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Circulation Research. 1996;78:482-491

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(Circulation Research. 1996;78:482-491.)
© 1996 American Heart Association, Inc.

Articles

Is a High Glycogen Content Beneficial or Detrimental to the Ischemic Rat Heart?

A Controversy Resolved

Heather R. Cross, Lionel H. Opie, George K. Radda, Kieran Clarke

From the Department of Biochemistry, University of Oxford (UK).

Correspondence to Heather R. Cross, University of Oxford, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
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Abstract A high glycogen level may be beneficial to the ischemic heart by providing glycolytic ATP or detrimental by increasing intracellular lactate and protons. To determine the effects of high glycogen on the ischemic myocardium, the glycogen content of Langendorff-perfused rat hearts was either depleted or elevated before 32 minutes of low-flow (0.5 mL/min) ischemia with Krebs-Henseleit buffer with or without 11 mmol/L glucose, followed by 32 minutes of reperfusion with buffer containing 11 mmol/L glucose. 31P nuclear magnetic resonance spectra were acquired sequentially throughout. Further experiments involved early reperfusion or the addition of HOE 694, a Na+-H+ exchange inhibitor, during reperfusion. When glucose was supplied throughout ischemia, no ischemic contracture occurred, and postischemic recovery of contractile function was highest, at 88% of preischemic function. In the absence of glucose, normal-glycogen hearts underwent ischemic contracture at 5 minutes, had an end-ischemic pH of 6.87, and recovered to 54%, whereas in high-glycogen hearts, contracture was delayed to 13 minutes, the end-ischemic pH was 6.61, and functional recovery decreased to 13%. Contracture onset coincided with the decrease in glycolysis, which occurred as glycogen became fully depleted. Functional recovery in the high-glycogen hearts increased to 89% when reperfused before contracture and to 56% when reperfused in the presence of HOE 694. Thus, during brief ischemia in the high-glycogen hearts, ischemic glycogen depletion and contracture were avoided, and the hearts were protected from injury. In contrast, during prolonged ischemia in the high-glycogen hearts, glycogen became fully depleted, and myocardial injury occurred; the injury was exacerbated by the lower ischemic pH in these hearts, leading to increased Na+-H+ exchange during reperfusion. The contradictory findings of past studies concerning the effect of high glycogen on the ischemic myocardium may thus be due to differences in the extent of glycogen depletion during ischemia.


Key Words: glycogenolysis • low-flow ischemia • 31P nuclear magnetic resonance spectroscopy • pHi • myocardial injury


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Controversy exists over the effect of high endogenous glycogen on the ischemic myocardium. Preischemic glycogen loading was beneficial in models of brief no-flow ischemia1 2 3 or hypothermic ischemic arrest.4 In contrast, preischemic glycogen depletion was protective in several studies of no-flow ischemia.5 6 7 Endogenous glycogen levels may also be important in preconditioning, because glycogen depletion by a short ischemic or hypoxic insult protects hearts from subsequent prolonged ischemia.8

During ischemia, anaerobic glycolysis from either glucose or glycogen results in the production of ATP and the intracellular accumulation of lactate and protons (H+) as metabolic end products.7 Glycolytic ATP production can be beneficial during myocardial ischemia9 10 and may play a role in ionic homeostasis.11 In contrast, elevated lactate and H+ during ischemia may exacerbate myocardial injury.7 Lactate may cause injury via feedback inhibition of glycolysis,12 13 whereas H+ may cause injury via increased Na+-H+ exchange, leading to elevated intracellular Na+ and Ca2+.14 15 The effect of high glycogen during ischemia may therefore depend on whether the advantage of increased glycolytic ATP production outweighs the detrimental effect of accumulation of lactate and H+.

We used a low-flow ischemia model, as opposed to the more commonly used total-ischemia model, which enables myocardial lactate production, a measure of anaerobic glycolysis, to be determined during ischemia. By relating changes in energetics, pHi, and myocardial lactate production to postischemic functional recovery in rat hearts of varying preischemic glycogen content, we aimed to determine the effect of high glycogen during low-flow ischemia and explain the apparently paradoxical findings of previous studies.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Heart Perfusion
Fed Male Wistar rats (n=32; body weight, 372±4 g) were anesthetized with an intraperitoneal injection of sodium pentobarbitone (75 mg/kg body wt). Hearts were rapidly excised and arrested in ice-cold Krebs-Henseleit buffer. Excess tissue was removed, and the hearts were cannulated, via the ascending aorta, for retrograde perfusion by the Langendorff method using Krebs-Henseleit buffer containing (mmol/L) NaCl 110, KCl 3.9, MgSO4·7H2O 1.2, CaCl2 · 2H2O 1.75, glucose 11, KH2PO4 2, NaHCO3 25, NaOH 8.4, phenylphosphonic acid 5, and dimethyl methylphosphonate 10. The buffer, aerated with 95% O2/5% CO2 to give a pH of 7.4 at 37°C, was perfused through the hearts at a constant pressure of 100 mm Hg. The pulmonary artery was incised to prevent venous pressure accumulation, and intraventricular pressure development was prevented by insertion of a drain through the apex of the left ventricle. A water-filled latex balloon, attached via polyethylene tubing to a Gould disposable pressure transducer, was inserted into the left ventricular cavity via the mitral valve and inflated sufficiently to result in an end-diastolic pressure of {approx}4 mm Hg. Left ventricular pressure and heart rate were recorded using a Lectromed MX216 recorder. Cardiac contractile function was expressed as a rate-pressure product, this being the product of left ventricular developed pressure (millimeters of mercury) and heart rate (beats per minute), where developed pressure is systolic pressure minus end-diastolic pressure.

