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Cardiovascular Research 1999 43(4):909-918; doi:10.1016/S0008-6363(99)00137-6
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Ischemic preconditioning and glucose metabolism during low-flow ischemia: Role of the adenosine A1 receptor

Robert de Jonge and Jan Willem de Jong*

Cardiochemical Laboratory, Thoraxcenter, EE2371, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands

* Corresponding author. Tel.: +10-408-8052; fax: +31-10-408-9494 j.w.dejong{at}tch.fgg.eur.nl

Received 21 September 1998; accepted 18 March 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Glycolysis-from-glucose may be more beneficial than glycogenolysis in protecting hearts against ischemia. We tested the hypothesis that ischemic preconditioning is mediated by increased exogenous glucose use during low-flow ischemia, an effect triggered by adenosine A1 receptor activation. Methods: Langendorff rat hearts were subjected to 25 min low-flow ischemia (0.6 ml/min) and 30 min reperfusion. Prior to underperfusion, hearts (n=6 per group) were subjected to two cycles of either preconditioning ischemia (PC), infusion of the adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.25 µmol/l), or PC in the presence of the adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT; 50 µmol/l). Glycolysis-from-glucose during underperfusion was measured using D-[2-3H]glucose. Results: At the end of reperfusion, recovery of rate-force product was enhanced in the PC and CCPA groups (62 and 67% of preischemic value) compared to the ischemic control hearts (IC, 32%; P<0.05), whereas protection was abolished in the SPT hearts (20%; P<0.05 vs. PC). PC improved total glycolysis-from-glucose during underperfusion by 31% (P<0.05 vs. IC); SPT abolished this increase. CCPA reduced total lactate release and glucose uptake during ischemia by 47% and 61%, respectively (P<0.05 vs. IC). Abolishment of the preconditioning-associated increase in glucose uptake during underperfusion, by switching to a low glucose buffer, resulted in a loss of functional protection. Conclusions: This study strongly suggests that increased exogenous glucose utilization during low-flow ischemia mediates ischemic preconditioning without increasing total anaerobic glycolytic flux. Although adenosine A1 receptor activation reduces ischemic injury, it does not facilitate the increased glucose uptake observed with ischemic preconditioning, suggesting a different mechanism of protection.

KEYWORDS Experimental; Heart; Organ; Biochemistry; Adenosine; Ischemia; Preconditioning; Receptors; Rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Opie [1] proposed the ‘glucose hypothesis’: enhanced uptake and metabolism of glucose during myocardial ischemia delays cellular damage. Many animal studies confirmed this hypothesis by showing that improved uptake and metabolism of exogenous glucose by the underperfused (ischemic) myocardium is associated with reduced diastolic and systolic dysfunction [2–4] and less release of cytosolic marker enzymes [3]. Beneficial effects of glucose–insulin–potassium infusions, stimulating glucose uptake, have been reported in patients after bypass graft surgery [5], after acute myocardial infarction [6,7], or during pacing stress testing [8]. Furthermore, glycolysis-from-glucose seems more effective than glycogenolysis in protecting hearts against myocardial ischemia [2,9]. The latter study showed that protection against the adverse effects of myocardial ischemia is more related to exogenous glucose utilization than to total glycolytic flux (glycolysis plus glycogenolysis). The protective effect of increased glucose uptake during ischemia supports the notion that glycolytically derived ATP maintains ionic homeostasis during ischemia and reperfusion [10–12]. The role of carbohydrate metabolism in the phenomenon of ‘ischemic preconditioning’ [13] is rather controversial [14]. However, the above mentioned results suggest that if ischemic preconditioning increases the rate of glycolysis-from-glucose during low-flow ischemia, this would reduce ischemic injury. We recently showed that preconditioning protection against severe underperfusion is associated with reduced glycogenolysis without affecting anaerobic glycolysis [15].

