INTRODUCTION

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GLYCOGEN is an important source of glucose for energy substrate metabolism and ATP generation. The contribution of glycogen as an endogenous source of glucose depends on both energy substrate availability as well as the metabolic status of the heart. The regulation of glycogen synthesis and degradation (turnover) has been extensively studied in the liver (1, 37, 39). Although the heart has a great potential for the synthesis and storage of glycogen (31), myocardial glycogen turnover is less well understood, particularly under ischemic or reperfused conditions.

When cardiac muscle is adequately perfused, exogenous glucose is transported into the myocyte, where it primarily either enters the glycolytic pathway or is stored as glycogen. This glycogen can also be subsequently mobilized to provide a source of endogenous glucose for glycolysis. During ischemia, when the supply of O₂ and exogenous substrates is impaired, fatty acid and glucose oxidation are inhibited and ATP generation from anaerobic glycolysis increases. Although glycolysis produces ATP in the absence of O₂, excessive rates of glycolysis may be deleterious during and after severe ischemia due to the production of protons from the hydrolysis of glycolytically derived ATP (7). During reperfusion of ischemic hearts, glycolytic rates continue to exceed glucose oxidation rates. This uncoupling of glycolysis from glucose oxidation continues to be an important source of protons and leads to intracellular acidosis, Na⁺ accumulation, and Ca²⁺ overload (21, 38). Recent data suggest that glucose, released from glycogen under conditions of glycogenolysis, is preferentially oxidized (16, 36). This important finding implies that endogenous glucose contributes less to proton production than exogenous glucose. Thus the preferential utilization of endogenous glucose, rather than exogenous glucose, may result in lower rates of proton production and Ca²⁺ overload.

Although the relationships between endogenous and exogenous glucose metabolism, proton production, and postischemic myocardial function have not been investigated, there is considerable evidence indicating that cardioprotection may arise from interventions that optimize energy substrate utilization both during and after ischemia. Depletion of glycogen by hypoxic preperfusion of hearts limits the potential for glycolysis during ischemia and is cardioprotective (28). Similarly, ischemic preconditioning, an adaptive process in which brief periods of ischemia protect the heart from a subsequent period of severe ischemia (17, 27), reduces glycogen content, inhibits glycolysis, and reduces proton production arising from glucose metabolism (12). Other cardioprotective interventions that improve the coupling of glucose metabolism include stimulation of glucose oxidation by dichloroacetate (23, 26) or inhibition of glycolysis by adenosine (11). These results support the hypothesis that the metabolic coupling of glycolysis to glucose oxidation is an important determinant of postischemic myocardial function.

Substrate availability as well as the activities of glycogen synthase and glycogen phosphorylase controls rates of glycogen synthesis and degradation. Although the relative activities of these enzymes are tightly coupled, simultaneous synthesis and degradation of glycogen (turnover) is demonstrable in isolated working rat hearts perfused under aerobic conditions with glucose as the sole exogenous energy substrate (14). Another study examining glycogen turnover under conditions of net glycogenolysis also showed that simultaneous synthesis and degradation of glycogen are still detectable (16). A limitation of most studies addressing glycogen metabolism is the inappropriate reliance on net changes in total glycogen content with little consideration for the rates of the simultaneous synthesis and degradation of glycogen that occur under aerobic and ischemic conditions. Consequently, there is a need to clarify the relative activities of glycogen synthase and glycogen phosphorylase and their contribution to rates of myocardial glycogen turnover and glucose metabolism. This is particularly true during ischemia and reperfusion, in which glycogen turnover has not been adequately addressed.

The present study was designed to measure glycogen turnover during aerobic conditions as well as during and following ischemia. The contribution of glycogen turnover to glucose metabolism and mechanical function postischemia was also assessed. A perfusion protocol utilizing dual-labeled glucose was designed to measure rates of glycogen turnover and the contributions of endogenous and exogenous glucose to glycolysis and glucose oxidation. Studies were performed under appropriate conditions of energy supply and demand in isolated working rat hearts perfused with both glucose and fatty acids.