For the NMR experiments, the perfused hearts were placed in a 20-mm-diameter NMR sample tube and buffer, containing 265 mmol/L mannitol, 3.5 mmol/L HEPES acid, and 6.5 mmol/L HEPES sodium salt, was pumped past the heart at a rate of 30 mL/min. Coronary effluent and mannitol buffer were evacuated from an overflow outlet above the heart, and the rate of their accumulation was used as a measure of coronary flow, after correction for mannitol buffer rate. Temperature was maintained at a constant 37°C using the Bruker variable temperature unit attached to the spectrometer and water-jacketed buffer reservoirs and perfusion lines. For the experiments that did not involve NMR spectroscopy, mannitol buffer was not used, and hearts were surrounded by a water-jacketed collection vessel from which coronary effluent was drained for determination of lactate concentrations.

NMR Spectroscopy
To measure tissue phosphorus metabolite concentrations, a fully relaxed spectrum was acquired for each heart using a Bruker AM-400 spectrometer with a 9.4-T superconducting magnet at the 31P resonance frequency of 161.92 MHz. A 90° pulse with an interpulse delay of 30 seconds was used, and each spectrum consisted of 64 summated transients, giving a total acquisition time of 32 minutes. Peak resolution was enhanced by reducing field inhomogeneities; this was achieved by shimming the proton signal to a line width of 18±1 Hz. The signal-to-noise ratio was increased by multiplying the 31P NMR free induction decays, before Fourier transformation, by an exponential function sufficient to generate a line broadening of 20 Hz.

To determine absolute intracellular concentrations of phosphorus metabolites from the 31P NMR spectra, an external standard of 20 µL (10 µmol) of a 500 mmol/L MPA solution was sealed in polyethylene tubing and placed adjacent to the heart during spectral acquisition. The areas of each of the spectral peaks were fitted to Lorentzian line shapes using a software program (NMR1 from NMRi), and the amount of the metabolites present in the hearts was calculated using the MPA standard. To allow these values to be converted to absolute intracellular concentrations, two phosphonate volume markers were included in the perfusate: 5 mmol/L phenylphosphonic acid was used as a measure of extracellular water volume, and 10 mmol/L dimethyl methylphosphonate was included as a measure of total water volume. The intracellular volume was calculated from the difference between the two measured volumes, allowing the expression of metabolite concentrations as millimoles per liter intracellular water.16

Relative changes in concentrations of phosphorus metabolites were observed during the time course of the experiments by acquiring consecutive 4-minute saturated 31P NMR spectra using a 60° pulse with an interpulse delay of 2.15 seconds. Each spectrum was obtained by acquisition of 104 transients and was line-broadened, Fourier-transformed, and quantified as described above. During ischemia, in the absence of contracture, there was <5% change in the MPA peak area, indicating that the sensitivity of the NMR coil did not change during the protocol in this case.17 However, when hearts underwent contracture, there was up to a 20% change in the MPA peak area. To compensate for such alterations in coil sensitivity, the area of each spectral peak was expressed relative to the MPA peak area throughout the protocol. pHi was estimated from the chemical shift of the Pi peak relative to PCr using previously obtained titration curves.18

Experimental Protocols
Standard Protocol
Ten hearts were perfused with glucose-containing Krebs-Henseleit buffer for 80 minutes before the start of low-flow ischemia. During this preischemic period, one fully relaxed and several saturated 31P NMR spectra were acquired, and coronary flow and contractile function were monitored. Five of the hearts were perfused with glucose-containing Krebs-Henseleit buffer during low-flow ischemia (+G condition), and five hearts were perfused with glucose-free Krebs-Henseleit buffer during low-flow ischemia (-G condition). The glucose-free Krebs-Henseleit buffer was introduced during a 3-minute perfusion period inserted between preischemic and ischemic periods to ensure that the perfusion lines and the extracellular space of the heart were free of glucose before the onset of ischemia. Low-flow ischemia was then initiated by diversion of the perfusion fluid and mannitol bathing solution through a separate Gilson Minipuls 3 pump operating at a flow rate of 0.5 mL/min to the heart and 1.3 mL/min to the sample tube. Ischemic coronary flow was monitored using the rate of accumulation of effluent as described above. The ischemic period continued for 32 minutes, during which eight saturated spectra were acquired. Ischemic contracture was observed as a sigmoidal increase in end-diastolic pressure, the onset and extent of which was recorded. Reperfusion, achieved in all hearts by restoring flow of the glucose-containing Krebs-Henseleit buffer at 100 mm Hg constant pressure, continued for 32 minutes, and eight saturated spectra were acquired. Coronary flow and contractile function were measured during acquisition of the final spectrum to determine the extent of functional recovery.