Triggers, identified to play a role in the mechanism of ischemic preconditioning, include adenosine, bradykinin, and catecholamines. Presumably, adenosine is the most important. Released during preconditioning ischemia, adenosine exerts its effects by binding to the adenosine A1 receptor located on the myocardial plasma membrane. However, the end-effector involved in the mechanism of preconditioning is unclear. The nucleoside has been shown to influence myocardial carbohydrate metabolism. Adenosine decreases glycolysis during normoxia [16], during low-flow ischemia [17,18], during no-flow ischemia [19,20], and during reperfusion following low-flow [18] or no-flow [16] ischemia. In contrast, increased glycolysis has been reported during normoxia [21] and low-flow ischemia [22]. Thus, adenosine could trigger the changes in carbohydrate metabolism observed in ischemically preconditioned hearts.

We hypothesized that a shift from endogenous carbohydrate to more advantageous exogenous glucose use during low-flow ischemia mediates preconditioning. Furthermore, we speculate that preconditioning effects on carbohydrate metabolism occur via adenosine A1 receptor activation. Although hardly studied [15], we used a model of preconditioning protection against low-flow ischemia, which we believe is clinically more relevant than the routinely used stop-flow set up. Part of this research has been published in abstract form [23,24].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
All animals were treated in conformity with the guiding principles in the Care and Use of Laboratory Animals as approved by the American Physiological Society. The Animal Welfare Committee, Erasmus University Rotterdam, approved the protocol.

2.1 Exclusion criteria
During stabilization, hearts were excluded if they met one of the following criteria: (1) unstable contractile function, (2) coronary flows outside the range of 9–19 ml/min, (3) severe arrhythmias, (4) myocardial temperature outside the range 37–39°C.

2.2 Isolated heart preparation
Fed, male Wistar rats (Wag/Rij inbred, weighing 280–330 g) were obtained from Harlan-CPB (Zeist, The Netherlands). They received a commercial rat chow (Hope Farms AM II, Woerden, The Netherlands) and tap water ad libitum. After anesthesia with an intraperitoneal injection of 0.6 ml sodium pentobarbital (Nembutal®, 60 mg/ml) supplemented with 0.1 ml heparin (Thromboliquine®, 5000 IU/ml), hearts were rapidly excised and arrested in saline (0°C) until beating ceased. Excess tissue was removed, and the hearts were cannulated within 1 min via the ascending aorta, for retrograde perfusion using a non-recirculating modified Krebs–Henseleit buffer containing (mmol/l): NaCl 118, KCl 5.6, CaCl2 2.4, MgCl2 1.2, NaHCO3 20, Na2HPO4 1.2 and D-glucose 10. Insulin (Sigma, St. Louis, MO, USA; from bovine pancreas, 1 U/l) was added to the buffer. Before use, the buffer was filtered through a 45-µm porosity filter to remove any particulate matter, and equilibrated with 5% CO2/95% O2, to give a pH of about 7.4 at 37°C. Myocardial temperature was kept at 37°C with a water-jacketed heart chamber and buffer reservoir, and regulated with an electric heating coil positioned around the aortic inlet line. The temperature of the outer ventricular wall was monitored with a thermocouple (A-F6, Ellab A/S, Roedovre, Denmark). Global, no-flow ischemia was induced by clamping the aortic line; low-flow ischemia was achieved using a perfusion pump (MV-MS3, Ismatec, Zurich, Switzerland) operating at a flow-rate of 0.6 ml/min.

Coronary flow was measured by timed collection of the pulmonary artery effluent. The hearts were allowed to beat spontaneously, unless otherwise indicated. Cardiac contractile function of isometrically beating hearts was estimated with a force-transducer (F5A-2, Koningsberg Instruments, Pasadena, CA, USA) connected to the apex of the heart [2,15]. The heart was pre-loaded with an initial resting tension of 2.5 g. Systolic tension and diastolic tension were continuously displayed on a recorder (Gould signal conditioner and Gould WindoGrafTM recorder, Valley View, OH, USA). Developed tension was calculated as systolic tension minus diastolic tension. Cardiac contractile function was expressed as rate-force product (RFP), the product of heart rate and developed tension. RFP at the end of reperfusion was compared to the pre-ischemic value after the 20-min stabilisation period and expressed as percentage recovery of RFP. Perfusion pressure was measured with a disposable pressure transducer (Braun Melsungen, Melsungen, Austria) and kept constant at 65 mmHg.