	METHODS

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Heart perfusions. Male Sprague-Dawley rats (327 ± 3 g), which were housed and treated according to the standards set by the Canadian Council of Animal Care, were anesthetized with pentobarbital sodium (60 mg/kg ip). After induction of anesthesia, hearts were rapidly removed and placed in ice-cold Krebs-Henseleit solution. The hearts were then cannulated as described previously (10) and perfused via the aorta during a 10-min equilibration period. Hearts were then switched to working mode and perfused at 37°C under aerobic conditions at a constant left atrial preload (11.5 mmHg) and aortic afterload (80 mmHg). Perfusate consisted of a modified Krebs-Henseleit solution containing 1.2 mM palmitate prebound to 3% BSA, 2.5 mM Ca²⁺, 100 µU/ml insulin, and 11 mM glucose and was oxygenated with carbogen (95% CO₂-5% O₂). Hearts were perfused under aerobic conditions for 60 min and then subjected to low-flow ischemia (0.5 ml/min) for 60 min, followed by 30 min of reperfusion (Fig. 1). Reperfusion was initiated by the removal of the clamps on the preload and afterload lines. This allowed flow into the left atrium at a rate that was influenced by the ability of each heart to eject fluid into the aortic afterload line. Afterload pressure was not maintained artificially during reperfusion and was also determined by the ability of each heart to eject perfusate. Hearts were paced at 300 beats/min throughout each phase of the perfusion protocol (voltage adjusted as necessary) with the exception of the initial 5 min of reperfusion, during which hearts were allowed to beat spontaneously. At the end of the perfusion protocol, hearts were rapidly frozen with the use of Wollenberger clamps cooled to the temperature of liquid N₂. Additional groups of hearts were also frozen at the start of the aerobic perfusion period (time 0) and immediately before or after the period of low-flow ischemia. Frozen tissues were pulverized and the resulting powders stored at 80°C.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Heart perfusion protocols for series I and II heart perfusions. In each series, hearts were frozen at the end of the aerobic, low-flow ischemic (LFI), and reperfusion periods. The 2 series represent a different order for radiolabeling glucose that was necessary to determine exogenous and endogenous contributions of glucose to glycolysis and glucose oxidation during LFI. Start of aerobic working period is represented at t = 0 min. Calculations and further details are presented in METHODS.

Myocardial mechanical function. Aortic systolic and diastolic pressures were measured with the use of a Gould P21 pressure transducer connected to the aortic outflow line. Cardiac output, aortic flow, and coronary flow (cardiac output  aortic flow) were measured (mean values, ml/min) with the use of in-line ultrasonic flow probes connected to a Transonic T206 ultrasonic flowmeter. Left ventricular minute work (LV work), calculated as cardiac output × (aortic systolic pressure  preload pressure), was used as a continuous index of mechanical function. Hearts were excluded if LV work decreased >20% during the 60-min period of aerobic perfusion.

Measurement of glycolysis and glucose oxidation. Rates of glycolysis were measured directly as previously described (33) from the quantitative determination of ³H₂O liberated from [5-³H]glucose at the enolase step of glycolysis. Glucose oxidation was also determined directly as previously described (10, 33) by measuring ¹⁴CO₂ liberated from [¹⁴C]glucose at the level of pyruvate dehydrogenase and in the tricarboxylic acid cycle. Perfusate samples were collected at various time points throughout the perfusion protocol. Average rates of glycolysis and glucose oxidation for each phase of perfusion are expressed as micromoles of glucose metabolized per minute per gram of dry weight.

Measurement of sources and fate of glucose during low-flow ischemia. A dual-label ([5-³H]- and/or [U-¹⁴C]glucose) protocol was designed to label the glycogen pool and then follow separately the fate of glucose (both glycolysis and glucose oxidation) arising from either exogenous or endogenous sources. Two series of identical perfusions were designed, except that the order and timing of isotope addition differed (Fig. 1).

Measurement of glycogen content and rates of glycogen turnover. Myocardial glycogen content (in µmol glucosyl units/g dry wt) was determined by measuring the glucose content in samples of frozen tissue that were subjected to alkaline extraction (30% KOH) to separate glycogen from exogenous glucose. This was followed by ethanol precipitation and acid hydrolysis (2 N H₂SO₄) to release endogenous glucose from glycogen; thereby, the radiolabeled content of the glycogen pool could be determined accurately without any contamination from free, unincorporated glucose. Glycogen extracts were also analyzed for [³H]glucose and [¹⁴C]glucose so that the specific activity of [³H]- and [¹⁴C]glycogen and the percentage of glycogen that became labeled with either [³H]glucose or [¹⁴C]glucose could be determined in hearts frozen at the end of each phase of the perfusion protocol.