Glycogen Depletion and Elevation Protocols
To deplete glycogen stores, five hearts were perfused with 7.6 nmol/L isoproterenol in glucose-containing Krebs-Henseleit buffer for 23 minutes before low-flow ischemia. Several saturated spectra were acquired during this time to ensure that PCr and ATP levels remained constant. To elevate glycogen levels, another five hearts were perfused with 24 nmol/L insulin in glucose-containing Krebs-Henseleit buffer for 80 minutes before ischemia. One fully relaxed and several saturated spectra were collected during this time. The ischemia/reperfusion protocol was then followed as described above for the -G condition. The low- and high-glycogen conditions are abbreviated to -G-gly and -G+gly, respectively.

Early Reperfusion Protocol
In four hearts, the -G protocol was repeated with reperfusion at 12 minutes of ischemia (-G, early rep). In four other hearts, the -G+gly protocol was repeated, also with reperfusion at 12 minutes of ischemia (-G+gly, early rep).

HOE 694 Protocol
In four hearts, the -G+gly protocol was repeated with 0.1 µmol/L HOE 694 (a Na+-H+ exchange inhibitor, kindly donated by Hoechst Pharmaceuticals) present in the reperfusion buffer (-G+gly+HOE 694).

Determination of Tissue [ATP] and Glycogen Content Before Ischemia
The standard, glycogen-depletion, and glycogen-elevation preischemic protocols were repeated (three hearts in each group). At the end of the preischemic period, the hearts were freeze-clamped and finely ground under liquid nitrogen. Heart tissue samples were then taken, and glycogen content was determined using an alkaline extraction method and spectrophotometric assay.19 Further heart tissue samples were taken and extracted in 5.6% perchloric acid. After neutralization, [ATP] of the extracts was determined using a spectrophotometric assay.20

Determination of Lactate Efflux During Ischemia and Tissue Lactate and Glycogen Content at the End of Ischemia
The preischemic and ischemic stages of the +G, -G, -G-gly, and -G+gly conditions were repeated (three hearts in each group) while coronary effluent was collected at 4-minute intervals. At the end of ischemia, the hearts were freeze-clamped and finely ground. Heart tissue samples were then taken, and glycogen content was determined as described above. Further heart tissue samples were taken and extracted in perchloric acid. The lactate concentrations of the effluent and neutralized tissue perchloric acid samples were determined using a spectrophotometric assay.21

Calculation of the Free Energy of ATP Hydrolysis
Free cytosolic [ADP] was calculated from the creatine kinase reaction as follows:


where CK is creatine kinase, and KCK is an equilibrium constant (1.66x109/mol).22 This allowed calculation of {Delta}GATP by the following equation:


At 37°C and [Mg2+] of 1 mmol/L, the standard free energy ({Delta}G°obs) was taken to be -30.5 kJ/mol. The gas constant (R) was 8.31 J/(K/mol), and the absolute temperature (T) was 310°K. For these calculations, ATP, PCr and Pi, observed by NMR, were considered to be unbound and within the cytosol. [Cr] was calculated from the observed changes in [PCr], assuming that the total creatine concentration ([PCr]+[Cr]) in heart was 28 mmol/L and that the Cr formed during ischemia remained within the cell.23

Expression of Results
Results are expressed as mean±SEM. Significance (P<.05) was determined by ANOVA and a modified t test where appropriate.24


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
Myocardial Glycogen Content
The preischemic and end-ischemic glycogen contents of low-, normal-, and high-glycogen hearts are shown in Table 1Down. By the end of ischemia, +G hearts had the highest glycogen content (P<.001), and in all other hearts, glycogen was almost fully depleted.


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  		Table 1. Myocardial Glycogen Utilization and Lactate Production During Low-Flow Ischemia

Contractile Function
Myocardial functional parameters for the four basic conditions are shown in Table 2Down, and examples of pressure traces are shown in Fig 1Down. During preischemia, there were no significant differences in rate-pressure products between the groups. When glucose was provided throughout low-flow ischemia (+G), no contracture was observed. However, in the absence of glucose during ischemia, contracture did occur. The onset of contracture was later, and the maximum contracture was lower, with higher preischemic glycogen content. By the end of reperfusion, recovery of contractile function was significantly greater in the +G hearts than in any hearts without glucose during ischemia (P<.001). Of the hearts without glucose, recovery of contractile function was lower with higher preischemic glycogen content, with the -G+gly hearts showing the lowest functional recovery and the highest end-diastolic pressure during reperfusion (P<.001). Coronary flow was not significantly decreased after low-flow ischemia in any hearts; therefore, the "no-reflow" phenomenon was not responsible for any reduction in functional recovery. In summary, in the presence of glucose during low-flow ischemia, hearts did not undergo contracture and remained essentially undamaged. In the absence of glucose during low-flow ischemia, high glycogen delayed the onset of ischemic contracture but had a detrimental effect on the recovery of contractile function during reperfusion.


