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J. Biol. Chem., Vol. 277, Issue 27, 24411-24419, July 5, 2002

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Regulation of Glycolytic Flux in Ischemic Preconditioning

A STUDY EMPLOYING METABOLIC CONTROL ANALYSIS^*

Achim M. Vogt^§, Mark Poolman^¶, Cordula Ackermann, Murat Yildiz, Wolfgang Schoels, David A. Fell^¶, and Wolfgang Kübler

From the  Medizinische Universitätsklinik (Ludolf-Krehl-Klinik), Abteilung Innere Medizin III (Schwerpunkt Kardiologie, Angiologie und Pulmologie), Bergheimer Straße 58, D-69115 Heidelberg, Germany and the ^¶ School of Molecular and Biological Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom

Received for publication, February 4, 2002, and in revised form, May 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Exact adjustment of the Embden-Meyerhof pathway (EMP) is an important issue in ischemic preconditioning (IP) because an attenuated ischemic lactate accumulation contributes to myocardial protection. However, precise mechanisms of glycolytic flux and its regulation in IP remain to be elucidated. In open chest pigs, IP was achieved by two cycles of 10-min coronary artery occlusion and 30-min reperfusion prior to a 45-min index ischemia and 120-min reperfusion. Myocardial contents in glycolytic intermediates were assessed by high performance liquid chromatographic analysis of serial myocardial biopsies under control conditions and IP. Detailed time courses of metabolite contents allow an in-depth description of EMP regulation during index ischemia using metabolic control analysis. IP reduced myocardial infarct size (control, 90.0 ± 3.1 versus 5.05 ± 2.1%; p < 0.001) and attenuated myocardial lactate accumulation (end-ischemic contents, 31.9 ± 4.47 versus 10.3 ± 1.26 µmol/wet weight, p < 0.0001), whereby a decrease in anaerobic glycolytic flux by at least 70% could constantly be observed throughout index ischemia. By calculation of flux:metabolite co-responses, the mechanisms of glycolytic regulation were investigated. The continuous deceleration of EMP flux in control myocadium could neither be explained on the basis of substrate availability nor be attributed to regulatory "key enzymes," as multisite regulation was employed for flux adjustment. In myocardium subjected to IP, an even pronounced deceleration of EMP flux during index ischemia was observed. Again, the adjustment of EMP flux was because of multisite modulation without any evidence for flux limitation by substrate availability or a key enzyme. However, IP changed the regulatory properties of most EMP enzymes, and some of these patterns could not be explained on the basis of substrate kinetics. Instead, other regulatory mechanisms, which have previously not yet been described for EMP enzymes, must be considered. These altered biochemical properties of the EMP enzymes have not yet been described.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Myocardial survival in states of supply/demand imbalance critically depends on cellular energy status (1). To limit energy deficit in conditions of energy shortage, e.g. hypoxia and ischemia, myocardial energy production switches from the preferential use of fatty acids to carbohydrates, thereby allowing maintenance of adequate ATP synthesis when decreased oxygen availability becomes the limiting factor (2-5). However, in zero-flow ischemia, experimental analysis has shown increased glycolysis to be a double-edged sword, as the accumulation of glycolytic end products (6, 7) outweighs the potential benefits of increased ATP synthesis.

In accordance with this paradigm, myocardial protection by prior exposure to ischemic preconditioning (IP)¹ employs a limitation in myocardial energy deficit (8, 9). Because IP tremendously reduces ischemic myocardial energy demands (8), energy deficit is largely decreased at even reduced rates of anaerobic glycolytic ATP formation in zero-flow ischemia. There is good evidence that this attenuation in ischemic lactate accumulation represents an important mechanism whereby IP myocardium better withstands the challenges of sustained ischemia. Although a decrease in anaerobic glycolytic flux is a consistent finding in myocardium protected by ischemic preconditioning (10-12), the precise mechanism used to adjust anaerobic glycolytic flux still remains unclear. The elucidation of this adaptive mechanism, which is not merely of theoretical interest, was the aim of our study.