2.3 Experimental protocol
After initial isolation and surgical preparation, all hearts were perfused with the modified Krebs–Henseleit buffer and allowed to equilibrate for 20 min followed by a 20-min treatment period. Thereafter, hearts were subjected to 25 min of low-flow ischemia (0.6 ml/min) followed by a reperfusion period of 30 min. The 20-min treatment period prior to low-flow ischemia consisted of: (1) normoxic perfusion (IC group; n=6); (2) preconditioning using two 5-min episodes of no-flow ischemia each interrupted by 5 min of reperfusion (PC group; n=6); (3) preconditioning with two 5-min infusions of 0.25 µmol/l of the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA group; n=6), interspersed by two 5-min periods of drug-free perfusion; (4) PC in the presence of 50 µmol/l of the non-selective adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT group; n=6) initiated 2 min prior to transient ischemia; and (5) PC followed by low-flow ischemia where the buffer was switched to one containing low glucose (5 mmol/l) and no insulin (LG group; n=6); during reperfusion the normal high-glucose and insulin containing buffer was used.

In addition, a set of CCPA treated hearts was paced at 350 beats per min (Grass S9 stimulator, Quincy, MA, USA) to investigate whether preconditioning effects of CCPA were due to negative chronotropic and dromotropic effects of this selective A1 agonist (CCPAp group; n=6). These hearts were paced throughout the protocol except during ischemia.

2.4 Glycolytic flux and glycogenolysis
The glycolytic flux from glucose during low-flow ischemia was quantitated by measuring 3H2O production from D-[2-3H]glucose in the reversible reaction catalyzed by glucose 6-phosphate isomerase [25,26]. Briefly, [2-3H]glucose and 3H2O in the coronary effluent samples were separated on 2x0.8-cm columns of Dowex 1 (1X2-400; Sigma), equilibrated with potassium tetraborate. Before use, the columns were washed with H2O. A 0.5-ml sample of the coronary effluent collected during low-flow ischemia was added to the column and eluted into scintillation vials with 1.2 ml H2O. The samples, collected in 10 ml scintillation mixture, were counted in a β-counter. For calculating glycolysis, 3H2O counts were corrected for the small amount (3%) of [2-3H]glucose not retained by the column. During ischemia, we assumed glucose uptake and phosphorylation to equal the anaerobic glycolytic flux from glucose giving rise to lactate. In fact, in isolated rat hearts, cross-over analysis of glycolytic intermediates [27] showed that the glycolytic flux in low-flow ischemia is determined by the rate of glucose delivery and subsequent transport into the cell and not by enzyme inhibition along the glycolytic pathway as previously suggested [26]. In line with these results, calculation of flux-control coefficients in isolated rat hearts [28] also showed that glucose uptake and phosphorylation dominated the control of glucose flux.

Anaerobic glycogenolysis during low-flow ischemia was estimated from the lactate in excess of that accounted for by glycolytic flux [4]. Lactate washout, glycolysis, and glycogenolysis were expressed as µmol 6-carbon (C6) units. Total glycogenolysis during 25 min underperfusion was calculated from the total lactate washout (lactate efflux during ischemia+first 2 min of reperfusion) minus the total glycolytic flux. We assumed that all glucose taken up during severe underperfusion was converted to lactate. However, a small part of exogenous glucose is oxidized during underperfusion (<3% [4]); hence, this method only slightly overestimates the actual contribution of glycolysis to total lactate production. Incorporation of labelled glucose into the glycogen pool and subsequent release of 3H2O is unlikely to occur during underperfusion when substantial glycogen breakdown occurs with little synthesis and amounts less than 5% in normoxic rats [25] and in hypoxic perfused rabbit intraventricular septum [9].

Anaerobic ATP production from both endogenous and exogenous glucose sources was calculated assuming 2 mol ATP per mol glucose taken up, and 3 mol ATP per mol glycosyl units of glycogen broken down.