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  		Table 2. Myocardial Function, Ischemic Contracture, and Coronary Flow Rates During the Various Protocols



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  		Figure 1. Myocardial contractile function during low-flow ischemia. Hearts contained low, normal, or high glycogen before ischemia and were perfused during ischemia with Krebs-Henseleit buffer with or without glucose. Hearts perfused without glucose showed a sigmoidal increase in end-diastolic pressure known as ischemic contracture, the onset of which occurred later with increasing preischemic glycogen content.

Phosphate Metabolite Concentrations and the Free Energy of ATP Hydrolysis
The preischemic intracellular volumes and phosphorus metabolite concentrations calculated from the fully relaxed 31P NMR spectra are given in Table 3Down. The estimated [ATP]s were the same as obtained in hearts freeze-clamped, extracted, and assayed using traditional methods.


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  		Table 3. Myocardial Phosphorus Metabolite Concentrations and Intracellular Volumes Before Ischemia

Changes in myocardial ATP levels during low-flow ischemia and reperfusion are shown in Fig 2Down, top left. During low-flow ischemia in the -G-gly, -G, and -G+gly hearts, loss of ATP occurred in three phases: a slow decrease until 4, 4, and 12 minutes, respectively, followed by a rapid decrease and then a plateau at a minimum of {approx}2.5 mmol/L, which started at 12, 15, and 24 minutes, respectively. The +G hearts showed a single slow phase of ATP depletion, and by the end of ischemia, ATP was higher than in the other three groups of hearts (P<.001). ATP levels were lower in the -G-gly than the -G hearts (P<.05) and in the -G than the -G+gly hearts (P<.01) until 18 minutes of ischemia. By the end of ischemia, there was no significant difference in ATP levels between the -G-gly, -G, and -G+gly hearts. During reperfusion, ATP levels increased in all hearts. By the end of reperfusion, ATP levels were highest in the +G hearts (P<.01) and lowest in the -G+gly hearts (P<.05). There was no significant difference in ATP levels between the -G-gly and -G hearts at this time.




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  		Figure 2. The effects of glucose or different levels of endogenous glycogen as ischemic substrates on myocardial intracellular concentrations of ATP (top left), PCr (top right), and Pi (bottom left) during low-flow ischemia and reperfusion. Points are mean±SEM (n=5). Hearts contained low (-gly), normal, or high (+gly) glycogen before ischemia. Hearts were perfused during ischemia with Krebs-Henseleit buffer with or without glucose (+G or -G) and during reperfusion with Krebs-Henseleit buffer containing glucose.

PCr decreased rapidly in all hearts at the onset of low-flow ischemia, with the majority of the ischemic decrease being observed in the first 2 minutes (Fig 2Up, top right). By the end of ischemia, the PCr level was highest in the +G hearts (P<.001), whereas the -G-gly, -G, and -G+gly hearts were not significantly different from each other. During reperfusion, PCr concentrations increased in all hearts. By the end of reperfusion, the PCr level was higher in the +G hearts than the -G and -G+gly hearts (P<.05). At this time, the -G-gly and -G hearts were not significantly different, and the -G+gly hearts had the lowest PCr level (P<.05).

Intracellular Pi increased at a fast rate in all hearts at the onset of ischemia (Fig 2Up, bottom left), presumably because of the rapid hydrolysis of PCr during the first 2 minutes of ischemia (Fig 2Up, top right). After 2 minutes, the +G hearts showed a slow rise in Pi and had the lowest Pi by the end of ischemia (P<.001). After 2 minutes, the Pi levels reached a plateau in the -G+gly hearts until 12 minutes, when a rapid increase occurred, followed by another plateau at 20 minutes, resembling the triphasic changes in ATP depletion over a similar time scale (Fig 2Up, top left). Pi increased rapidly in the -G-gly hearts until 12 minutes, followed by a slower increase. By the end of ischemia, all groups of hearts had different Pi levels (P<.01). [Pi] decreased during reperfusion in all hearts. At the end of reperfusion, the Pi level in the +G hearts was lowest (P<.05), whereas there were no significant differences among the other groups of hearts.

Preischemic {Delta}GATP was calculated to be -56.4±0.1 kJ/mol in low- and normal-glycogen hearts and -59.6±0.2 kJ/mol in high-glycogen hearts. {Delta}GATP remained most negative (P<.001) throughout low-flow ischemia in the +G hearts (Fig 3Down). The -G-gly, -G, and -G+gly hearts differed from each other by the end of ischemia (P<.05). Reperfusion recovery of {Delta}GATP was highest in the +G hearts (P<.001) and lowest in the -G+gly hearts (P<.01). There was no significant difference in {Delta}GATP values at the end of reperfusion between the -G-gly and -G hearts.