To analyze the regulation of metabolic pathways, traditional concepts mostly imply that control over a pathway is achieved by action on a single pathway enzyme, which is often assumed to be a nonequilibrium step near the beginning of the pathway subjected to feedback inhibition (13, 14). However, because the rise of metabolic control analysis (MCA) (15) there is growing evidence that these comfortable, time honored concepts may mislead more than enlighten. Hence, the tools of MCA were used to obtain a state-of-the-art analysis of glycolytic regulation in ischemic myocardium protected by preceding ischemic preconditioning.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The experimental protocol described in this study was approved by the Bioethical Committee of the District of Karlsruhe, Germany. All animals in this study were handled in accordance with the guiding principles in care and use of animals as approved by the American Physiological Society and the investigation conformed with the Guide for Care and Use of Laboratory Animals, United States National Institutes of Health.

Animal Preparation-- 12 castrated German domestic pigs with body weights between 25.0 and 32.5 kg (27.8 ± 0.32 kg) were used in this study employing an established model of coronary occlusion and reperfusion as described in detail previously (16).

Experimental Groups-- The experimental protocol is shown in Fig. 1. In all animals, myocardial ischemia causing infarction was induced by a 45-min LAD occlusion (index ischemia). The animals of the control group (C, n = 6) were only subjected to index ischemia followed by 120 min reperfusion. In the IP group, the animals were subjected to two ischemic episodes (10 min each, with 30 min reperfusion between) before the onset of the 45-min index ischemia (IP long; n = 6).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Control animals (C), following a stabilization period of 2 h, were subjected to 45 min of index ischemia and 120 min of reperfusion. In the IP group, a preconditioning cycle of brief LAD occlusion (10 min) and reperfusion (30 min) was performed twice. LAD occlusions are indexed by thick-lined boxes, reperfusion periods by white bars. The numbers indicate duration of these periods in minutes. The arrows mark the time points when myocardial biopsies were taken (before as well as at 2, 5, 10, 20, and 45 min after onset of index ischemia).

Myocardial drill biopsies were taken at the end of the stabilization period, i.e. before any experimental intervention, from virgin, non-ischemic myocardium, and directly before occluding the LAD at the onset of index ischemia. During the 45-min LAD occlusion, biopsies were taken from the center of the ischemic area after 2, 5, 10, 20, and 45 min.

Determination of the Infarcted Area and Quantification of Myocardial Protection-- Determination of myocardial infarct size was determined according to standard methods by identifying ischemic myocardium using fluorescent microspheres and detecting infarcted myocardium following incubation in triphenyltetrazolium chloride. Quantification was performed by normalizing infarcted myocardial mass (infarcted region) to left ventricular myocardium or to the mass of ischemic myocardium (risk region). These ratios are given in percent.

Biopsies and Metabolite Analysis-- Left ventricular drill biopsies (20 mg each) were taken at various time points (Fig. 1). The biopsies were immediately frozen (within 5 s) and kept in liquid nitrogen until further use. Homogenization and deproteinization were performed in ice-cold 60% acetonitrile (v/v) using a Branson sonifier. Using two different high performance liquid chromatography protocols, the myocardial contents in ATP, ADP, and AMP, and the intermediary metabolites and end products of glycolysis were determined (17, 18). The injury caused by the biopsies did not interfere with the triphenyltetrazolium chloride stainings. Myocardial glycogen contents was assessed according to Ref. 19.

Data Analysis-- Dihydroxyacetone phosphate (DHAP) and 1,3-bisphosphoglycerate could not be detected. The DHAP content was, therefore, calculated from the glyceraldehyde 3-phosphate content assuming the equilibrium state of the triose-phosphate isomerase reaction (20). Two- and 3-phosphoglycerate (2-PG and 3-PG, respectively) were not always clearly separated in the chromatograms, although its common peak could be easily detected. The individual proportions were calculated according to the equilibrium of the phosphoglyceromutase (PGM) reaction as shown.

(Eq. 1)

The equilibrium of isomerase and phosphoglyceromutase are known to be unaffected by ischemia (13). Free myocardial ADP content was calculated from the myocardial contents of ATP and AMP, assuming an equilibrium state of the adenylate kinase reaction (21). Glycolytic flux (J) (in micromole of C6-units/g wet weight/min) during ischemia was calculated from the accumulation of lactate and pyruvate and glyceraldehyde 3-phosphate (GA3P) (22).

(Eq. 2)

Our experimental animal model employs total myocardial ischemia. As a consequence, the use of extracellular glucose, which is taken up by the cell via glucose transporters and enters glycolysis after phosphorylation to glucose 6-phosphate (Glu-6-P) by hexokinase can be quantitatively neglected. In total myocardial ischemia, fuel for glycolysis is only provided by the glycosyl units resulting from glycogen breakdown that enter glycolysis via glucose 1-phosphate (Glu-1-P), and to a lesser extent, free glucose.