2.5 Analysis of coronary effluent
During ischemia and reperfusion, coronary perfusate samples were continuously collected at 2-, 3-, 5-, or 10-min intervals, depending on the changes expected. Prior to ischemia, several 1-min samples were taken. Lactate in the samples (0°C) was determined enzymatically with an Elan auto-analyzer (Eppendorf, Merck, Amsterdam, The Netherlands) according to Sigma procedure 735.

2.6 Chemicals
D-[2-3H]glucose (17.0 Ci/mmol) was supplied by Amersham (U.K.). The drugs 2-chloro-N6-cyclopentyladenosine and 8-(p-sulfophenyl)theophylline were obtained from RBI (Natick, MA, USA). Freshly prepared SPT was directly dissolved in the Krebs–Henseleit buffer. A stock solution of CCPA, dissolved in deionized water, was further diluted in the buffer. Stock solutions of CCPA were discarded after two days storage at 4°C.

2.7 Statistical analysis
The data are expressed as means±S.E.M., with n=number of hearts. Summary measures were constructed for hemodynamic parameters and carbohydrate fluxes according to published recommendations [29]: Recovery of rate-force product was expressed as a percentage of baseline value; from the recording of resting tension, the magnitude of peak contracture during ischemia was compared between groups (statistics presented in Fig. 1b). The sum of carbohydrate fluxes during low-flow ischemia was calculated as described in the Methods section. One-way analysis of variance with subsequent Student–Newman–Keuls post-hoc tests were used for comparisons between groups. If values were not normally distributed or variances between groups were unequal, Kruskal–Wallis ANOVA on ranks was used. Values of P<0.05 (two-tailed test) were regarded as significant.


Figure 1
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  		Fig. 1 Changes in rate-force product (a) and resting tension (b). After 20 min stabilization, hearts were preconditioned with two 5-min periods of the following interventions interspersed by two 5-min periods of normal perfusion: no-flow ischemia (PC, {circ}), infusion of 0.25 µmol/l of the selective adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA, {blacktriangleup}), or PC in the presence 50 µmol/l of the non-selective adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT, {bigtriangleup}), given 2 min prior to transient ischemia. Control hearts (IC, bullet) underwent an extra 20-min period of normoxic perfusion following stabilization. A group of CCPA hearts was paced to prevent the fall in rate-force product during drug infusion (CCPAp, {diamond}). Thereafter, all hearts were exposed to 25 min of low-flow ischemia (0.6 ml/min) and 30 min of reperfusion. In one group of PC hearts during low-flow ischemia, the perfusion buffer was switched at the start of ischemia to one containing low glucose (5 mmol/l) and no insulin (LG, {square}). Pretreatment with ischemia or CCPA improved recovery of rate-force product compared to control hearts whereas protection was abolished in LG and SPT hearts. Peak ischemic contracture was significantly lower in CCPA hearts compared to controls whereas contracture was increased in LG hearts. Fig. 1b *P<0.05 vs. all other groups; #P<0.05 vs. PC, IC, and LG (one-way ANOVA). Means±S.E.M. (n=6). For clarity reasons, error bars are only presented for IC hearts; error bars for the other groups were the same order of magnitude as those in control hearts. Summary statistics error bars for rate-force product and resting tension are given in Fig. 2 and in the results section, respectively.

 

   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Contractile function
Fig. 1 presents time-dependent changes in rate-force product and resting tension, reflecting ventricular systolic and diastolic functioning, respectively. Rate-force product, resting-tension, and coronary flow were not different between groups after the 20-min stabilization period. Mean coronary flow amounted to 13.5±0.5 ml/min after stabilization. Rate-force product rapidly fell to zero during the short bouts of preconditioning ischemia in the PC, SPT, and LG groups. After transient ischemia, rate-force product recovered to 75% in PC and LG groups and to 65% in SPT hearts. Infusion of CCPA resulted in a 79% decline in myocardial function, mainly due to the negative dromotropic and chronotropic effects of the drug. Rate-force product of all groups of hearts fell to 0 within 5 min after the onset of low-flow ischemia. Low-flow ischemia amounted to 4% of baseline flow. Recovery of rate-force product expressed as a percentage of baseline (Fig. 2) was improved in PC and CCPA hearts (62±6% and 67±7%, respectively) compared to control hearts (32±9%; P<0.05). SPT and LG abolished preconditioning protection (20±12% and 30±7% recovery; P<0.05 vs. PC). Pacing (CCPAp) prevented the decline in rate-force product during CCPA infusion but did not diminish the protection by CCPA against prolonged ischemia (68±5%; P<0.05 vs. IC; Figs. 1 and 2Go).


