This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Weiss, R. G. Articles by Gerstenblith, G. Search for Related Content PubMed PubMed Citation Articles by Weiss, R. G. Articles by Gerstenblith, G.

(Circulation Research. 1996;79:435-446.)
© 1996 American Heart Association, Inc.

Articles

Attenuated Glycogenolysis Reduces Glycolytic Catabolite Accumulation During Ischemia in Preconditioned Rat Hearts

Robert G. Weiss, Cicero P. de Albuquerque, Koenraad Vandegaer, V.P. Chacko, Gary Gerstenblith

the Peter Belfer Laboratory of the Cardiology Division (R.G.W., C.P. de A., K.V., G.G.), Department of Medicine, and the Division of NMR Research (V.P.C.), Department of Radiology, The Johns Hopkins Hospital, Baltimore, Md.

Correspondence to Robert G. Weiss, MD, Carnegie 584, The Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287-6568. E-mail rgweiss@rad.jhu.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Appendix: Evaluation of... References

 
Prior transient episodes of ischemia ("ischemic preconditioning") reduce lactate accumulation and attenuate acidosis during a subsequent prolonged ischemic insult. The mechanisms responsible for attenuated glycolytic catabolite accumulation have not been established but may include earlier exhaustion of glycogen stores, slowed glycogenolysis before complete glycogen depletion, and/or inhibition of glycolysis. Simultaneous repeated measures of myocardial glycogen and the rates of glycolysis, glycogenolysis, glucose utilization, and glycolytic ATP production were obtained during total ischemia by ¹³C nuclear magnetic resonance spectroscopy in control and ischemia-preconditioned isolated rat hearts. Both [¹³C]glycolytic and [¹³C]glycogenolytic rates were significantly lower during total ischemia in preconditioned compared with control hearts (0.77±0.04 versus 1.06±0.06 µmol/min per gram wet weight [P<.01] for glycolysis and 0.15±0.07 versus 0.78±0.12 µmol/min per gram wet weight [P<.001] for glycogenolysis, respectively, at 2.5 minutes of ischemia). Slowed glycolysis was present even during the early minutes of ischemia, when significant amounts of available [¹³C]glycogen were still present. Importantly, the reduction in the rate of glycogenolysis was larger and out of proportion to the reduction in glycolysis and occurred despite an increase in glucose utilization in preconditioned hearts (2.23±0.15 versus 1.5±0.10 µmol/min per gram wet weight at 1.25 minutes, P<.01). During early ischemia, conversion of glycogen phosphorylase to the a or "active" form was less in preconditioned than in control hearts (29.1±2.6% versus 41.2±9.8%, respectively; P<.05). Taken together, these findings demonstrate that ischemic preconditioning significantly depresses glycolytic catabolite accumulation during sustained ischemia not by more severe glycolytic inhibition or exhaustion of glycogen stores but by depressed glycogenolysis from the onset of ischemia.

Key Words: glycogen • glycolysis • carbon-13 nuclear magnetic resonance • lactate • phosphorylase

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Appendix: Evaluation of... References

 
Transient ischemic episodes (ischemic preconditioning) reduce the ultrastructural, metabolic, contractile, and arrhythmic consequences of a subsequent prolonged total ischemic period.^{1 2} The precise mechanisms underlying ischemic preconditioning have not been identified, although several have been proposed, including attenuation of glycolytic catabolite accumulation,^{2 3 4} high-energy phosphate preservation,^{2 5} adenosine receptor stimulation,⁶ changes in transsarcolemmal ionic gradients,⁷ ATP-dependent K⁺ channel activation,⁸ and others. These factors are not mutually exclusive, and it is likely that more than one may contribute to the preconditioning phenomena. It has recently been recognized that ₁-adrenergic stimulation before ischemia mimics preconditioning benefits, whereas blockade reduces protection in both rats⁹ and rabbits.¹⁰ More recent studies suggest that ₁-adrenergic–mediated activation of protein kinase C (PKC) and its translocation are important events in preconditioning in several species.^{9 11} Since several of these proposed mechanisms increase intracellular pH (pH_i),^{12 13} the effects of ischemic preconditioning interventions to attenuate ischemic acidosis and alter glycolysis may be one mechanism by which these signaling pathways contribute to the protection of ischemic myocardium.