As 1,3-bisphosphoglycerate could not be detected, glyceraldehyde dehydrogenase and phosphoglycerate kinase were considered as a single glycolytic step. As 2- and 3-PG, as well as of DHAP, contents were calculated the given values are not mathematically independent. Therefore, also GAPDH and 3-PGK as well as PGM and enolase were also put together.

On basis of these calculations, anaerobic glycolytic regulation was investigated using flux:metabolite co-responses as described in detail previously (23).

(Eq. 3)

Note that it follows from the definition of a co-response O^J,S that J = cS^O, where c is a constant. Where an enzyme converts metabolite S to metabolite P the co-responses O^J,S and O^J,P involve the same flux J, so O^J,S/O^J,P = lnP/lnS, and if the enzyme had no change in its mass:action ratio (P/S) when lnP = lnS then the co-responses will be equal. If O^J,S is greater than O^J,P then there has been a relatively larger increase in product concentration, and the reaction has moved closer to equilibrium, whereas if O^J,S is smaller than O^J,P, the reaction has moved away from equilibrium. Where only a single enzyme in a pathway is altered, flux:substrate co-response coefficients are likely to be less than 1 (or less than the Hill coefficient for cooperative enzymes) (23). Under several known physiological conditions (e.g. hypoxia) (23), observed co-response coefficients are larger than this; exact mechanistic explanations of this are not known, but in theory, parallel activation of several steps (the "universal method" (24), multisite modulation (25, 26), or proportional activation (27)) can account for this. Enzymes that are extremely close to equilibrium (25) can show large co-responses during passive adaptation to a change in metabolic flux, but this would imply virtually no detectable change in the displacement from equilibrium, and hence ln (P/S)  0, with virtually identical substrate and product co-responses as described above. A negative flux:substrate co-response corresponds to a classic crossover effect, where activation of an enzyme has induced a decrease in its substrate. Thus the co-response coefficients provide insight into the events associated with a change in flux.

Assumptions-- Our study is based on two major assumptions. (i) Essentially maintained tissue integrity during index ischemia and (ii) constant levels and activities in the enzymes of anaerobic glycolysis, which are both supported by a vast body of evidence. (i) Lethal myocardial injury is initialized and determined during ischemia, whereas the demarcation of necrosis happens during reperfusion. In this context, it is one of the seminal findings of myocardial pathophysiology that "myocytes have suffered severe injury before reperfusion, and that the dramatic consequences of reperfusion are simply postmortem manifestations of lethal injury made possible by the sudden availability of large volumes of plasma water, calcium, or both. In other words, reperfusion accelerates the undertaking events associated with cell death" (28). (ii) Because protein synthesis and degradation are inhibited in myocardial ischemia (29), and because of the short time course of index ischemia, constant levels and unchanged activities in the enzymes of the Embden-Meyerhof pathway can be assumed.

In accordance to these basic findings, we feel justified to perform our analysis of anaerobic glycolytic regulation in myocardium with an unchanged enzymatic composition and an essentially maintained tissue integrity prior to reperfusion. Thus, lactate accumulation could be used as a measure of anaerobic glycolytic flux.

Statistical Analysis-- Comparisons of metabolite levels between groups were performed using ANOVA, for comparisons within groups repeated measures of ANOVA (Scheffé-test) was used. Analysis of variance was also employed to calculate regression differences. The obtained correlations were compared using the Spearman's rank correlation coefficient. Statistical differences were assumed at p values smaller than 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Myocardial Protection-- At constant areas at risk (C, 17.2 ± 0.12%; IP, 15.8 ± 0.72%), IP limits infarct size (C, 90.0 ± 3.1 versus 5.05 ± 2.1%; p < 0.001).

Anaerobic Glycolytic Flux-- As ischemia in control animals proceed, anaerobic glycolytic flux is slowed down. In preconditioned myocardium, glycolytic flux is decreased at the onset of index ischemia, showing an even more pronounced deceleration during the subsequent phase (Fig. 2). Glycogen content at the onset of index ischemia did not differ between normal and IP myocardium (control, 99.0 ± 8.09 µmol of C6-units/g wet weight; IP, 96.2 ± 16.7).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Anaerobic glycolytic flux in control (filled) and IP myocardium (unfilled).