Figure 2
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  		Fig. 2 Summary of the recovery of rate-force product obtained from the time-dependent changes in myocardial function depicted in Fig. 1a. See legend to Fig. 1 for protocol explanation. An extra group of CCPA-treated hearts was paced throughout the experiment (CCPAp; n=6) except during low-flow ischemia. Recovery of rate-force product expressed as a percentage of baseline value was significantly improved in PC, CCPA, and CCPAp hearts compared to controls. The supply of low glucose during underperfusion or SPT during pretreatment to ischemic preconditioned hearts abolished protection completely. * P<0.05 vs. PC, CCPA, and CCPAp (one-way ANOVA).

 
No functional recovery was observed (0%) in a set of ischemic control hearts supplied with low glucose (5 mM) during underperfusion (IC-low glucose group; n=4, data not shown).

Prior to underperfusion, resting tension was not significantly affected by any of the interventions. During low-flow ischemia, resting tension gradually increased towards a maximum at 25 min of ischemia (indicated as peak ischemic contracture, statistics in Fig. 1b) except in CCPA and CCPAp hearts where contracture was suppressed. Peak ischemic contracture was lower in CCPA (0.2±0.2 g) hearts compared to PC (6.0±0.7 g), IC (6.0±1.4 g), SPT (3.6±1.3 g), and LG (9.7±0.9 g) groups (P<0.05). Peak ischemic contracture was also lower in CCPAp hearts (1.7±0.5 g; P<0.05 vs. PC, IC, LG). It was similar in PC, IC, and SPT hearts. LG resulted in a greater peak contracture compared to all other groups (P<0.05).

3.2 Lactate
The release of lactate in the coronary effluent was taken as a measure of anaerobic glycolysis both from exogenous (glucose) and endogenous (glycogen) sources. In PC, SPT and LG hearts, the short bouts of no-flow ischemia induced a large efflux of lactate (Fig. 3). Lactate release during ischemia and reperfusion was similar in the PC, IC, SPT, and LG groups; CCPA pretreatment resulted in reduced lactate efflux during underperfusion and reperfusion. The sum of lactate released during low-flow ischemia and reperfusion is depicted in Fig. 4. Total lactate released during low-flow ischemia was not different between groups except for CCPA which reduced lactate release by 47% (P<0.05 vs. all groups). Reduced lactate release during underperfusion was also observed in paced CCPA hearts (Fig. 3). Hence, less lactate production in CCPA pre-treated hearts was not due to reduced pre-ischemic contractility (see Fig. 1a).


Figure 3
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  		Fig. 3 Release of lactate in the coronary effluent. See legend to Fig 1 for protocol explanation. Lactate release during low-flow ischemia and reperfusion was similar in the PC, IC, SPT and LG groups. However, CCPA decreased lactate release during ischemia and reperfusion. For CCPAp hearts, n=3.

 

Figure 4
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  		Fig. 4 Total lactate release, total glycolysis-from-glucose, and estimated total glycogen breakdown during 25 min of underperfusion. Total lactate release reflects total anaerobic glycolysis from both endogenous and exogenous glucose sources and was calculated based on the data in Fig. 3. Total glycolysis-from-glucose was obtained from the data in Fig. 5. Total glycogen breakdown was estimated from the lactate in excess of that accounted for by glycolytic flux. See legend to Fig. 1 for protocol explanation. Total lactate release was not different between groups except for CCPA-treated hearts, which depressed lactate release by 47% compared to control hearts. PC increased and CCPA decreased glycolysis from exogenous glucose compared to controls. * P<0.05 vs. all other groups (one-way ANOVA).