1

1

(1)

(2)

Glucose uptake and extraction. Glucose uptake in aerobic and ischemic hearts was calculated from the sum of the actual rates of glycogen synthesis (G_in) and glycolysis from exogenous glucose. Glucose extraction (%) under these conditions was calculated from rates of glucose uptake as a percentage of glucose delivery (glucose concentration × coronary flow).

Measurement of activities of glycogen synthase and glycogen phosphorylase. The activities of glycogen synthase and glycogen phosphorylase were determined in tissue samples frozen at the end of each phase of the perfusion protocol. Glycogen phosphorylase activity, expressed as phosphorylase a as a percentage of total, was determined as described previously (8) by measuring the formation of glucose-6-phosphate in the presence of excess glycogen and in the absence and presence of AMP. Glycogen synthase activity, expressed as percent active, was determined as previously described (30) by measuring the consumption of UDP in the absence and presence of glucose-6-phosphate.

Drugs and reagents. D-[5-³H]glucose and D-[U-¹⁴C]glucose were purchased from NEN (Boston, MA). BSA (fraction V) was obtained from Boehringer Mannheim (Indianapolis, IN). Hyamine hydroxide (methylbenzethonium; 1 M in methanol solution) and Ecolite counting scintillant were obtained from ICN Biomedicals (Mississauga, ON, Canada). Dowex 1 × 4 anion exchange resin (200-400 mesh chloride form) was obtained from Bio-Rad (Richmond, VA). Lactate dehydrogenase, pyruvate kinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase were obtained from Boehringer Mannheim. Glucose assay kits were obtained from Sigma Chemical (St. Louis, MO). Other chemicals were reagent grade.

Statistical analysis. Data are expressed as means ± SE. Comparisons between groups were performed using the unpaired Student's t-test. Multiple comparisons were made using ANOVA followed by the Student-Newman-Keuls post hoc test. Differences were judged to be significant if P < 0.05.

	RESULTS

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Mechanical function of aerobic, ischemic, and reperfused hearts. LV work, which was used as an index of mechanical function, was stable throughout the 60-min period of aerobic perfusion (Fig. 2). All measurable LV work ceased during low-flow ischemia and recovered to 20 ± 5% of preischemic levels by the end of the 30-min period of aerobic reperfusion. Other indexes of myocardial function, including cardiac output, aortic flow, and coronary flow, were also constant during aerobic perfusion and recovered with a similar time course to 25 ± 5, 9 ± 3, and 57 ± 3%, respectively, by the end of reperfusion (Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Left ventricular (LV) work during aerobic, LFI, and reperfusion phases of perfusion in isolated working rat hearts. Solid bar represents period of LFI (0.5 ml/min coronary flow). Data are means ± SE for 17 hearts.