Flux:Metabolite Co-responses-- Myocardial metabolite contents are given in Table I. Flux:metabolite co-responses are illustrated in Figs. 3 and 4, whereby the co-responses of metabolites within glycolysis itself are depicted in Fig. 3, and the co-responses of non-glycolytic metabolites are shown in Fig. 4. The corresponding regression data are given in Table II, significant differences could be attributed to every alteration in the flux:metabolite co-response between control and IP myocardium. As kinetic properties of an enzyme may depend on both substrate as well as product levels, the following description of glycolytic regulation during acute myocardial ischemia and its modulation by preceding ischemic preconditioning focuses on the enzymes of the Embden-Meyerhof pathway.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Flux:metabolite graphs for glycolytic metabolites (mean ± S.E.). Filled, control; unfilled, ischemic preconditioning.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Flux:metabolite graphs for non-glycolytic metabolites (mean ± S.E.). Filled, control; unfilled, ischemic preconditioning.

Glycogen Phosphorylase/Synthase-- Glycogen is the source of glucose 6-phosphate during ischemia, so the reducing glycolytic flux corresponds to a reduced net flux through this potential substrate cycle.

In control myocardium, Glu-1-P levels decrease as the net flux through the enzymes breaking down glycogen to Glu-1-P increases. The co-response (in the range 0.5 to 1) could easily be consistent with a slowing of this step by product inhibition of the glycogen phosphorylase and substrate activation of glycogen synthesizing reactions (UDP-glucose phosphatase/synthase). In IP myocardium, the decrease in flux for a given Glu-1-P level is significantly more pronounced. Because the graph for IP does not represent an extension of the graph for controls the altered flux:metabolite co-responses indicate changed biochemical properties of these enzymes.

The myocardial contents in its co-substrate P_i and allosteric activator AMP only showed poor correlations (not significant) with glycolytic flux under control conditions. Thus, it is unlikely that they contributed to flux regulation at this level. Although there was a better formal correlation between P_i (p < 0.05) and AMP (not significant) following IP, decreases in P_i and AMP with increasing flux still do not seem sufficient to account for the change in enzyme activity observed.

Phosphoglucomutase-- In control animals, the decrease in glycolytic flux during index ischemia is associated with an increase in the substrate (G1P) content of PGM. This occurs at basically stable levels in the product of PGM, Glu-6-P.

These findings indicate that the observed inhibition of glycolytic flux during acute myocardial ischemia in control myocardium is not because of limited G1P availability. Substrate accumulation at increasing net flux, corresponding to a classical crossover effect, suggests a regulatory step downstream from the Embden-Meyerhof pathway, causing a passive increase in Glu-1-P. However, as changes in net flux were independent of Glu-6-P levels, product inhibition of PGM may not account for this alteration. Hence, other regulatory mechanisms must be considered. Moreover, the observation of decreasing substrate contents at stable product levels indicates that the displacement from equilibrium must be decreasing detectably during ischemia, even though this reaction is commonly viewed as close to equilibrium.

Following IP, comparable increases in Glu-1-P content during ischemia were associated with even more inhibition of flux through PGM. Correspondingly, the correlation between flux inhibition and substrate accumulation shows a significantly steeper slope in IP myocardium. Moreover, the graph for preconditioned myocardium does not represent an extension of the correlation, which has been calculated for control myocardium. This altered correlation between PGM and changing substrate concentrations indicates a different kinetic behavior of this enzyme. Moreover, as substrate content changed independently of product levels, a shift in the PGM mass:action ratio also occurred in this case, although these shifts were in an opposite direction as observed in control myocardium.

Also following IP, the decrease in glycolytic flux throughout index ischemia occurred at basically stable Glu-6-P levels. However, there was a significantly negative flux-Glu-6-P co-response. So although there was potentially an increase in product inhibition by Glu-6-P, it is doubtful whether it could counteract the larger increase in Glu-1-P.

As a consequence, the kinetic response of this enzyme does not seem accounted for by the changes in its substrate and product according to classical substrate and product kinetics. Together with the fact that the response following IP is clearly different from that of controls, suggests that preconditioning has had an effect on this step by an unknown mechanism, reinforced by the fact that for a given Glu-1-P concentration the flux is much lower in IP hearts than in controls.