 
3.3 Glycolysis-from-glucose and glycogenolysis
Glucose uptake and phosphorylation was monitored using [2-3H]glucose present in the perfusion medium during low-flow ischemia. Anaerobic glycolysis-from-glucose during underperfusion (Fig. 5) reached a plateau after about 15 min of ischemia (at 52.5 min). Fig. 4 presents the sum of glycolysis-from-glucose during ischemia. PC increased glycolysis from exogenous glucose by 31% (P<0.05 vs. IC) whereas CCPA reduced this flux by 61% (P<0.05 vs. IC). Increased total glycolysis in PC hearts was mainly due to increases during the first 15 min of ischemia whereas CCPA reduced glycolysis throughout the ischemic period (Fig. 5). Total glycogen breakdown during low-flow ischemia was estimated from the lactate in excess of that accounted for by glycolytic flux-from-glucose (Fig. 4). Estimated glycogen breakdown was not different between groups but tended to be lower in PC hearts.


Figure 5
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  		Fig. 5 Glycolytic flux (from glucose) during underperfusion as estimated from the release of tritiated water from labelled glucose. See legend to Fig. 1 for protocol explanation. Glycolysis-from-glucose was increased in PC hearts during the first 15 min of ischemia compared to control hearts. This increase was abolished in the SPT and LG groups. CCPA treatment resulted in depressed glucose uptake throughout the period of underperfusion. Same time scale as other figures.

 
3.4 ATP production
Total ATP production during 25 min low-flow ischemia from the anaerobic breakdown of glucose and glycogen (Fig. 6) was calculated based on the total fluxes from glucose and glycogen during ischemia presented in Fig 4. PC increased ATP production from exogenous glucose by 31% compared to controls (P<0.05) whereas CCPA reduced glycolytic ATP by 61% (P<0.05 vs. IC). ATP production from endogenous glycogen during ischemia was similar in all experimental groups.


Figure 6
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  		Fig. 6 ATP production during 25 min low-flow ischemia from both anaerobic breakdown of exogenous glucose ({blacksquare}) and endogenous glycogen ({square}). Values were calculated from the total glycolytic flux and the estimated total glycogen breakdown during underperfusion as depicted in Fig. 4. See legend to Fig. 1 for protocol explanation. PC increased and CCPA decreased ATP production from exogenous glucose; ATP production from glycogen during underperfusion was not different between groups. *P<0.05 vs. all other groups (one-way ANOVA).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Carbohydrate metabolism during ischemia
Values of glucose utilization and calculated glycogen breakdown (Fig. 4) in this study correspond to those obtained in a study using a similar level of underperfusion [4]. Furthermore, the present results using direct quantitation of glucose flux (Fig. 5) and indirect calculation of glycogen breakdown confirm a previous study of our group that assessed glycogen in freeze-clamped hearts [15]. In that study, glycogen breakdown was reduced in preconditioned hearts during underperfusion despite similar lactate production, suggesting increased glycolysis-from-glucose. Although not significant in the present study, calculated glycogen breakdown was less than half in PC hearts compared to IC. Ischemic preconditioning increased glycolysis early during ischemia compared to controls (Fig. 5). This corresponds to the observation of Runnman et al. [9] that the cardioprotective effects of increased glucose flux occurred early during hypoxia. To exclude the possibility that increased glycolysis-from-glucose during underperfusion is just an epiphenomenon of ischemic preconditioning, we reduced glycolysis-from-glucose during ischemia in preconditioned hearts by switching to a buffer with lower glucose and zero insulin (note: glucose supply to the underperfused ischemic heart is the rate-limiting step in glycolytic flux from glucose [27]). This resulted in a reduction of glycolysis to values similar to the control group. The fact that these hearts did not show improved contractile recovery upon reperfusion with normal buffer suggests that ischemic preconditioning-induced protection against underperfusion is mediated by increased glycolytic flux early during ischemia. A study in isolated rabbit hearts reached similar conclusions [30]. Furthermore, we observed no functional recovery (0%) in a set of ischemic control hearts supplied with low glucose (5 mM) during underperfusion (IC-low glucose group; data not shown). Thus, this confirms the hypothesis that (1) glycolytic flux controls myocardial viability during underperfusion (please, compare IC vs. IC-low glucose and PC vs. LG), and (2) suggests that ischemic preconditioning is mediated by increased glycolysis during low-flow ischemia (please, compare PC vs. IC and LG vs. IC-low glucose). Beneficial effects of increased glycolytic flux by various means during underperfusion/hypoxia have been well documented in animal experiments [2–4] and also in clinical trials [7,31]. Vanoverschelde et al. [32] observed a linear relation between glucose uptake during ischemia and functional recovery after reperfusion in isolated rabbit hearts. We add to these observations that the mechanism of protection by ischemic preconditioning against low-flow ischemia may involve increased glucose uptake.