Although attenuation of ischemic acidosis in preconditioned hearts is a consistent finding from studies of different species,^{3 4 5 7 14} the mechanism(s) providing for attenuation of glycolytic catabolite accumulation remains unclear. Since increased ischemic buffering capacity is not present in preconditioned hearts,¹⁵ likely explanations include inhibition of a specific reaction of glycolysis, reduced stimulation of glycolysis, reduced glycogen breakdown by prior depletion of glycogen stores, and reduced glycogen breakdown due to modulation of glycogenolysis before exhaustion of glycogen stores. Distinguishing among these is important for understanding the underlying mechanisms and targeting pharmacological interventions to mimic the benefit. After their original description of ischemic preconditioning,¹ Murry et al² measured myocardial lactate levels in separate dogs at different times in ischemia and reported that ischemic preconditioning reduces glycolytic catabolite accumulation. Glycogen levels were not directly measured, but estimates suggested reduced glycolysis in preconditioned hearts during total ischemia, and the authors concluded that this was not due to exhaustion of glycogen stores or to inhibition of glycogenolysis.² More recent studies by Wolfe et al³ indicate that glycogen depletion due to ischemic preconditioning is associated with attenuation of ischemic acidosis and that glycogen repletion parallels the loss of protection from ischemic injury as the time from the preconditioning episodes to the prolonged ischemic insult increases. Such findings are consistent with the hypothesis that ischemic preconditioning episodes deplete glycogen and thereby limit the primary glycolytic substrate during ischemia.

To understand the regulatory mechanisms underlying reduced catabolite accumulation during ischemia in preconditioned hearts, we exploited the unique nondestructive properties of ¹³C nuclear magnetic resonance (NMR) spectroscopy to serially and simultaneously quantify the rates of glycolysis, glycogenolysis, and glucose utilization as well as catabolite accumulation during total ischemia in intact rat hearts. These combined NMR and biochemical studies were designed to test two hypotheses: (1) Reduced catabolite accumulation during prolonged ischemia in preconditioned hearts is due to the reduced availability of glycolytic substrate (eg, glucose and glycogen) rather than the accentuated inhibition of glycolytic reactions. (2) Reduced glycolytic substrate is due to the attenuation of glycogenolysis via the phosphorylase reaction before the exhaustion of glycogen stores rather than to the depletion of available glycogen.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Appendix: Evaluation of... References

 
Glycolytic Rates During Total Ischemia
Glycolysis is typically defined as a metabolic pathway common to glucose and glycogen breakdown that yields pyruvate and, during anaerobic conditions, lactate and alanine.¹⁶ During aerobic conditions, pyruvate is converted to acetyl coenzyme A by pyruvate dehydrogenase before tricarboxylic acid cycle oxidation. That pathway is negligible during total ischemia, when tricarboxylic acid cycle oxidation rapidly stops. If glycogen and glucose stores are ¹³C-enriched before ischemia, then the rate of appearance of ¹³C label in glycolytic end products, corrected for any isotopic dilutional effects, measures the glycolytic rate at various times during total ischemia. The experimental strategy was to perfuse hearts with [1-¹³C]glucose (99% enriched) before ischemia to replace intracellular unlabeled glucose with [1-¹³C]glucose and to accumulate [1-¹³C]glycogen. Prior reports indicate that glycogen metabolism in the heart follows the "last on, first off" hypothesis of glucosyl unit addition/deletion.^{17 18} Therefore, after [1-¹³C]glycogen formation in the above protocol, any subsequent degradation during ischemia will not involve the [¹²C]glucosyl moieties of glycogen until the outer [¹³C]glucosyl units are first depleted. The mean glycolytic rate during total ischemia between sequential NMR acquisitions, n and n+1, can be described by the following relationship:

where Glycolysis_nn+1 is the mean [¹³C]glycolytic rate during continuous NMR acquisitions n to n+1; metabolite_n+1-metabolite_n is the increase in [3-¹³C]metabolite (µmol/g) between the n and n+1 NMR acquisitions for lactate (lac), alanine (ala), and pyruvate (pyr); and time interval_nn+1 is the time (minutes) for each NMR acquisition. Since each glucosyl unit is labeled with a single ¹³C nucleus in the C1 position and its glycolytic metabolism produces a single ¹³C-enriched pyruvate, lactate, or alanine molecule and since changes in these metabolites can be measured in µmol/g, the glycolytic rate in the Equation can be expressed as µmol of glucosyl units/g per minute. This approach assumes that oxidative metabolism is rapidly inhibited by total ischemia and that no pyruvate enters the tricarboxylic acid cycle (see below). During ischemia, this relationship can be empirically simplified by eliminating the pyruvate term, since pyruvate is rapidly converted to lactate and alanine under these conditions and the myocardial concentration of pyruvate is very low relative to that of lactate and alanine and does not increase during ischemia.^{19 20} If significant amounts of unlabeled glycogen are metabolized during late ischemia, after consumption of [1-¹³C]glycogen, the Equation can be modified to account for dilutional effects from [¹²C]glucosyl units. Because this technique allows repeated metabolic measures in the same heart, it has considerable theoretical advantages over conventional biochemical techniques, which rely on detecting differences in lactate and alanine pools among different hearts with variable preischemic glycogen and glucose stores and which are then frozen after different ischemic durations.