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  		Figure 3. The effects of glucose or different levels of endogenous glycogen as ischemic substrates on myocardial free energy of ATP hydrolysis during low-flow ischemia and reperfusion. Points are mean±SEM (n=5). For experimental conditions, see the legend to Fig 2Up.

pHi
pHi was 7.12±0.02 during the preischemic period in all hearts and decreased during ischemia. In the -G+gly hearts, pHi decreased rapidly until 4 minutes, followed by a plateau until 14 minutes and a further decline until 22 minutes, after which the pH remained unchanged (Fig 4Down). In the +G, -G-gly, and -G hearts, pH decreased until 6, 6, and 10 minutes, respectively. By the end of ischemia, pHi differed between the -G-gly, -G, and -G+gly hearts (P<.001). There were no significant pH differences between +G and -G+gly hearts at the end of ischemia. pHi returned to preischemic levels in all hearts during the first 6 minutes of reperfusion (Fig 4Down).



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  		Figure 4. The effects of glucose or different levels of endogenous glycogen as ischemic substrates on myocardial pHi during low-flow ischemia and reperfusion. Points are mean±SEM (n=5). For experimental conditions, see the legend to Fig 2Up.

Myocardial Lactate Production During Ischemia
Lactate efflux, 0.14±0.02 µmol·min-1·g wet wt-1 during the preischemic period, increased in all hearts during the first 2 minutes of ischemia (Fig 5Down). In the hearts without glucose, lactate efflux later decreased. The decrease coincided with the onset of contracture, which, in these hearts, occurred at 5.0±0.2, 6.5±1.3, and 20±0.4 minutes in the -G-gly, -G, and -G+gly conditions, respectively. In the +G hearts, lactate efflux reached {approx}2.7 µmol·min-1·g wet wt-1 by 6 minutes and remained at this level throughout the rest of ischemia; no contracture was observed. Total myocardial lactate production during ischemia (total lactate efflux plus end-ischemic tissue lactate content) was the highest in +G hearts (P<.001). Total lactate production differed among the -G-gly, -G, and -G+gly hearts (P<.001) and was approximately twice the amount of glycosyl units used (Table 1Up).



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  		Figure 5. The effects of glucose or different levels of endogenous glycogen as ischemic substrates on myocardial lactate efflux during low-flow ischemia. Points are mean±SEM (n=3). For experimental conditions, see the legend to Fig 2Up. Vertical parallel lines indicate time of onset of contracture.

Early Reperfusion Experiments
Reperfusion of -G+gly hearts after 12 minutes, instead of 32 minutes, of low-flow ischemia resulted in a higher functional recovery by the end of reperfusion (P<.001, Table 2Up). This higher recovery was not significantly different from that of +G hearts after 32 minutes of ischemia. The higher recovery in -G+gly, early rep hearts was not due simply to the shorter duration of ischemia, because reperfusion of -G hearts after 12 minutes of ischemia did not improve functional recovery. In fact, recovery of -G, early rephearts was lower than in -G hearts. This lower recovery may be related to the fact that the hearts were reperfused during contracture, but the parameters measured in our experiments do not provide sufficient evidence to propose a mechanism.

The ATP values and pHi were not significantly different at 12 minutes of low-flow ischemia between the -G+gly hearts and the -G+gly, early rephearts (Figs 6Down and 7Down). By reperfusing early, ischemic contracture and the rapid phases of both the pHi and ATP decrease were avoided in the -G+gly, early rep hearts, and by the end of reperfusion, [ATP] was higher than in the -G+gly hearts (P<.001). PCr and {Delta}GATP values were higher in the -G+gly, early rep hearts than in the -G+gly hearts (P<.001), reaching 15.3±0.7 mmol/L and -56.5±0.7 kJ/mol, respectively. In contrast, the recoveries of ATP, PCr, and {Delta}GATP were not greater in the -G, early rep hearts than in the -G hearts (not shown). As with functional recovery, there were no significant differences in any measurements at the end of reperfusion in the -G+gly, early rephearts compared with the +G hearts.



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  		Figure 6. The effect of early reperfusion (early rep) on myocardial [ATP]i. Points are mean±SEM (n=4). Hearts contained normal or high glycogen (+gly) before ischemia. Hearts were perfused during ischemia with Krebs-Henseleit buffer without glucose (-G) and during reperfusion with Krebs-Henseleit buffer containing glucose. Double vertical line indicates onset of low-flow ischemia. Single vertical lines indicate onset of reperfusion, which occurred either after 12 minutes (early rep) or 32 minutes of low-flow ischemia.



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  		Figure 7. The effect of early reperfusion (early rep) on myocardial pHi. Points are mean±SEM (n=4). For experimental conditions, see the legend to Fig 6Up. Double vertical line indicates onset of low-flow ischemia. Single vertical lines indicate onset of reperfusion, which occurred either after 12 minutes (early rep) or 32 minutes of low-flow ischemia.

Addition of HOE 694 During Reperfusion
Addition of HOE 694 during reperfusion of -G+gly hearts resulted in a higher functional recovery by the end of reperfusion (Table 2Up). The functional recovery of these -G+gly+HOE 694 hearts was not significantly different from the -G-gly hearts without HOE 694.