In our study, total anaerobic glycolysis (lactate production) was similar in preconditioned and control hearts (Fig. 3) in line with other reports [15,33]. This contrasts with no-flow ischemic models of ischemic preconditioning where lactate production is reduced [34]. However, in the latter 13C-NMR study, glucose utilization actually increased in preconditioned hearts during the first five min of ischemia despite reduced glycogenolysis and lactate production.

Contrary to our expectations, pretreatment with CCPA protected contractile function despite reduced lactate production (Fig. 3) and reduced glycolysis-from-glucose during underperfusion (Fig. 5). This was not due to the negative chronotropic effect associated with CCPA infusion since less ischemic lactate production was also observed in paced CCPA hearts (Fig. 3). Other studies described similar effects of adenosine infusion on lactate production [18–20] and on glycolysis-from-glucose [18] during low-flow or no-flow ischemia. However, increased lactate production [35] and glycolysis-from-glucose [22] with adenosine treatment have also been observed during low-flow ischemia. These discrepancies on the effects of adenosine on glycolysis during ischemia may be related to the preischemic metabolic status of the heart [16]. Thus, although glucose uptake may mediate ischemic preconditioning in this study, the beneficial effect of CCPA on mechanical recovery was associated with depressed glucose flux. This could indicate that the observed increase in glucose uptake in ischemic preconditioned hearts is not mediated by adenosine A1 receptor activation. However, care should be taken since opposite effects of the adenosine A1 agonist N6-cyclohexyladenosine and adenosine itself on glycolysis have been reported [16]. In that study, adenosine pretreatment decreases glycolysis whereas adenosine combined with brief ischemia increases glycolysis. Moreover, N6-cyclohexyladenosine, a compound similar to CCPA, also decreases glycolysis, in line with the results presented in this report [16]. Thus, it is possible that adenosine released during brief ischemia mediates the increase in glycolysis-from-glucose during underperfusion in this study (Fig. 5). The fact that the effects of ischemic preconditioning on mechanical recovery and increased glucose uptake during ischemia were abolished when the nonspecific adenosine antagonist SPT was present during the pretreatment supports this notion. Paradoxically, in the present study, CCPA induced pronounced cardioprotective effects (no ischemic contracture and improved functional recovery) despite depressed glycolysis-from-glucose. We do not have an suitable explanation for this observation. Myocardial protection against ischemia and reperfusion induced by pretreatment with ischemia or CCPA may involve different mechanisms. Ischemic preconditioning is a highly redundant phenomenon: it can take place by many alternative routes of which increasing glycolysis-from-glucose during low-flow ischemia may be one.

4.2 Recovery of function
Most ischemic preconditioning experiments have examined no-flow ischemia after pretreatment and consistently shown increased protection. In clinical practice, it is more likely that low-flow conditions will prevail, due to partial coronary occlusion and/or collaterals present. However, ischemic preconditioning-induced protection against low-flow ischemia is rather controversial. The few studies conducted so far have obtained both positive [15,30,36–38] and negative results [33]. In line with a recent report of our group [15], ischemic preconditioning protected against contractile dysfunctioning arising from severe underperfusion in the present study (Figs. 1 and 2Go). We showed before that preconditioning also reduces irreversible injury after low-flow ischemia [15]. Furthermore, ischemic preconditioning exists in species having collateral flow. We [15] and others [33] have suggested that there could be a critical flow above which preconditioning effects are lost.