The rate of glycogenolysis can be directly calculated during total ischemia by quantifying the changes in the area of the glycogen resonance between sequential ¹³C NMR acquisitions.²¹ This is valid, despite the large size of the glycogen molecule, because several studies,²² including those using direct measures in isolated hearts,²³ demonstrate that [¹³C]glycogen is 100% NMR visible. A similar approach can be used to estimate the rate of glucose utilization during total ischemia if the perfusate is not removed or circulated. It is important to emphasize that the ¹³C peaks at 97 and 93 ppm represent both the and ß forms of glucose in the intracellular and extracellular spaces and in the space surrounding the heart. These peaks also overlap the much smaller peaks of the C1 of the and ß anomers of glucose-6-phosphate (data not shown), which accumulate far less than does lactate during ischemia.¹⁹ Nevertheless, decreases in the [1-¹³C]glucose peaks, especially during early ischemia (before significant amounts of glucose-6-phosphate accumulate), can be quantified and represent myocardial glucose metabolism. In addition, the rate of glycolytic ATP formation can be estimated from such data, since it is known that each glucosyl unit derived from glycogen generates three ATP molecules, each unit from glucose generates two ATP molecules, and net glycogen synthesis consumes 1 ATP molecule per glucosyl molecule.

Isolated Rat Heart Preparation
Nonfasting, retired-breeder, male Wistar rats (body weights, 500 to 700 g) were anesthetized with 100 to 150 mg intraperitoneal pentobarbital, and the hearts were rapidly excised and retrogradely perfused at a constant flow of 20 mL/min via a peristaltic pump with oxygenated solution at 37°C, as previously described.^{24 25 26 27} The perfusate contained (mmol/L) sodium 144, potassium 5, calcium 1.5, bicarbonate 17.5, magnesium 1.2, and chloride 134, along with 5 µg/mL lidocaine. This was equilibrated with a gas mixture of 95% O₂/5% CO₂, resulting in a perfusate pH of 7.4. Hearts were paced at a rate of 240 bpm using a Grass SD-9 stimulator via a KCl wick electrode placed into the right ventricle. A polyvinyl chloride balloon attached to PE-190 tubing was inserted into the left ventricle through the mitral valve and was filled in increments so as to obtain the maximum isovolumic developed pressure, which typically occurred at an end-diastolic pressure of 8 to 12 mm Hg. The balloon solution contained either [6-¹³C]hexanoate or [2-¹³C]acetate, which served as ¹³C NMR standards. The other end of the PE tubing was connected to a Gould P23Db transducer for continuous measurements of left ventricular pressure. [2-¹³C]Acetate (99% enriched) was obtained from Isotec; [1-¹³C]glucose (99% enriched), from Cambridge Isotopes; [6-¹³C]hexanoate (99% enriched), from MSD Isotopes; and other chemicals, from Sigma Chemical Co.

NMR Spectroscopy
The hearts were positioned in a broad-band 20-mm commercial probe of a Bruker MSL 500 wide-bore NMR spectrometer (field strength, 11.75 T) in a fashion similar to that described previously at a lower magnetic field strength.^{24 25 26} Magnetic field homogeneity was optimized while observing the water proton signal using the decoupler coil. Proton-decoupled minimally saturated ³¹P spectra were obtained with a 1.19-second delay between pulses of 50-microsecond duration (45°) using a 2000-point data table, and proton-decoupled ¹³C spectra were collected with a 1.16-second delay between pulses of 45-microsecond duration (45°) using 2000 points and zero-filled to 8000 before Fourier transformation. A temperature of 37°C was maintained within the NMR probe with a thermocouple-controlled air-flow device, which is part of the NMR spectrometer system. A temperature of 37°C was confirmed in the perfusate in the NMR tube surrounding the heart before and during ischemia by measurements with a fiberoptic device (Luxtron Inc) during preliminary experiments. Serial ¹³C NMR spectra were acquired continuously without delay between spectra by writing to a memory buffer at the end of each spectrum. Free induction decays were multiplied by an exponential function, which introduced a line broadening of 15 to 20 Hz. pH_i was measured by the ³¹P chemical shift of the inorganic phosphate peak relative to the creatine phosphate peak before ischemia or by the ¹³C chemical shift of the [3-¹³C]glycerol 3-phosphate resonance.²⁸ Metabolite concentrations were measured by integrating areas under individual peaks by an automated time-domain–fitting algorithm.⁴ A calibration graph for determination of absolute metabolite concentrations was constructed for each heart by obtaining NMR spectra as the intraventricular balloon volume containing the ¹³C standard was varied by a known amount. Integrated peak areas of the ¹³C spectra were determined from difference spectra between the natural abundance and enriched acquisitions. Corrections for partial saturation and nuclear Overhauser enhancement (NOE) allowed quantification of the amounts of ¹³C-containing substances. These were calculated by comparing, in separate hearts, spectra acquired with the described parameters with fully relaxed spectra acquired without NOE. Hearts were weighed in air on a Mettler scale at the beginning of the experiments, and the weight of excised noncardiac tissue was subtracted. Results are expressed as micromoles metabolite per gram wet weight.

