for biopsy weight. The results of both measurements were averaged and taken as an estimate of free oxyradical production in the myocardium. Peak-to-peak evaluation was done by importing the numerical data of the electron spin resonance spectrum into a standard spreadsheet program. The peak-to-peak amplitude [in arbitrary units (AU)] was divided by the sample weight (in mg) to obtain a measure for the ascorbyl radical concentration (in AU/mg).


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  		Fig. 1.   Two representative electron spin resonance (ESR) spectra obtained from frozen myocardial biopsies of similar weight. Sample 1 (gray line) was taken at baseline, and sample 2 (black line) was taken at 1-2 min of reperfusion after the preconditioning ischemia. Peak-to-peak amplitude: sample 1, 880 arbitrary units (AU); sample 2, 1,670 AU.

Experimental Protocols

A schematic of the experimental protocols is shown in Fig. 2.


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  		Fig. 2.   Schematic diagram of the four experimental protocols. Group 1 (n = 9): 90 min of ischemia and 120 min of reperfusion. Group 2 (n = 9): 10 min of ischemia, 15 min of reperfusion, 90 min of ischemia, and 120 min of reperfusion. Group 3 (n = 6): ascorbic acid with 90 min of ischemia and 120 min of reperfusion. Group 4 (n = 9): ascorbic acid with 10 min of ischemia, 15 min of reperfusion, 90 min of ischemia, and 120 of min reperfusion.

Group 1; n = 9. After baseline measurements of systemic hemodynamics, regional myocardial function, and blood flow, coronary inflow was reduced to achieve an 85-90% reduction in regional myocardial function. At 5 and 85 min ischemia measurements were repeated; thereafter, the myocardium was reperfused for 2 h before infarct size was determined by TTC staining.

Group 2; n = 9. After baseline measurements of systemic hemodynamics, regional myocardial function, and blood flow, the myocardium was subjected to one cycle of 10 min of preconditioning ischemia with an 85-90% reduction in regional myocardial function and 15 min of reperfusion. During reperfusion, coronary perfusion pressure was maintained at the level measured before ischemia by the continuous adaptation of coronary inflow with the roller pump. After reperfusion, coronary inflow was once again reduced to the same level as during the preconditioning ischemia. Thereafter, the protocol of this group was identical to that of group 1.

Group 3; n = 6. The protocol of group 3 was identical to that of group 1, except that 30 min before ischemia, a bolus of ascorbic acid (2 g iv) dissolved in saline was slowly administered and was followed by a continuous infusion of ascorbic acid (25 mg/min iv) until the end of the sustained ischemia.

Group 4; n = 9. The protocol of group 4 was identical to that of group 2, except that 30 min before the preconditioning ischemia, a bolus of ascorbic acid (2 g iv) dissolved in saline was slowly administered and was followed by a continuous infusion of ascorbic acid (25 mg/min iv) until the end of the sustained ischemia. Myocardial biopsies were taken at baseline and at 1-2 min of reperfusion after the preconditioning ischemia.

Data Analysis and Statistics

There were no exclusions of animals from the study. Data are reported as means ± SE. Statistical analysis for groups 1-4 comprised two-way ANOVA for repeated measures and Fisher's least-square difference (LSD) post hoc tests when significant overall effects were detected. Subendocardial blood flow during the sustained ischemia, infarct size, and area at risk were analyzed by one-way ANOVA and Fisher's LSD tests.

Electron spin resonance spectroscopy data obtained at baseline and during the first minutes of reperfusion were compared by paired t-test. A P value <0.05 was taken to indicate a significant difference.

In groups 1-4, linear regression analyses between subendocardial blood flow at 5 min of ischemia in the LV area at risk and infarct size (expressed as percentage of area at risk) were performed. Regression lines were compared by analysis of covariance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamics

Heart rate, systemic hemodynamics, regional myocardial function, and blood flow at baseline were not different between groups (Table 1). Regional myocardial function of the posterior control wall remained stable throughout the experimental protocol in each group. With the onset of the preconditioning or sustained ischemia, respectively, regional myocardial function (Table 1) and regional myocardial blood flow (Table 2) in the anterior wall were significantly reduced in all four groups.