Content and percentage labeling of glycogen in aerobic, ischemic, and reperfused hearts. Normal levels of glycogen in the heart in vivo are 120-150 µmol/g dry wt (41). As expected, removal of the heart and perfusion for a short period (10 min) without fatty acids in the Langendorff mode resulted in a decrease in glycogen to 74 ± 9 µmol/g dry wt. Glycogen content of hearts increased toward in vivo levels when hearts were perfused for 60 min under aerobic conditions (Fig. 3), and, during this period, 52 ± 3 or 61 ± 3% of the glycogen pool became labeled with [³H]- or [¹⁴C]glucose, respectively. Because glycogen labeling is independent of the nature of the isotope, values for percent labeling are presented as an average of values obtained with each isotope in each perfusion series. After 60 min of aerobic perfusion, 56 ± 4% of the glycogen was labeled with [³H]- or [¹⁴C]glucose. During low-flow ischemia, there was a predictable glycogenolysis, and, after 60 min, glycogen content had markedly decreased relative to values immediately before the onset of ischemia (Fig. 3). Although total glycogen decreased during low-flow ischemia to values less than those observed before exposure to either [³H]- or [¹⁴C]glucose, the isotope that was used to label the glycogen during aerobic perfusion was not entirely depleted. In fact, during low-flow ischemia, the percentage of glycogen labeled with this initial isotope increased further to 76 ± 10% (Fig. 3). In addition, there was a net incorporation (38 ± 4%) of the second isotope that was added at the onset of low-flow ischemia. Glycogen contents of hearts after reperfusion were not significantly different from those measured at the end of low-flow ischemia (Fig. 3). However, in reperfused hearts, a further loss of unlabeled glycogen was observed such that the extent of glycogen labeling was 87 ± 16 and 68 ± 9%, respectively, with the first and second isotopes (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Glycogen content and percent labeling of glycogen at the end of aerobic, LFI, and reperfusion periods. Values are means ± SE of 7-17 hearts in each group. Working rat hearts were perfused as described in legend to Fig. 1. t = 0 represents glycogen content before aerobic working period. Solid bars represent glycogen labeled with first isotope, shaded bars represent glycogen labeled with second isotope, and open bars represent unlabeled glycogen. * Significant difference from t = 0 (min); # significant difference from aerobic values.

Rates of glycolysis, glucose oxidation, and proton production in aerobic, ischemic, and reperfused hearts. Similar to previous studies in fatty acid-perfused working hearts (11, 12, 23), the rate of glycolysis during the initial aerobic perfusion period was greater than that of glucose oxidation (Fig. 4). This uncoupling of glycolysis from glucose oxidation resulted in proton production rates attributable to glucose metabolism of 7.5 ± 0.9 µmol · min¹ · g dry wt¹. During low-flow ischemia, the sum of the rates of glycolysis, glucose oxidation, and proton production arising from both exogenous and endogenous sources of glucose (indicative of total rates) were similar to those measured during the initial aerobic perfusion. In the reperfusion period, when LV work had recovered to only 20% of aerobic values, flux of glucose from both endogenous and exogenous sources was slightly depressed. Glucose oxidation was unaffected, and this resulted in a slight improvement in the coupling of glycolysis to glucose oxidation and a slight inhibition in proton production.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Average rates of glycolysis (top), glucose oxidation (middle), and proton production (bottom) from glucose metabolism during aerobic, LFI, and reperfusion periods. Values are means ± SE of 7-10 hearts in each group. Endogenous (Endo) and exogenous (Exo) contributions of glucose to glycolysis, glucose oxidation, and proton production were determined during LFI. Hearts were perfused as described in Fig. 1. * Significant difference from total during LFI; # significant difference between Exo and Endo during LFI; $ significant difference from aerobic period.

Relative contributions of endogenous and exogenous glucose to rates of glycolysis, glucose oxidation, and proton production during low-flow ischemia. Despite the dramatic decrease in coronary flow during low-flow ischemia, rates of glycolysis using exogenous glucose were significantly higher than for glycolysis using endogenous sources. However, the contributions of exogenous and endogenous glucose to rates of glucose oxidation were not significantly different. This indicates a preferential oxidation of the endogenous glucose liberated from glycogen, resulting in a lower rate of proton production from endogenous, relative to exogenous, sources of glucose (Fig. 4).

Glucose uptake and extraction. Rates of glucose uptake were inhibited by 32% during ischemia and further decreased to 53% of aerobic values during the reperfusion period (Table 2). Glucose extraction values indicate that glucose availability was not rate limiting, because even at maximal rates of glucose uptake, which were observed during low-flow ischemia, only 21% of the available glucose was extracted from the low-flow perfusate (11 mM glucose).

Rates of glycogen turnover (G_in and G_out) in aerobic, ischemic, and reperfused hearts. Apparent rates of glycogen synthesis and degradation (G'_in and G'_out), which were based on net changes in total glycogen content, indicated that synthesis predominated during aerobic perfusion (Table 2). As expected, degradation predominated during low-flow ischemia. During reperfusion, G_in and G_out were essentially similar. Calculations that measured the average rates of the simultaneous synthesis and degradation of glycogen indicated that G_in was 2.3-fold higher than G_out during the initial aerobic perfusion, whereas, during low-flow ischemia, G_out was 3.9-fold higher than G_in. Although there was no net change in glycogen content during the reperfusion period and the rates of synthesis and degradation were not significantly different, the high values for G_in and G_out indicated that there was considerable glycogen turnover during this period (Table 2). Glycogen synthesis was suppressed by 2.3-fold during low-flow ischemia but recovered to preischemic values during reperfusion. During low-flow ischemia, glycogenolysis was stimulated fourfold relative to aerobic rates and remained elevated (3-fold) during reperfusion (Table 2).