Protocols
The perfusate also contained [1-¹³C]glucose (5 mmol/L) with insulin (0.05 U/mL) and was not recirculated. At no time was nonenriched glucose supplied to the hearts studied with ¹³C NMR spectroscopy. All hearts were perfused with this solution for 45 to 60 minutes during magnetic field shimming and acquisition of calibration spectra while [1-¹³C]glycogen accumulated. Baseline metabolic (³¹P and ¹³C) and contractile parameters were obtained after stabilization.

Before the prolonged ischemic episode, eight hearts underwent two cycles of 5 minutes of total no-flow ischemia followed by 5 minutes of reperfusion at the baseline coronary flow rate. We have recently shown in glucose-perfused isolated rat hearts that two such 5-minute ischemic episodes provide an ischemic preconditioning benefit after a subsequent prolonged 30-minute episode of total ischemia, as evidenced by improved contractile recovery (developed pressure, 54±6% versus 8±2% of baseline; end-diastolic pressure, 32±4 versus 74±3 mm Hg; for preconditioned and control hearts, respectively; P<.001 for both) and increased metabolic recovery (creatine phosphate, 90±4% versus 52±4% of initial values for preconditioned and control hearts, respectively; P<.001).⁴ The perfusate surrounding the heart in the NMR tube was evacuated after each brief ischemic period in order to remove residual [3-¹³C]lactate produced at that time and then refilled with perfusate. This takes 90 seconds, and NMR data were then acquired during the last 2.5 minutes of the reperfusion period.

Total global ischemia was induced in these and nine control hearts by turning off the pump and clamping the perfusion lines. The duration of total ischemia varied between 14 and 22 minutes to be certain that [¹³C]glycogen was still detectable in each heart during the glycolytic rate measurements. The perfusate surrounding each heart was not exchanged during ischemia, as in some prior reports,⁴ so that accumulation of diffusible catabolites (lactate and ⁺H) remained within the NMR tube and could be analyzed serially.

Tissue Extracts
Several hearts were frozen by rapid compression between tongs cooled to the temperature of liquid nitrogen. For lactate assay, the frozen tissue was pulverized in a mortar under liquid nitrogen in 18% perchloric acid and centrifuged for 10 minutes at 50 000g. The supernatant was neutralized with 2 mol/L potassium hydroxide and recentrifuged. The fractional ¹³C enrichment of lactate was determined from ¹H NMR spectra after lyophilization and resuspension in ²H₂O (99%). In several other hearts, glucose-6-phosphate was measured by assessing the reduction of NADP in the presence of glucose-6-phosphate dehydrogenase under conditions identical to an ATP assay,²⁹ but with glucose and hexokinase omitted. In hearts assayed for total glycogen, the frozen tissue was placed in 5 mol/L potassium hydroxide and dissolved, and the glycogen was precipitated in 70% ethanol according to the method of Walaas and Walaas.³⁰ Portions of other hearts were frozen in liquid nitrogen and extracted in a glycerol mixture for assay of glycogen phosphorylase activity. The relative portion of phosphorylase in the a or active form was determined by methods detailed before.²¹

Statistics
Data distributions were examined for both scale and normality. They were found to be essentially normally distributed and therefore allowed for parametric statistical analysis. The primary statistical tool was repeated measures ANOVA, which compared results of the two groups over time. A value of P<.05 was considered significant.