                              
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  		Table 1.   Hemodynamic data from groups 1-4 during time course of experiments


                              
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  		Table 2.   Regional subendocardial blood flow at 5 min of sustained ischemia, infarct size, and area at risk

Infarct Size

The area at risk was comparable between groups (Table 2). After 90 min of severe myocardial ischemia and 120 min of reperfusion, infarct size averaged 26.9 ± 3.9% (group 1; n = 9). Ischemic preconditioning by one cycle of 10 min of ischemia and 15 min of reperfusion reduced infarct size to 6.4 ± 2.4% (group 2; n = 9; P < 0.05 vs. other groups). The relationship between infarct size and subendocardial blood flow was significantly shifted downward compared with group 1 (Fig. 3). With ascorbic acid, infarct size after 90 min of ischemia and 120 min of reperfusion (group 3; n = 6) was 22.6 ± 6.5% and not different from group 1, whereas ascorbic acid largely abolished the infarct size reduction by ischemic preconditioning; i.e., infarct size was 19.1 ± 5.4% (group 4; n = 9). The relationships between infarct size and subendocardial blood flow in groups 3 and 4 were not different from group 1.


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  		Fig. 3.   Relationship between infarct size and subendocardial blood flow at 5 min of ischemia (regression line 1) was shifted downward by ischemic preconditioning (regression line 2). This effect was largely attenuated by ascorbic acid (regression line 4). Ascorbic acid per se had no influence on the relationship (regression line 3). Symbols (bottom) indicate group numbers.

Ascorbyl Radical Concentration

The ascorbyl radical concentration was determined in all nine animals of group 4 at baseline and at 1-2 min of reperfusion after the preconditioning ischemia. The weight-corrected peak-to-peak amplitude of the electron spin resonance spectrum of the ascorbyl radical increased significantly from 69.7 ± 8.3 at baseline to 82.7 ± 4.9 AU/mg at reperfusion after the preconditioning ischemia (P < 0.05). The increase of the ascorbyl radical concentration during reperfusion after the preconditioning ischemia was also related to the severity of the preconditioning ischemia (Fig. 4); such analysis revealed that a lower residual myocardial blood flow during the preconditioning ischemia was associated with a higher ascorbyl radical concentration during reperfusion.


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  		Fig. 4.   Relationship between the increase in ascorbyl radical concentration from baseline to reperfusion after the preconditioning ischemia and subendocardial blood flow during the preconditioning ischemia.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies in small animals (rats and rabbits) have demonstrated the involvement of free oxyradicals in the signal transduction of ischemic preconditioning (24, 33). In the present study, we have shown that free oxyradicals are also involved in ischemic preconditioning in pigs, which are larger mammals with coronary anatomy and infarct size development that are closer in similarity to those of man. Ascorbic acid, a natural radical scavenger, largely abolished the infarct size reduction by ischemic preconditioning.

The ascorbyl radical, which is the reaction product of ascorbic acid and free oxyradicals, may be proapoptotic at higher concentrations (17, 26) and may therefore increase irreversible tissue damage. In the present study, the ascorbic acid concentration, which was calculated on the assumption of uniform dispersion in total body water (0.56 mM) was, however, only half of that minimally required for apoptosis induction in cancer cell lines (26). Therefore, a direct impact of ascorbic acid on infarct development appears unlikely. Importantly, in accordance with previous results in rabbits (4), ascorbic acid per se in fact did not increase infarct size after 90 min of ischemia in pigs (see Fig. 3).

In anesthetized dogs (31) and in the present study, the ascorbyl radical was detectable already under baseline conditions, which suggests a basal free radical production that might be favored by the open-chest preparation (16). Additionally, due to a rate constant for ascorbic acid to scavenge free oxyradicals that is some magnitudes higher than the rate constant for the dismutation of the ascorbyl radical (8), an accumulation of the ascorbyl radical may occur, and the concentration of free oxyradicals at baseline may be overestimated.