Activities of glycogen synthase and glycogen phosphorylase in aerobic, ischemic, and reperfused hearts. At the end of the period of low-flow ischemia, glycogen phosphorylase and glycogen synthase activities were increased significantly compared with values measured at the end of aerobic perfusion (Table 3). During reperfusion, glycogen phosphorylase activity returned to aerobic values, whereas glycogen synthase activity remained significantly elevated.

	DISCUSSION

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Although it has been suggested that glycogen metabolism possesses only a minor role in myocardial energy substrate metabolism (29), glycogen metabolism has recently been shown to contribute significantly to overall myocardial ATP production (14, 16). This study developed a new approach to determine directly the contributions of glycogen turnover to overall glucose metabolism in aerobic, ischemic, and reperfused working rat hearts. The data demonstrate that there is a substantial turnover of glycogen not only under aerobic conditions but also during low-flow ischemia and during aerobic reperfusion. Despite the marked acceleration of glycogenolysis during ischemia, glycogen synthesis was still demonstrable. Furthermore, although the total myocardial glycogen pool was markedly reduced, glycogen turnover persisted during reperfusion. Direct measurements of radiolabeled glucose in the glycogen pool revealed that glucose, which became incorporated during the initial aerobic perfusion, was retained within the glycogen pool after ischemia despite marked glycogenolysis and glycogen depletion. This result suggests that the "last on, first off" hypothesis of glycogen metabolism (3, 19) at the molecular level does not apply to the intact organ. Finally, our finding that the preferential oxidation of glucose derived from glycogen during low-flow ischemia was associated with a lower rate of proton production suggests that optimization of glycogen turnover may be a useful therapeutic strategy for improving the recovery of mechanical function during reperfusion of postischemic hearts.

The isolated perfused working rat heart provides an ideal system for studying myocardial energy metabolism because hearts can be exposed to an appropriate array of energy substrates (fatty acid and carbohydrates) as well as to a physiological workload and energy demand. There are several reasons why the presence of exogenous fatty acid was critical for this study. First, when present at concentrations (1.2 mM) observed in patients suffering myocardial ischemia or undergoing cardiac surgery (22), fatty acid oxidation contributes 90% of total myocardial ATP production (33). Fatty acids also influence glucose and glycogen metabolism, and their presence in the perfusate of isolated hearts maintains myocardial glycogen contents close to levels measured in vivo (20, 41). This is in marked contrast to the majority of studies using isolated hearts in which the absence of fatty acids in the perfusate leads to abnormally low glycogen levels (18, 35). Furthermore, because of their marked inhibition of glucose oxidation (32), fatty acids result in a metabolic uncoupling between rates of glycolysis and glucose oxidation (34, 40), a condition that leads to a significant rate of proton production (10, 21).

LV work was used in this study as an index of mechanical function. During aerobic perfusion, LV work was stable, thereby facilitating measurement of glycogen and exogenous glucose metabolism under constant levels of energy demand. Total myocardial glycogen increased during aerobic perfusion, indicative of net glycogen synthesis in response to abundant energy supply. During low-flow ischemia, measurable LV work ceased, as would be expected at this level of severe ischemia. Hearts were perfused at a low coronary flow to mimic severe ischemia due to coronary occlusion, with some residual perfusion as might be associated with collateral flow. Ischemia stimulated a net depletion of glycogen to levels lower than those measured at the beginning of aerobic perfusion. Aerobic reperfusion of hearts after low-flow ischemia resulted in a gradual and only partial recovery of LV work and no net change in glycogen content. This provided conditions for the study of glycogen turnover and glucose metabolism in the postischemic heart in which mechanical function was severely compromised by ischemia-induced myocardial damage. Relative to baseline aerobic conditions, coronary flow and, hence, delivery of energy substrates (oxygen, glucose, fatty acids) were more than adequate for the level of LV work during reperfusion.