Two variables, pH and [¹³C]glycogen content, had initial nonzero values and were therefore examined separately at time 0 with one-way ANOVA. For these variables over time and for other variables, two-way ANOVA was performed over the entire ischemic time interval. For two-way analyses, groupxtime interaction was also examined.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Appendix: Evaluation of... References

 
Preischemic Contractile and Metabolic Function
Initial heart weights were similar in control and preconditioned hearts (1.76±0.09 versus 1.73±0.07 g, respectively; P=NS). During the baseline period before any interventions, the mean initial developed pressure (peak systolic pressure-end-diastolic pressure) was 146±8 mm Hg (mean±SEM), and the end-diastolic pressure was 10±1 mm Hg in the 17 hearts and did not differ significantly between the two groups. Under baseline preischemic conditions, both ³¹P and ¹³C NMR spectra were acquired, and representative examples of ¹³C NMR spectra are shown in Fig 1. These demonstrate relatively high levels of glycogen and low levels of lactate in the ¹³C spectrum under preischemic conditions. The mean ratio of creatine phosphate to ATP, uncorrected for saturation and NOE effects, was 2.70±0.10 for all hearts and did not differ between hearts later randomized to the control and ischemia-preconditioned groups. The mean amount of [1-¹³C]glycogen was 14.7±0.8 µmol/g wet wt and also did not differ between the groups. This value of accumulated [¹³C]glycogen determined by NMR agrees with biochemical estimates of total glycogen accumulated during this perfusion interval, as measured by comparing glycogen levels in six other hearts (12.7±1.4 µmol/g wet wt) frozen immediately after excision and in four other hearts (25.0±2.0 µmol/g wet wt) after 60 minutes of glucose perfusion (net accumulated total glycogen, 25.0±2.0-12.7±1.4=12.3 µmol/g wet wt). [3-¹³C]Lactate was not detected in most hearts before ischemia. Mean pH_i, determined by ³¹P NMR, was 7.17±0.01 and did not differ between the groups. Mean pH_i under preischemic conditions was also determined by ¹³C NMR in the 13 hearts in which the [3-¹³C]glycerol phosphate peak was detectable and was 7.17±0.01. During subsequent ischemia, -glycerol phosphate concentrations increased rapidly, and pH_i could be determined in all hearts by ¹³C NMR spectroscopy. These baseline indices are consistent with those in prior reports of isolated perfused rat hearts^{21 28 31 32} and support the contractile and metabolic integrity of the preparation routinely obtained.

View larger version (17K): [in this window] [in a new window]   Figure 1. Representative 13C NMR spectra acquired from a rat heart under preischemic conditions (bottom panel) and after 13 minutes of total ischemia (top panel). The abbreviations denote [1-13C]glycogen (G), the ß and anomers of [1-13C]glucose (ß and ), [3-13C]glycerol-3-phosphate (GP), [3-13C]lactate (L), and [3-13C]alanine (A). The prominent unmarked peak at 24 ppm is from [2-13C]acetate contained within the intraventricular balloon. During ischemia, there is a decrease in the glycogen and glucose resonances and an increase in the -glycerol phosphate, lactate, and alanine resonances. In addition, the chemical shift position of the GP resonance changes in response to pHi.28

Effects of Ischemia-Preconditioning Episodes
Systolic pressure fell rapidly with the termination of coronary flow in all preconditioned hearts during the two 5-minute episodes of total ischemia. The developed pressures at 5 minutes into each subsequent reperfusion period (92±8 and 96±7 mm Hg, respectively) did not recover to levels observed before the preconditioning ischemia (148±9 mm Hg). Mean end-diastolic pressure, however, was not elevated (10±1 mm Hg) when compared with preischemic values. The incomplete recovery of developed pressure at only 5 minutes of reperfusion has been observed before^{33 34 35} and was due to the short reperfusion interval, since in six separate hearts reperfused for 10 to 20 minutes developed pressure achieved 107±2% of initial values. [1-¹³C]Glycogen decreased modestly during the short ischemic periods, and [3-¹³C]glycerol phosphate, [3-¹³C]alanine, and [3-¹³C]lactate increased. The mean [3-¹³C]lactate concentration and pH_i were 2.8±0.1 µmol/g wet wt and 6.56±0.03, respectively, at the end of the first ischemic episode, and 2.2±0.3 µmol/g wet wt and 6.72±0.04 at the end of the second ischemic episode. ¹³C NMR spectra were not obtained during the earliest 2 minutes of reperfusion while the perfusate around the heart was drained to remove residual [3-¹³C]lactate. However, subsequent acquisitions showed that [1-¹³C]glycogen declined further during the early minutes of reperfusion, as previously demonstrated.²¹ At the end of the ischemic preconditioning protocol and just before prolonged ischemia, [1-¹³C]glycogen had decreased to 6.9±0.7 µmol/g wet wt, while pH_i and [3-¹³C]lactate had returned to preischemic values.