Glucose-phosphate Isomerase-- In control myocardium, the decrease in flux through glucose-phosphate isomerase is only very weakly correlated to substrate content, and no significance can be attached to the co-response coefficient with glucose 6-phosphate. Hence, the behavior of this enzyme cannot be explained by substrate kinetics. However, as Fru-6-P content was increased at reduced flux levels, product inhibition may contribute to enzymatic regulation. As substrate levels change independently of product concentration, a changing mass:action ratio (closer to equilibrium) must be assumed for glucose-phosphate isomerase.

In IP myocardium, the changes in substrate and product co-response coefficients are indicative of a shift of the mass:action ratio away from equilibrium as ischemia progresses. However, because of the large negative co-responses, mechanisms distinct from classical substrate or product-dependent enzyme kinetics are apparently operative in IP.

Phosphofructokinase-1-- In control myocardium, the decrease in glycolytic flux is accompanied with an increase in the substrate Fru-6-P for PFK-1. Fru-1,6-P₂ increases with increasing flux, showing a positive flux co-response and a change in mass:action ratio away from the equilibrium. Strictly, the ADP:ATP ratio should be included in this calculation, but as it changes in the same sense as the Fur-1,6-P₂:Fru-6-P ratio (from the relative sizes of the ATP and ADP co-response coefficients), it reinforces rather than counteracts the change.

These observations imply that under control conditions an inhibition of glycolytic flux was not because of limited Fru-6-P availability. Moreover, Fru-6-P accumulation suggests a regulatory step in downstream glycolysis, causing a passive increase in Fru-6-P. This finding is consistent with an inhibitory role of PFK-1 under these conditions.

AMP, ADP, citrate, and especially Fru-2,6-P₂ are potent activators of PFK-1. Hence, the finding of decreased contents in these metabolites at decreased net fluxes through PFK-1 may account for the behavior of this enzyme, assuming that low concentrations of these stimulatory factors have more impact on regulation of PFK-1 than its inhibitor ATP, for which a decreased content was found. However, control myocardium shows a reduced glycolytic rate with increased Fru-6-P, and little change in allosteric effector AMP or co-substrate ATP (at least relative to K_m for ATP).

In IP myocardium, the decrease in glycolytic flux is associated with a steep increase in Fru-6-P content, whereas the decrease in flux through PFK-1 occurs independent of Fru-1,6_-P₂ levels. For this enzyme under IP conditions, substrate and product contents are indicative of a change in mass:action ratio away from equilibrium.

According to the altered flux/substrate relationship, the mechanism involved in downstream regulation has apparently changed following IP. Under this condition, PFK-1 regulation may not be explained on the basis of co-response, as large positive co-responses of PFK flux to Fru-6-P are not consistent with an effect of substrate kinetics. Moreover, the change in flux through PFK-1 relative to controls occurred at largely unchanged contents in citrate, AMP, and Fru-2,6-P₂. Again, mechanisms distinct and independent from substrate levels and contents of effector metabolites are apparently operative.

Phosphofructokinase 2/Fructose-2,6-bisphosphatase-- As PFK1/Fru-2,6-P₂ase represents a dead-end side branch of the Embden-Meyerhof pathway, flux through this enzyme differs from net glycolytic flux. However, Fru-2,6-P₂ has been shown to be important for regulating the biochemical characteristics of PFK-1.

As PFK-1/Fru-2,6-P₂ase is known to be inhibited by Fru-6-P, its decreased content as glycolytic flux increases may well account for the increased contents in Fru-2,6-P₂ in control myocardium. In addition to the pattern of regulation found for PFK-1, this finding is in good agreement with a regulatory role for PFK-1 under control conditions. Thus, an increased content in the positive regulator of Fru-2,6-P₂ may be involved in adjusting glycolytic flux.

In IP myocardium, a significant correlation between Fru-2,6-P₂ content and glycolytic flux was not observed. This finding fits well to the altered pattern for PFK-1 (see above) with respect to flux adjustment following IP, which appears to be independent of its "traditional" regulators.

Aldolase-- In control myocardium, a decrease in aldolase flux was accompanied by a decrease in Fru-1,6-P₂ content. Hence, for aldolase, the positive co-response observed indicates that changes in substrate content may well account for changes in aldolase flux. At aldolase, the courses of substrate and product content indicate a shift of the mass:action ratio toward equilibrium conditions.