In anesthetized dogs, the ascorbyl radical concentration (determined by electron spin resonance spectroscopy) in coronary venous blood was increased by 27 ± 14% during the first minutes of reperfusion that followed 5 min of ischemia (31). In the present study, we measured a comparable 28 ± 32% (mean ± SD) increase in the ascorbyl radical concentration in myocardial biopsies at 1-2 min of reperfusion after the preconditioning ischemia.

In dogs undergoing 15 min of coronary occlusion, the amount of collateral blood flow to the ischemic region correlated inversely with the free radical production during subsequent reperfusion, which was estimated by alpha -phenyl-N-tert-butyl nitrone adduct release in coronary venous blood (5, 6). Similarly, in the present study, the increase in the ascorbyl radical that was determined during reperfusion was related to the severity of the preconditioning ischemia, i.e., the strength of the preconditioning stimulus.

Whether the increase in the myocardial ascorbyl radical concentration occurred during the preconditioning ischemia and/or during the subsequent reperfusion after the preconditioning ischemia could not be differentiated in the present study. Radical formation during both ischemia and reperfusion have been described in isolated buffer-perfused rat hearts (11, 13) and in anesthetized cats in vivo (22).

In the present study, we did not determine the exact step in which free oxyradicals interact with other signal transduction elements of ischemic preconditioning. However, there are established interactions between certain elements of the signal transduction cascade of ischemic preconditioning with free oxyradicals. G proteins are activated by free radicals (21), and free radicals also interact with phospholipase C (19), although it is unclear how such an interaction would promote the protection of ischemic preconditioning. Generation of free oxyradicals by hypoxanthine and xanthine oxidase in isolated rabbit hearts induced protection through protein kinase C, and this protection was accordingly inhibited by polymyxin B (3). Also, p38 mitogen-activated protein kinase, which is a key element in the signal transduction of ischemic preconditioning (32) also in pigs (28), is activated by free oxyradicals (38); accordingly, the resulting protection is abolished by dimethylthiourea, a radical scavenger (10).

During ischemic preconditioning, mitochondria appear to be the major source of free oxyradicals. Not only the mitochondrial ATP-sensitive potassium (KATP) channel agonist diazoxide (23), but also activation of the membrane receptors for acetylcholine, bradykinin, and opioids opens mitochondrial KATP channels, subsequently signals through free oxyradical generation, and ultimately induces cardioprotection in isolated rabbit hearts (9). Notably in this study, the adenosine-induced protection was not signaled through the opening of mitochondrial KATP channels (9). This, however, is in contrast with previous findings. In rabbit hearts in situ, ischemic preconditioning was blocked by N-2-mercaptopropionyl glycine despite an unaltered increase in adenosine concentration in myocardial microdialysate (12). In dogs (2, 14) and pigs (35), the adenosine-induced protection was lost by unspecific blockade of KATP channels with glibenclamide. Also, in our model in pigs, the protection of ischemic preconditioning is prevented by both the attenuation of the increase in endogenous adenosine (30) and the scavenging of free oxyradicals (present study), which suggests that either adenosine signals through free oxyradicals or the concerted action of both adenosine and free oxyradicals is required to induce protection of ischemic preconditioning. Both potential explanations need to be addressed in additional studies.

Our results also suggest a more critical view on the use of ascorbic acid in humans. Many people use ascorbic acid as a dietary supplement in doses comparable to those used in our experiments. Free oxyradicals seem to play an ambivalent role: they are both harmful chemical compounds and important signaling molecules. This means that the positive effects of scavenging free oxyradicals, e.g., inhibition of lipid peroxidation to prevent atherosclerosis (18), are inevitably accompanied by adverse effects in that an endogenous protective mechanism such as ischemic preconditioning is disturbed. Such controversial effects of ascorbic acid may also be responsible for the inconclusive results from cohort studies and clinical trials on the effects of ascorbic acid in the prevention of cardiovascular diseases (1).


    FOOTNOTES

Address for reprint requests and other correspondence: G. Heusch, Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstraße 55, 45122 Essen, Germany (E-mail: gerd.heusch{at}uni-essen.de).

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.

First published October 10, 2002;10.1152/ajpheart.00693.2002

Received 14 August 2002; accepted in final form 3 October 2002.


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METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 284(2):H698-H703
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