Effects of Prolonged Total Ischemia on Metabolites and Metabolic Rates in Control and Preconditioned Hearts
Total ischemia rapidly abolished measurable systolic pressures in all control and ischemia-preconditioned hearts. Fig 2 presents an expanded portion of the glycogen, glucose, lactate, and alanine regions from representative ¹³C NMR spectra acquired every 2.5 minutes during total ischemia. Immediately after the onset of total ischemia, an increase in the [3-¹³C]lactate as well as a decrease in the [1-¹³C]glucose resonances were observed. Further reductions in the glucose peaks were generally not seen after 3 minutes, but a progressive decrease in the intensity of the [1-¹³C]glycogen peak occurred. The increase in the lactate and alanine peaks from one spectrum to the next indicates the glycolytic rate during that time (see Equation) and demonstrates that the higher glycolytic rates (ie, the largest changes) occur during early ischemia. The rates of glycogenolysis and glucose utilization are likewise reflected in the interval reductions in the glycogen and glucose peaks and demonstrate that glucose utilization is maximal during the first minute of ischemia and that glycogenolysis is the prime substrate source for glycolysis during subsequent ischemia.

View larger version (23K): [in this window] [in a new window]   Figure 2. Expanded portions of the glycogen (G) and glucose resonances (ß and ) (all top panel) and of the lactate (L) and alanine (A) regions (bottom panel) of 13C NMR spectra acquired every 2.5 minutes in the same control heart. The numbers below each spectrum indicate the time in minutes during total ischemia, when the spectrum was acquired with time 0, indicating the preischemic spectra. The increase in peak areas between sequential spectra of the bottom panel reflect the [13C]glycolytic flux occurring during those acquisitions. The progressively smaller differences in lactate and alanine peaks from one acquisition to the next during later ischemia demonstrate the rate at which glycolytic flux slows during total ischemia. The top panel displays the time course of the sources of that glycolytic flux and demonstrates that nearly all glucose utilization occurs during the first minutes of ischemia and that glycogen utilization begins after 1 minute of ischemia.

Mean levels of [1-¹³C]glycogen, [3-¹³C]lactate, and pH_i during ischemia are shown in Fig 3. In control hearts, glycogen levels began to decrease after 2 to 3 minutes of ischemia and fell steadily thereafter. Lactate concentrations increased with the onset of ischemia and continued to increase even after 15 minutes of total ischemia, consistent with persistent ongoing glycolysis. [3-¹³C]Alanine concentrations at the end of ischemia were <5% of [3-¹³C]lactate concentrations. In contrast, [¹³C]glycogen levels were significantly lower at the onset of ischemia in ischemia-preconditioned hearts than in control hearts (P<.01), showed a nonsignificant increase during the first 3 minutes of ischemia, and remained significantly lower, but still detectable, at 21 minutes of ischemia (P<.05). [3-¹³C]Lactate increased throughout ischemia but increased significantly less in preconditioned than in control hearts (P<.001). For example, [3-¹³C]lactate at 14 minutes of ischemia was 9.0±0.4 µmol/g wet wt in control and 6.3±0.3 µmol/g wet wt in preconditioned hearts (n=8 each) and 10.9±0.6 and 7.8±0.4 µmol/g wet wt at 22 minutes of ischemia (n=4 and 6, respectively). The mean pH_i determined by ¹³C NMR was significantly higher during 21 minutes of ischemia in preconditioned hearts than in control hearts (P<.001) and was, for example, 6.13±0.03 and 5.93±0.02 at 14 minutes of ischemia and 5.91±0.02 and 5.77±0.02 at 21 minutes of ischemia in preconditioned and control hearts, respectively. Repeated measures two-way ANOVA demonstrated significant differences in lactate and pH_i between control and preconditioned hearts during the entire ischemic interval (P<.001), and significant differences in both were evident as early as 3.75 minutes of ischemia (P<.02). Thus, pH_i decreased less during ischemia in preconditioned hearts than in control hearts, as previously observed.^{3 4 5 7}

View larger version (17K): [in this window] [in a new window]   Figure 3. Mean levels of [1-13C]glycogen, [3-13C]lactate, and pHi during ischemia in control hearts (open circles, dotted line) and ischemia-preconditioned hearts (filled inverted triangles, solid line). [13C]Glycogen levels are lower and decline more slowly throughout ischemia in ischemia-preconditioned hearts compared with control hearts. During ischemia, ischemia-preconditioned hearts accumulate progressively less lactate and protons than do control hearts.