Pharmacological preconditioning with CCPA, a selective adenosine A1 agonist, effectively mimicked ischemic preconditioning by improving contractile recovery after low-flow ischemia whereas protection was abolished in hearts ischemically preconditioned in the presence of the non-selective adenosine antagonist SPT. We showed before, using the same drugs, that the adenosine A1 receptor is involved in protection by ischemic preconditioning in a no-flow ischemic model [39]. This sharply contrasts reports that adenosine is not involved in ischemic preconditioning of the rat heart (for reviews, see [40,41]). The protective effect of pretreatment with CCPA was not due to negative chronotropic and dromotropic effects of the drug since pacing, which kept myocardial contractility constant during drug infusion, did not abolish protection by CCPA. Moreover, the different metabolic response in CCPA-treated hearts is unlikely to be the cause of incomplete washout prior to ischemia and consequent negative chronotropic effects during ischemia since the time from onset ischemia to complete contractile arrest was not statistically different between groups (IC: 7.2±4.1 min; PC: 3.5±0.5 min; LG: 5.3±1.7 min; CCPA: 4.1±1.2 min; SPT: 2.4±0.4 min; CCPAp: 7.6±3.9 min). Furthermore, in a previous study [39], we showed that the effects of CCPA are also independent of changes in coronary flow since in that study flow was held constant. CCPA infusion did not result in negative inotropic effects (see CCPAp group in Fig. 1a). Adenosine A1 receptors have been shown to couple to ATP-sensitive K+ channels via G-proteins in membrane patches of rat ventricular myocytes [42], which could lead to negative inotropic effects. However, in intact guinea pig ventricular myocytes, adenosine does not affect potassium membrane current [43] in line with our observation that CCPA induced negative chronotropic/dromotropic effects without affecting the inotropic state (Fig. 1a). With some exceptions, most rat-heart studies using adenosine or adenosine antagonists have failed to mimic or abolish preconditioning, respectively. However, the relatively high adenosine levels in this species may require increased antagonist concentrations [44]. The fact that the endothelium forms an active barrier for the transport of adenosine to the interstitium and the especially high activities of adenosine degrading enzymes in the rat heart may limit the efficacy of exogenous adenosine administration. In this respect, it is interesting to note that studies using adenosine A1 agonists, like the one used in this investigation, have obtained positive results in rat hearts [44,45].

4.3 Possible mechanisms of action
The cause of the increase in exogenous glucose utilization in preconditioned hearts was not the subject of this study. However, transient ischemia may result in translocation of heart GLUT4 glucose transporters [46]. The fact that preconditioning increased glucose uptake especially during early ischemia in this study could indicate that glucose transporters were already/more rapidly translocated to the sarcolemma due to the short antecedent ischemia. However, this explanation seems unlikely since in the presence of insulin, glucose uptake is not a rate-limiting step in glucose metabolism [28]. Alternatively, preconditioning may affect key glycolytic enzymes inhibiting glycogenolysis [34] and stimulating glycolysis. Moreover, the reason for the superiority of glycolysis over glycogenolysis in reducing ischemic injury is unclear, but sustained glycolysis-from-glucose during ischemia may support ionic homeostasis and thereby reduce ischemic injury [10–12].

In summary, this study strongly suggests that ischemic preconditioning is mediated by enhanced utilization of exogenous glucose during low-flow ischemia without increasing total anaerobic glycolytic flux. Adenosine A1 receptor stimulation attenuated ischemic injury in rat hearts but did not mediate the increase in glucose utilization observed in ischemically preconditioned hearts. Therefore, ischemic and pharmacological preconditioning may involve different pathways.

Time for primary review 22 days.


   Acknowledgements
 
We gratefully acknowledge the support of the Netherlands Heart Foundation (NHS 94.043) and the Trust Fund of the Erasmus University Rotterdam, The Netherlands.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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