The measurement of time-dependent changes in glycogen content only provides information on the relative rates of glycogen synthesis and degradation. Consequently, a perfusion protocol was designed that utilized both [³H]- and [¹⁴C]glucose to enable the measurement of the average rates of the simultaneous synthesis and degradation of glycogen during each phase of the perfusion protocol. This allowed for a detailed assessment of glycogen turnover during aerobic ischemic and postischemic conditions. Another advantage of the dual-label protocol is that rates of glycolysis and glucose oxidation for endogenous as well as exogenous glucose during low-flow ischemia could be calculated.

Glycogen turnover has been studied previously in aerobic hearts in the absence and presence of catecholamines and other hormones (13, 14, 16, 36). However, the present study is the first to directly investigate glycogen turnover under aerobic conditions as well as under pathophysiological conditions of low-flow ischemia and reperfusion. In addition, previous studies have failed to account for any simultaneous synthesis and degradation of glycogen or have studied hearts using perfusates devoid of fatty acids. Rates of glycogen synthesis and degradation were calculated in two ways. First, apparent rates (G'_in and G'_out) were calculated simply on the basis of relative time-dependent changes in glycogen content. However, these apparent rates represent an underestimation of the rates of glycogen synthesis and degradation because they do not account for any simultaneous synthesis and degradation of glycogen (14). Consequently, a new calculation was adopted that accounted for the potential of radiolabeled glucose to be released after its incorporation into glycogen. With this approach, higher rates of glycogen synthesis (G_in) and degradation (G_out), indicative of substantial turnover, were observed for periods of aerobic perfusion, low-flow ischemia, and aerobic reperfusion compared with calculated apparent rates. A limitation of both these calculations of glycogen turnover is the assumption that the rates of synthesis and degradation are constant throughout each phase of perfusion. Although rates of myocardial glycolysis and glucose oxidation were indeed stable throughout each phase, further studies evaluating other time intervals are required to assess time-dependent alterations in glycogen turnover within each phase of perfusion.

During low-flow ischemia, despite net glycogenolysis and marked glycogen depletion, radiolabeled glucose that was added to the perfusate only at the onset of ischemia became incorporated into glycogen. Thus, even during severe ischemia, a significant rate of glycogen synthesis was still detectable. Glycogen synthesis has been hypothesized to proceed by the successive additions of glucosyl units to an internal protein core, whereas degradation releases these most recently incorporated glucosyl units. This concept has been termed the last on, first off hypothesis (3, 19). Interestingly, although ischemia stimulated the depletion of glycogen to a level below that measured in aerobic hearts, the proportion of glycogen labeled with this isotope was greater than that labeled with the isotope that was added only at the onset of ischemia. Thus a component of the radiolabeled glucose that had become incorporated in glycogen during aerobic perfusion was retained at the end of ischemia. This result suggests that the last on, first off hypothesis of glycogen metabolism at the molecular level does not apply to the intact working heart during conditions of marked glycogenolysis.

Interestingly, during reperfusion, rates of synthesis as well as degradation were high, indicating a marked increase in glycogen turnover in the postischemic heart. Measurements of glycogen content alone would have suggested that glycogen turnover did not occur during reperfusion, because glycogen levels did not change. However, direct measurements of the rates of synthesis and degradation determined that this was not the case and that glycogen did not recover during reperfusion, because rates of synthesis and degradation were equal. This result emphasizes the need to measure glycogen turnover directly when glucose metabolism is measured in the postischemic heart.