In contrast to the control myocardium, no significant correlations between substrate or product content and net flux could be observed following IP. This finding is not in favor of a flux-regulatory role for aldolase under these conditions.

GAPDH and 3-PGK-- In control myocardium, the observed reductions in flux occurs with poor correlations with glyceraldehyde 3-phosphate and 3-PG content. To the extent that the co-responses with respect to substrate and product are comparable, the pattern observed indicates a similar inhibition of substrate supply and product demand, implying that these enzymes apparently do not play major roles in flux regulation. No relevant shift in mass:action ratio could be seen.

In IP myocardium, again no significant correlation between substrate content and net flux was observed. For 3-PG content, a significant correlation was calculated. However, the large value for the co-response might indicate that in addition to product concentration, additional modifications of the enzyme activities themselves may be responsible for the altered behavior of these glycolytic steps in adjusting flux in IP myocardium. Mass:action ratio shifted away from equilibrium.

PGM/Enolase-- In control myocardium, the change in flux was poorly correlated with the substrate and product levels. This pattern does not suggest a regulating role for these enzymes, and the mass:action ratios remained largely unchanged.

In IP myocardium, the biochemical properties of these enzymes have changed as different correlations between enzyme fluxes and substrate content were seen in the co-response values. The values are large and positive for the substrate but not significant for the product. Because changes in flux occur at almost unchanged substrate and product contents, the behaviors of these enzymes may show that they are very close to equilibrium.

Pyruvate Kinase-- In control myocardium, the decrease in glycolytic flux was not accompanied by statistically significant changes in substrate content. Although for its product pyruvate a significant co-response could be observed, the value was too large to explain the behavior of this enzyme on the basis of product inhibition, so other mechanisms must be considered.

In IP myocardium, flux through PK again changes at basically stable levels for substrate content. For its product pyruvate, again significant co-responses could be observed, although this correlation did differ with controls. For both experimental conditions, there was little evidence for a change in the mass:action ratio of the reaction, because the co-responses to PEP and pyruvate are comparable within experimental error. These observations imply that this enzyme takes part in flux adjustment under both experimental conditions, although its role could not be attributed to substrate or product kinetics and distinct mechanisms must be considered.

Lactate dehydrogenase-- As flux through LDH decreases, pyruvate content remains basically stable, whereas lactate content increases. These data not only indicate a shift in mass:action ratio away from equilibrium but suggest that product inhibition of LDH may be a contributory factor.

In IP myocardium, the substrate for LDH again remained at basically stable levels, but the correlation between lactate content and glycolytic flux has lost its statistical significance. The slope of the regression graph was much lower than 1.0, indicating that regulatory mechanisms distinct from classical metabolite-flux kinetics must occur.

Response of Glycolytic Flux to Changes in Non-glycolytic Metabolites-- As changes in glycolytic flux may occur in response to alterations in contents of non-glycolytic metabolites acting as modulators of certain glycolytic enzymes, their possible contribution to flux adjustment was also investigated.

In control myocardium, a decrease in glycolytic flux with decreasing ATP content could be seen. Although there was a comparable response of glycolytic flux at high ATP concentrations in IP myocardium, the deceleration in glycolytic flux with decreasing ATP content was even more pronounced. A comparable pattern was found for myocardial- free ADP content, although the flux:metabolite co-responses for free ADP were not significant in IP myocardium. For AMP content, no significant correlation to glycolytic flux was observed under control and IP conditions. In control myocardium, inorganic phosphate content did not significantly correlate to glycolytic flux. Although this correlation formally improved in IP myocardium, flux increased as P_i remained stable, indicating an independence of flux adjustment from P_i levels. In control myocardium, anaerobic flux decelerated with decreasing citrate contents, whereas for IP myocardium, no significant correlation could be observed.

Summarizing Considerations-- Under both experimental conditions, glycolytic flux slows down as index ischemia precedes. However, this inhibition is more pronounced in IP myocardium and employs distinct mechanisms of flux adjustment (Table III).

In control myocardium, decreasing substrate concentrations account for decreased fluxes at PFK-1 and aldolase levels. The regulating properties of glycogen phosphorylase, glucose-phosphate isomerase, and lactate dehydrogenase could be attributed to product inhibition. For phosphoglucomutase, GAPDH/3-PGK, PGM/enolase, and PK, the mechanisms employed for flux adjustment were found to be independent of traditional substrate kinetics. Of these enzymes, a regulating role could be found for phosphoglucomutase and PK, whereas the block consisting of GAPDH/3-PGK and PGM/enolase showed a rather passive behavior.