The rates of glycolysis, glycogenolysis, and glucose utilization were calculated throughout total ischemia in each heart using the methods described above, and the results are shown in Fig 4. In control hearts, the mean [¹³C]glycolytic rate was high (1 to 1.2 µmol/min per gram wet weight) during the earliest 4 minutes of ischemia and decreased thereafter. Although prior reports suggested that glycolysis is nearly completely inhibited during early severe ischemia,^{19 36 37} continued [¹³C]glycolysis of 0.5 µmol/min per gram wet weight was observed through at least 15 minutes of ischemia. Mean rates of [¹³C]glycogenolysis in control hearts increased from undetectable amounts at 1.25 minutes of ischemia to 0.85±0.09 µmol/min per gram wet weight at 5 minutes and decreased slowly thereafter to 0.3 µmol/min per gram wet weight after 15 minutes of ischemia. Glucose utilization occurred only during the first 4 minutes of total ischemia and decreased rapidly to undetectable levels. The effect of ischemic preconditioning was to slow glycolytic rates during both the early and late stages of total ischemia. During the first 2.5 minutes of ischemia, the mean glycolytic rate of preconditioned hearts was significantly lower than that of control hearts (0.77±0.04 versus 1.06±0.06 µmol/min per gram wet weight, P<.05) and remained significantly lower throughout ischemia (P<.05). Preconditioned hearts had significantly higher rates of glucose utilization during early ischemia than did control hearts (P<.05) but much slower rates of glycogenolysis throughout 21 minutes of ischemia (P<.05). In fact, early in ischemia, all preconditioned hearts exhibited some transient glycogen synthesis, followed by a slower rate of glycogen breakdown during ischemia. For example, glycogen was the source of 66±7% and 97±2% of glucosyl units entering glycolysis in control hearts at 2.5 and 5 minutes of ischemia, respectively, whereas glycogen only accounted for 17±6% and 76±12% at the same times in preconditioned hearts. By 7.5 minutes of ischemia, glycogen accounted for the vast majority of glucosyl units entering glycolysis in both groups. The inhibition of glycogenolysis, in absolute terms, during early ischemia was actually greater than the reduction in glycolysis (Fig 4). Since glycogen and glucose compete for the glycolytic pathway, a primary reduction in glycogenolysis would be expected to increase glucose utilization, which was observed. The rate of glycolytic ATP production throughout total ischemia was also significantly lower in preconditioned hearts (P<.05). For example, glycolytic ATP production rates were 3.5±0.4, 2.7±0.3, and 2.3±0.3 µmol/min per gram wet weight at 1.25, 2.5, and 5 minutes of ischemia in control hearts but were only 3.0±0.2, 1.0±0.2, and 0.8±0.2 µmol/min per gram wet weight in preconditioned hearts at the same times. At 21 minutes of ischemia, average glycolytic ATP production rates of 0.6 to 1.0 µmol/min per gram wet weight were still detectable.

View larger version (19K): [in this window] [in a new window]   Figure 4. Rates of [13C]glycolysis, [13C]glycogenolysis, and [13C]glucose utilization during ischemia in control hearts (open circles, dotted line) and ischemia-preconditioned hearts (filled inverted triangles, solid line) expressed as micromoles per minute per gram wet weight. In control hearts, glycolytic rates fall after several minutes of ischemia as glucose utilization rates are declining rapidly and glycogenolytic rates are rising, but persistent glycolysis is readily detectable for at least 20 minutes of total ischemia. Glycolytic rates are significantly lower throughout ischemia in ischemia-preconditioned hearts than in control hearts. Higher glucose utilization is observed at 1.25 minutes in preconditioned hearts, which coincides with brief glycogen synthesis. The rate of glycogenolysis is significantly lower in ischemia-preconditioned than in control hearts throughout ischemia.