The ATP, PCr, Pi, {Delta}GATP, and intracellular pH values (not shown) were not significantly different at the end of low-flow ischemia in the -G+gly+HOE 694 hearts compared with the -G+gly hearts, as expected due to the identical protocols up to this point. On reperfusion, the -G+gly+HOE 694 hearts showed a greater increase in ATP than the -G+gly hearts (P<.01), reaching 6.4±0.8 mmol/L. PCr and {Delta}GATP recovered better during reperfusion in the -G+gly+HOE 694 hearts than the -G+gly hearts (P<.01), reaching 13.0±1.0 mmol/L and -55.5±0.8 kJ/mol, respectively. The -G+gly and -G+gly+HOE 694 hearts had the same pHi throughout the protocol (not shown), with the exception of the first 6 minutes of reperfusion. During this time, the pHi was significantly lower in the -G+gly+HOE 694 hearts at 6.98±0.02 (34 minutes) and 7.03±0.02 (38 minutes, P<.05). As with functional recovery, there were no significant differences in any measurements at the end of reperfusion in the -G+gly+HOE 694 hearts compared with the -G-gly hearts without HOE 694.

Metabolic Correlates of Contracture and Postischemic Recovery of Contractile Function
The various metabolic changes that occurred during ischemia and reperfusion were correlated with onset of ischemic contracture and recovery of contractile function for all 32 hearts. Although accurate correlates of the onset of ischemic contracture were difficult to determine because of the 4-minute temporal resolution required for NMR acquisition, it was noted that contracture occurred only in hearts in which [ATP] fell below 7.0 mmol/L, lactate efflux fell below 1.75 µmol·min-1·g wet wt-1, [Pi] rose above 17.5 mmol/L, and {Delta}GATP became less negative than -51.2 kJ/mol. It was found that no end-ischemic values correlated absolutely with functional recovery on reperfusion. At the end of reperfusion, [ATP], [PCr], and -{Delta}GATP correlated with recovery of function (r>.95). There was no correlation between recovery of function and pHi or [Pi] during ischemia or reperfusion.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
The Mechanism Underlying Ischemic Contracture
Ischemic contracture is thought to be initiated by a decrease in ATP and/or an increase in cytosolic Ca2+.25 26 27 28 29 However, because of the different experimental models or conditions used, the relative contribution of these two possible mediators and the precise time scales and threshold levels required have not been established unequivocally. In the present study, a large decrease in ATP before contracture was not observed, although contracture occurred only in hearts in which [ATP] fell below 7 mmol/L. A more striking finding was that a decrease in lactate efflux, a measure of anaerobic glycolysis, coincided with the onset of contracture, which was consistent with the observations of Owen et al30 and Kingsley et al25 and indicates that ATP production rates may be more important than absolute [ATP] in initiating contracture. Increasing preischemic glycogen content allowed lactate efflux to be maintained for longer and delayed the onset of contracture. Because end-ischemic glycogen levels were negligible in all cases in which glucose was absent during ischemia, the decrease in lactate efflux probably represented the point at which glycogen became fully depleted. When glucose was provided throughout low-flow ischemia, glycogen was not fully depleted, lactate efflux was maintained, and no contracture occurred. Because glycolytic ATP is thought to be used preferentially for ionic homeostasis in the myocardium,11 31 the decrease in glycolytic flux may be linked to an increase in cytosolic Ca2+ and the initiation of contracture.

The onset of ischemic contracture in the hearts without glucose was followed by a rapid decrease in ATP and pHi, reflecting net hydrolysis of ATP in response to contracture, as predicted by Allshire et al28 and observed in the low-flow ischemic rabbit heart by Vanoverschelde et al.32 It has been suggested that this ATP consumption is attributable to either rigor activation of myosin-ATPase28 or Ca2+-induced activation of Ca2+-ATPases.25 27 29 In the present study, the cessation of the fall in ATP and pHi coincided with the contracture maximum, which occurred at ventricular pressures ranging from 40 to 132 mm Hg. [ATP] at this point was 2.5 mmol/L in all hearts that underwent contracture. This observation is more consistent with ATP consumption caused by activation of myosin-ATPase, which concomitantly increases contracture. We speculate that once triggered by decreased glycolytic flux and increased cytosolic Ca2+, contracture continued until all ATP available to the myosin-ATPase had been consumed, with the residual inaccessible ATP being 2.5 mmol/L in the present study. This hypothesis is supported by our observation that in hearts in which the onset of contracture was earlier, [ATP] at onset was greater, and this resulted in higher maximum contracture. A correlation between the amount of ATP loss and the degree of cell shortening has been observed in ischemic isolated myocytes.33

The Effect of High Glycogen on the Ischemic Myocardium
In the absence of glucose during ischemia, hearts with high preischemic glycogen levels had low recoveries of contractile function, ATP, PCr, and {Delta}GATP during reperfusion. At the end of ischemia, ATP and PCr levels were not significantly different between the low-, normal-, and high-glycogen hearts. Pi was lower and {Delta}GATP was more negative in the high-glycogen hearts, but this should have been beneficial, not detrimental, to the ischemic myocardium. End-ischemic tissue lactate was significantly higher in the high-glycogen hearts, but because lactate-induced ischemic injury is mediated by inhibition of glycolysis12 and glycolysis had already ceased in these hearts, this is unlikely to have contributed to myocardial injury. Consequently, it was the lower ischemic pH in these hearts that we further investigated as a potential mediator of the detrimental effect of high glycogen.