In IP myocardium, the pattern of flux regulation has changed. Decreasing substrate concentrations do not account for decreased fluxes through PFK-1 and aldolase. Here, the characteristics of these enzymes have obviously changed, and for both enzymes, mechanisms distinct to traditional substrate kinetics, such as covalent modification, must be considered. For adjustment of flux by glycogen phosphorylase/synthase and LDH, product inhibition could also be shown in IP myocardium. However, the sensitivities of these enzymes were modulated by IP, and for LDH, an additional mechanism appeared. Aldolase has lost its importance for flux adjustment following IP, whereas formerly insignificant enzymes (block from GAPDH to enolase) play active roles in modulating flux in IP myocardium.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Metabolic Control Analysis-- For almost every known enzyme, numerous data describing their biochemical characteristics are readily available (30). On the basis of these kinetic and thermodynamic properties, various concepts were developed to explain metabolic regulation (13, 14). These traditional concepts mostly convey that control over a pathway is achieved by action on a single pathway enzyme, which is often assumed to be a non-equilibrium step near the beginning of the pathway being subjected to feedback inhibition. However, starting with the rise of MCA, there is growing evidence that these comfortable, traditional concepts may mislead more than enlighten (23). Therefore, MCA was first applied to obtain a state-of-the-art analysis of glycolytic flux, and its regulation in myocardium was subjected to zero flow ischemia with and without prior ischemic preconditioning.

Regulation of Anaerobic Glycolytic Flux during Ischemia in Control Myocardium-- In control myocardium, a continuous decrease in anaerobic glycolytic flux was observed. As regulatory roles for most pathway enzymes could be shown, our data strongly contradict concepts implying glycolytic flux to be adjusted by only one or two regulatory sites ("key enzymes"). Rather, as the tools for altering glycolytic flux were distributed along the entire Embden-Meyerhof pathway, the findings of our study are in good agreement with the concept of multisite modulation, enabling rapid and tight adjustments of glycolytic flux to changing demands (23). This cannot be achieved if only one regulatory site is operative. Moreover, as Glu-1-P content did not decrease but increased with decelerating glycolytic flux, the concept of substrate availability as regulator of flux through glycolysis in acute zero flow myocardial ischemia (31) is not supported by our findings.

Of the steps of the glycolytic chain, only the downstream block from GAPDH to PK was shown not to play dominating roles in flux adjustment. For the remaining enzymes, the flux-substrate (product) analyses revealed an active participation, whereas the mechanism used for regulating enzymatic properties were different. Product inhibition accounted for the behaviors of glycogen phosphorylase/synthase, glucose-phosphate isomerase, and LDH, whereas for PFK-1 a regulatory pattern consistent with traditional substrate kinetics could be observed. Moreover, the in vivo properties of PFK-1 for flux adjustment as documented by MCA show a good correlation to the known biochemical in vitro properties of this enzyme, such as its modulation by changing concentrations in adenine nucleotides, Fru-2,6-P₂, and citrate. For phosphoglucomutase, however, MCA suggests that this step may no longer be seen only passively linking glycogen phosphorylase to glucose-phosphate isomerase. Moreover, the mechanism of the regulation of this enzyme could not be explained on the basis of substrate kinetics, so that other regulatory mechanisms must be taken into account.

According to traditional concepts of glycolytic regulation, it was assumed that decreasing concentrations in ATP and increasing levels in ADP, AMP, and citrate, indicating energy shortage, will increase flux through glycolysis. In our analysis, this concept was supported by considering the co-responses of glycolytic flux to citrate and ADP, whereas for AMP, regression analysis could not prove a dependence of net flux from this metabolite. Moreover, glycolytic flux decreased as ATP levels declined, contradicting traditional models. However, as lactate accumulation appears to be a stronger noxious stimulus for ischemic myocardium than ATP depletion under the conditions of zero flow ischemia (32), this finding might indicate a useful adaptive myocardial mechanism.