Glycogen turnover is tightly controlled by the activities of two key enzymes, glycogen synthase and glycogen phosphorylase. At the end of low-flow ischemia, a marked increase in glycogen phosphorylase activity was detectable, and this correlated with the stimulation of glycogen degradation that occurred during this period. Although an inverse relationship between the activities of glycogen synthase and phosphorylase has been reported (5), a decrease in glycogen synthase activity was not detected in hearts at the end of ischemia. This result agrees with that of McNulty and Luba (25), who showed that glycogen mobilization early in ischemia also activates glycogen synthase. During reperfusion, net glycogen synthesis did not occur even in the presence of decreased glycogen phosphorylase activity and increased glycogen synthase activity. Both G_in and G_out increase during reperfusion to a similar extent, explaining the lack of any net change in glycogen content. This suggests that it is not simply the activities of these two enzymes that control glycogen turnover during reperfusion. For example, substrate supply may influence glycogen synthesis in addition to the activity of glycogen synthase. Alternatively, this apparent discrepancy may be explained by the fact that the rates of G_in and G_out are averages based on the entire period of reperfusion, whereas the activities of these two enzymes are determined at a single time point (end of reperfusion).

The rate of glycolysis in working hearts perfused with glucose and fatty acids normally exceeds that of glucose oxidation even under aerobic conditions (10, 11, 33). This so-called "metabolic uncoupling of glucose metabolism" is an important source of protons (7) that, in the absence of adequate perfusion, leads to acidosis and impaired mechanical function. This is supported by our studies showing that improvement in the coupling between glycolysis and glucose oxidation caused by either inhibition of glycolysis (9, 10) or stimulation of glucose oxidation (23, 26) will attenuate proton production and acidosis. This effect is associated with an improved recovery of mechanical function and cardiac efficiency in the postischemic heart (21), possibly due to a decrease in the supply of protons for the Na⁺/H⁺ exchange that limits the potential for Ca²⁺ overload (38). Recently, it has been shown that endogenous glucose, namely that derived from glycogen, is preferentially oxidized in aerobic hearts (13, 16, 36). In this study, we demonstrate that this preferential oxidation of endogenous glucose also occurs during low-flow ischemia. This improvement in the metabolic coupling of glycolysis to glucose oxidation resulted in a lower rate of proton production from endogenous (glycogen) relative to exogenous sources.

The rate of overall proton production from glucose metabolism in aerobic hearts was 7.52 ± 0.87 µmol · min¹ · g dry wt¹. In ischemic hearts, the decrease in the total rate of glucose oxidation, i.e., from both endogenous and exogenous sources, in combination with an unaltered rate of total glycolysis, increased the degree of metabolic uncoupling and, therefore, further increased proton production. Because there is preferential oxidation of endogenous glucose, proton production from the metabolism of exogenous glucose was significantly greater than that from endogenous sources. The lower rate of metabolism of exogenous glucose was not due to a limitation in glucose delivery, because glucose extraction was only 21% during low-flow ischemia. In addition, ischemia did not cause a complete inhibition of glucose oxidation, suggesting that the availability of O₂ and glucose was sufficient to maintain a degree of oxidative metabolism.

During reperfusion, the uncoupling of glycolysis from glucose oxidation persisted. This continued production of protons during the critical early period of reperfusion limits the recovery of mechanical function. The importance of proton production during reperfusion is supported by previous studies in which inhibition of proton production only during the reperfusion phase results in an improved recovery of postischemic mechanical function (21). This finding highlights the need to study glycogen turnover in the actual reperfusion period. The relative contributions of the metabolism of endogenous and exogenous sources of glucose to proton production during reperfusion could not be determined in the present study, because both isotopic labels had been incorporated into glycogen by the end of ischemia. This prevented discrimination between the sources of glucose in the reperfusion period. However, because glycogen content was stable during reperfusion, it appears that most of the glucose was derived from exogenous sources.

There are important relationships between glycogen turnover, glycolysis, glucose oxidation, and postischemic function (6). Glycogen depletion by anoxic preperfusion (28) or ischemic preconditioning (4, 27, 41) improves recovery of postischemic hearts and reduces infarct size (2). Conversely, elevation of preischemic glycogen content is protective under some conditions because it enhances substrate availability for glycolysis (15, 24). Thus the demonstration that the metabolism of endogenous glucose generates fewer protons than the metabolism of exogenous glucose indicates that regulation of the source of glucose may be an important target for strategies to reduce acidosis and, therefore, elicit cardioprotective actions. The demonstration that there is substantial turnover of glycogen during ischemia and reperfusion suggests that strategies designed to alter turnover and, in particular, increase the oxidation of glucose from glycogen, as opposed to extracellular glucose, may reduce ischemia-induced acidosis and provide therapeutic benefit.