The Role of H+
In the ischemic and reperfused myocardium, evidence exists for H+ efflux via the Na+-H+ exchanger, resulting in high [Na+]i that has been related to Ca2+ influx and myocardial damage.5 14 15 34 35 Na+-H+ exchange is particularly important on reperfusion, when rapid washout of H+ from the extracellular space leads to a steep [H+] gradient across the sarcolemma, activation of Na+-H+ exchange, and an influx of Na+.5 36 Na+-H+ exchange inhibitors improve postischemic myocardial function and energetics when provided during reperfusion.37 38 To test the hypothesis that the detrimental effect of high glycogen was due to the lower ischemic pH in these hearts, leading to increased Na+-H+ exchange on reperfusion, a Na+-H+ exchange inhibitor, HOE 694, was supplied throughout reperfusion to a second group of high-glycogen hearts. HOE 694 was chosen because, unlike many other Na+-H+ exchange inhibitors, it is both potent and highly specific.37 During reperfusion in these hearts, contractile function, ATP, PCr, and {Delta}GATP increased to levels not significantly different from those observed in hearts depleted of glycogen before ischemia and reperfused without HOE 694. Consistent with inhibition of Na+-H+ exchange,37 pHi was lower during the first 6 minutes of reperfusion in high-glycogen hearts when HOE 694 was present. Thus, the exacerbation of myocardial injury associated with a high preischemic glycogen content was abolished by inhibition of Na+-H+ exchange during reperfusion. We conclude that in the absence of glucose during ischemia, the lower postischemic recovery of high-glycogen hearts was due to the higher ischemic [H+], leading to increased Na+-H+ exchange on reperfusion.

The Role of Glycolysis and Contracture
Of all the conditions without glucose during low-flow ischemia, the highest functional recovery observed, 56% of preischemic contractile function, was still lower than the 88% achieved when glucose was supplied throughout ischemia. Because a decrease in glycolysis always occurred in the absence, but not in the presence, of glucose during low-flow ischemia, we propose that myocardial damage was exacerbated by lack of glycolysis,30 either acting directly or as a result of the concomitant initiation of ischemic contracture.39 To test this hypothesis, a group of high-glycogen hearts perfused without glucose during low-flow ischemia were reperfused early at 12 minutes, before the expected onset of contracture. To prove that any increase in functional recovery was not due simply to the shorter duration of ischemia, a group of normal-glycogen hearts perfused without glucose during low-flow ischemia was also reperfused after 12 minutes; these hearts had already undergone contracture. In the early reperfused high-glycogen hearts, contractile function, ATP, PCr, and {Delta}GATP increased to levels not significantly different from those observed in hearts supplied with glucose and reperfused at 32 minutes. In contrast, early reperfusion of normal-glycogen hearts did not increase postischemic recovery of contractile function or metabolites. Thus, reperfusion before decreased glycolysis and initiation of contracture increased recovery, implying that these phenomena exacerbate myocardial injury. These results also show that before its depletion during ischemia, endogenous glycogen was as effective as exogenous glucose in preserving myocardial integrity. This observation can be reconciled with the study of Apstein et al,40 who used the low-flow ischemia model, in which glucose, insulin, and K+ treatment during ischemia only decreased injury relative to the control condition if ischemia was sufficiently long to deplete endogenous glycogen. In summary, in conditions in which glycolysis was maintained throughout ischemia, myocardial injury was prevented.

The Mechanism Underlying the Detrimental Effect of High Glycogen
The results of the present study have outlined some of the factors contributing to the low postischemic recovery observed in high-glycogen hearts. When glycolysis was not maintained throughout ischemia, the low pH arising from the initial glycogenolysis led to reperfusion injury via increased Na+-H+ exchange. This H+ effect could be alleviated by inhibition of Na+-H+ exchange, but recovery was still significantly impaired. In contrast, when glycolysis was maintained throughout ischemia, regardless of its source, the concomitant H+ production was tolerated by the myocardium, and postischemic recovery was high. A hypothesis explaining these observations is outlined in Fig 8Down and described as follows: we propose that the decrease in glycolysis, and therefore ATP production, in the high-glycogen hearts prevented maintenance of sufficient Na+,K+-ATPase activity to remove Na+ imported by Na+-H+ exchange. This will cause an increase in [Na+]i, especially at the low pH seen in the high-glycogen hearts, which leads to elevated [Ca2+]i and myocardial damage.5 Narita et al41 demonstrated a correlation between elevated end-diastolic pressure and high cytoplasmic Ca2+. The coincidence of high end-diastolic pressure during reperfusion and low recovery of contractile function in the high-glycogen hearts in the present study, therefore, supports the existence of Ca2+-mediated damage. This proposal is also supported by reports of glycolytic ATP being used preferentially for ionic homeostasis in the myocardium.11 31 Changes in {Delta}GATP observed in the present study suggest a thermodynamic restriction of Na+,K+-ATPase activity in the high-glycogen hearts,42 43 which may be further reduced by direct inhibition due to the increased Pi and ADP.44 45 The hypothesized mechanism is further substantiated by other studies from our laboratory, using an identical experimental model, which showed that Na+,K+-ATPase activity is inhibited in the absence of glucose during low-flow ischemia and that, when Na+,K+-ATPase activity is inhibited by ouabain during low-flow ischemia, glucose fails to prevent ischemic contracture and myocardial injury.46 This latter finding implies that the initiation of ischemic contracture is also mediated by a decrease in glycolytic flux, leading to attenuation of Na+,K+-ATPase activity and a concomitant increase in cytosolic Ca2+. Contracture may exacerbate the injury caused by a decrease in glycolysis.39 The contribution of contracture to ischemic injury may be mediated by mechanical stress or vascular constriction,39 47 although these factors have been demonstrated by Vanoverschelde et al32 to be irrelevant when constant flow is imposed during ischemia, as in the present study. Also, the high postischemic coronary flow observed in these experiments indicated that the reperfusion no-reflow phenomenon did not contribute to myocardial injury. It is more likely that in this experimental model, any exacerbation of injury by contracture is mediated by the concomitant decrease in ATP and pH, the increase in Pi, and changes in {Delta}GATP,32 all of which contribute to inhibition of Na+,K+-ATPase activity.44 45