Regulation of Anaerobic Glycolytic Flux during Ischemia in Myocardium Protected by Ischemic Preconditioning-- Following ischemic preconditioning, the deceleration of anaerobic glycolytic flux was even more marked. As it was shown for control myocardium, the adjustment of anaerobic glycolytic flux could neither be attributed to substrate availability (myocardial glycogen content was unchanged in control and IP myocardium) nor to one certain flux regulating the key enzyme. Again, multisite modulation could be observed, whereas the pattern observed along the glycolytic chain has been altered by the preceding proconditioning protocol.

Although the regulation at the glycogen phosphorylase/synthese level was also characterized by product inhibition, the sensitivity of this enzyme to react to changing product concentrations has been changed, indicating a modification of this enzyme. As it was seen in control myocardium, the properties of PGM could not be explained on the basis of classical substrate kinetics. However, the much better correlation observed between flux and substrate content indicates an alteration in the biochemical property of the enzyme. Also for glucose-phosphate isomerase, a change in enzyme characteristics could be documented, as the regulatory mechanism following IP could no longer be explained by product inhibition. Comparable findings were obtained for PFK-1 and aldolase, whereby the latter enzyme has lost its active role for flux regulation in IP myocardium. As their biochemical properties had changed, the formerly rather passive enzymes downstream from aldolase are now actively involved in flux adjustment. For LDH, an increase in its degree of product inhibition could be shown, whereas additional regulatory mechanisms must be considered. Unlike in control myocardium, the adjustment of glycolytic flux following IP was independent of the contents in citrate.

As the biochemical behaviors and the regulatory properties of most glycolytic enzymes have changed following IP, the description of the mechanisms responsible for these partially unexpected alterations is an important issue. In this context, the method used for analyzing glycolytic regulation validly allows to differentiate whether an enzyme employs substrate/product kinetics in classical terms or uses distinct regulatory mechanisms. Although the latter mechanisms cannot be specifically characterized using the co-response approach, a differentiation whether or not a change in the regulating mechanism occurs can be performed.

Anaerobic glycolysis was always assumed to be a metabolic pathway, which is exclusively regulated on the basis of substrate kinetics or allosteric mechanisms (13, 33-35). Our data indicate that in addition to these traditional mechanisms, other principles must also be considered to explain flux adjustment by glycolytic enzymes. For glycogen phosphorylase, its regulation by enzyme phosphorylation is firmly established. Therefore, this mechanism must also be considered by analyzing the alterations in enzyme behavior following IP. Interestingly, there is increasing evidence that also the kinetic behavior of glycolytic enzymes may be influenced by enzyme modification, which must not only be because of phosphorylation (36-44). As it could already be seen in control myocardium, glycolytic flux decreased as ATP levels declined, although the sensitivity of changing glycolytic flux to alterations in ATP content was even increased, supporting the view that decelerating lactate accumulation rather than ATP depletion indicates a useful adaptive mechanism to severe myocardial ischemia.

Summary and Possible Implications-- The deceleration of glycolytic flux in myocardium protected by ischemic preconditioning is achieved by multisite modulation, whereby the regulatory mechanism of the glycolytic enzymes could only partly be explained by classical enzyme kinetics. Moreover, previously unknown patterns of flux adjustment were observed. Because the attenuation of lactate accumulation represents a major protective mechanism of IP, the analysis of these regulatory mechanisms might have therapeutic implications.

	FOOTNOTES

^* This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany, within the Sonderforschungsbereich 320, "Herzfunktion und ihre Regulation," and the University of Heidelberg (Teilprojekt C14), Germany (to A. V.).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.

^§ To whom correspondence should be addressed. Tel.: 49-6221- 56-8611; Fax: 49-6221-56-5515; E-mail: Achim_Vogt@med.uni- heidelberg.de.

Supported by Wellcome Trust, UK, Showcase awards 048728 and 056275.

Published, JBC Papers in Press, May 2, 2002, DOI 10.1074/jbc.M201138200

	ABBREVIATIONS

The abbreviations used are: IP, ischemic preconditioning; MCA, metabolic control analysis; LAD, left anterior descending coronary artery; 2-PG, 2-phosphoglycerate; 3-PG, phosphoglycerate; DHAP, dihydroxyacetone phosphate; PGM, phosphoglyceromutase; GAPDH, gluceraldehyde-3-phosphate dehydrogenase; ANOVA, analysis of variance; PFK-1, phosphofructokinase-1; LDH, lactate dehydrogenase.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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