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  		Figure 8. Summary of the proposed intracellular processes occurring during low-flow ischemia in high-glycogen hearts. After cessation of glycolysis, {Delta}GATP becomes less negative, ATP decreases, and ADP and Pi increase, all leading to inhibition of Na+,K+-ATPase activity. In the absence of Na+,K+-ATPase activity, H+ efflux via Na+-H+ exchange leads to increased [Na+]i and myocardial injury. In contrast, when glycolysis continues throughout ischemia, Na+,K+-ATPase activity is maintained, allowing removal of the Na+ entering via Na+-H+ exchange and preventing increased [Na+]i and myocardial injury. In this way, continued glycolysis throughout ischemia allows tolerance of high [H+].

A Controversy Resolved?
The observations made in these experiments may explain some of the paradoxical findings of previous studies with regard to the effects of high glycogen on the ischemic myocardium. In the experiments in which high glycogen was found to be beneficial, in contrast to our initial findings, glycogen may not have been fully depleted by the end of ischemia, thus maintaining glycolysis and avoiding the detrimental effects of concomitant H+ production. This may occur with shorter durations of ischemia, as in the studies of Goodwin and Taegtmeyer1 and Schneider and Taegtmeyer,3 with slower glycogenolysis, such as during ischemic arrest and/or hypothermia, as in the studies of McElroy et al,4 or with a higher preischemic glycogen content. Indeed, in many of these studies,1 3 4 glycogen was not fully depleted by the end of ischemia. If glycogen was to become fully depleted, removal of H+ by a higher flow may alleviate the detrimental effects of high glycogen. This may explain why increased glycogen levels were found to be protective in the study by Scheuer and Stexoski,48 who used a model of hypoxia with sustained coronary flow. Another contribution may be species-specific differences in myocardial glycogen metabolism; eg, ischemic glycogen depletion was less in rabbits49 than in rats7 during identical protocols.

The detrimental effect of high glycogen observed in these experiments may be relevant to preconditioning, because glycogen depletion by a short ischemic or hypoxic insult protects hearts from subsequent prolonged ischemia.8 The dependence of the effect of high glycogen on experimental conditions may explain why, in other studies, depletion of glycogen did not seem to contribute to the protective effect of preconditioning, as in the studies of Doenst et al,2 who used only a brief period of total ischemia. Interestingly, pretreatment with adenosine, a popular contender for the mediator of preconditioning, delays ischemic glycogen depletion.50

In summary, by observing differences in myocardial energetics and postischemic functional recovery in rat hearts of varying preischemic glycogen content, we have shown that during brief low-flow ischemia when glycogen was not fully depleted and contracture was avoided, high endogenous glycogen allowed maintenance of sufficient glycolysis to outweigh any detrimental effect of increased H+. However, during prolonged ischemia when glycogen became fully depleted, myocardial injury occurred, with the injury being exacerbated by the lower ischemic pH in the high-glycogen hearts, leading to increased Na+-H+ exchange during reperfusion. The contradictory findings of past studies concerning the effect of high glycogen on the ischemic myocardium may thus be due to differences in the extent of glycogen depletion during ischemia.


*    Selected Abbreviations and Acronyms
 
Cr = free creatine
early rep = early reperfusion
{Delta}GATP = free energy of ATP hydrolysis
+G = presence of glucose
-G = absence of glucose
+gly = high glycogen
-gly = low glycogen
MPA = methylphosphonic acid
NMR = nuclear magnetic resonance
PCr = phosphocreatine


*    Acknowledgments
 
This study was funded by the Medical Research Council of Great Britain and the British Heart Foundation. Ms Cross thanks the Medical Research Council of Great Britain for a Research Studentship, and Professor Opie thanks the British Heart Foundation for a Senior Visiting Fellowship and Lincoln College, Oxford, for facilities. Hoechst Pharmaceuticals kindly donated the HOE 694.

Received August 16, 1995; accepted November 8, 1995.


*    References
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*References
 

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