The amount of [3-¹³C]lactate accumulated (mean values of 9 and 6.3 µmol/g wet wt at 13 minutes of ischemia) was more than the amount of [¹³C]glycogen degraded (8.6 and 2.9 µmol/g wet wt) during the same interval in both control and preconditioned hearts, respectively. This "excess" [3-¹³C]lactate in control hearts (9-8.6=0.4 µmol/g wet wt) and preconditioned hearts (6.3-2.9=3.4 µmol/g wet wt) was likely derived from [1-¹³C]glucose itself. The amounts of [1-¹³C]glucose used during the same interval, measured from the decrease in the [1-¹³C]glucose peaks, in control and preconditioned hearts (2.9±0.2 and 5.1±0.2 µmol/g wet wt, respectively) were more than sufficient to account for this lactate, with the excess (1.7-2.5 µmol/g wet wt) appearing in [3-¹³C]glycerol phosphate (0.5 µmol/g wet wt), [3-¹³C]alanine (0.7 µmol/g wet wt), [1-¹³C]glucose-6-phosphate, and all of the other glycolytic intermediates. These glucose utilization observations could be further validated if lactate derived from [1-¹³C]glycogen and [1-¹³C]glucose could be measured separately. It was not possible with the original experimental design to differentiate lactate derived from glucose and glycogen, since both sources were labeled with [1-¹³C]glucose. Therefore, additional experiments were performed to lend further support to the original glucose utilization rate measures. Four control hearts were initially perfused with [1-¹³C]glucose in order to accumulate [1-¹³C]glycogen, but 4 minutes before ischemia, all [1-¹³C]glucose was replaced with equimolar [2-¹³C]glucose. This time was sufficient to replace the intracellular glucose pools but insufficient to synthesize measurable [2-¹³C]glycogen (Fig 5, top panel). During subsequent ischemia, glucose utilization was directly observed in the appearance of [2-¹³C]lactate (Fig 5, top panel; 69 ppm) and could be distinguished from [3-¹³C]lactate (Fig 5, top panel; 21 ppm) derived from [1-¹³C]glycogen. The amount of [¹³C]glucose used during this ischemic interval (Fig 5, middle panel on left) was 2.8±0.3 µmol/g wet wt and agreed well with the mean amount calculated in the prior experiments over the same interval (2.9±0.2 µmol/g wet wt). A similar approach was also used in four preconditioned hearts, with the [2-¹³C]glucose given after the second preconditioning episode and for the last 4 minutes before total ischemia. In these preconditioned hearts (Fig 5, middle panel on right), a profound depression of early glycogen degradation (C3) and increased glucose utilization (C2) was observed. Thus, excess lactate production not accounted for by glycogen degradation in preconditioned hearts can be accounted for by glucose utilization. Likewise, the [¹³C]glucose utilization rates (Fig 5, bottom panel) measured in this direct fashion in these hearts agree closely with those measured by the disappearance of the [1-¹³C]glucose peaks in the original control hearts (compare with Fig 4). These data provide additional evidence that the proposed methods of quantifying glucose utilization are accurate, that glucose can provide an important glucosyl source during the first minutes of total ischemia in this model, and, importantly, that glycogenolysis is profoundly depressed while glucose utilization is increased in preconditioned hearts during early ischemia.

View larger version (25K): [in this window] [in a new window]   Figure 5. Representative 13C NMR spectra (top panel), [13C]lactate levels (middle panel, µmol/g wet wt), and [13C]glucose utilization rates (bottom panel, µmol/min per g wet wt) acquired from control and preconditioned (IPC) hearts (n=4, each) with glycogen synthesized from [1-13C]glucose and extracellular and intracellular glucose pools subsequently replaced with [2-13C]glucose before total ischemia. The peaks denote [1-13C]glycogen (GG1), the and ß anomers of [2-13C]glucose (G2ß,), the intraventricular balloon [2-13C]acetate standard (S), [2-13C]lactate (L2), [3-13C]lactate (L3), and [3-13C]alanine (A3). During ischemia (middle panel), [2-13C]lactate (C2; filled inverted triangles, solid line), derived from [2-13C]glucose, appears rapidly and achieves maximal levels within 3 minutes. [3-13C]Lactate (C3; open circles, dotted line), derived from [1-13C]glycogen, appears more gradually but accounts for nearly all of the lactate formed after the first minute or so of ischemia in control hearts. In preconditioned hearts (middle panel, right), depressed glycogen utilization and increased glucose utilization are observed during early ischemia, in agreement with the earlier experiments (Figs 3 and 4). [13C]Glucose utilization rates (bottom panel), derived directly from the appearance of [2-13C]lactate in these experiments, trend higher in preconditioned hearts and also agree well, although with more variability, than similar measures derived from the disappearance of the [1-13C]glucose peaks (open circles, dotted line) described before (see Fig 4 for comparison).

Reduced Ischemic Glycolysis in Preconditioned Hearts
Glucose-6-phosphate levels were measured in separate hearts frozen during early ischemia, when the most significant differences in glycolytic rates between control and preconditioned hearts were observed. Before ischemia, glucose-6-phosphate concentrations were 0.17±0.01 µmol/g wet wt (n=3). At 4 minutes of ischemia, glucose-6-phosphate levels increased to 0.40±0.04 µmol/g wet wt in 10 control hearts, which were significantly higher than those in 10 preconditioned hearts (0.18±0.01 µmol/g wet wt, P<.05). Since glucose-6-phosphate concentrations are increased in the presence of inhibitors of glycolysis but reduced when glycogenolysis is slowed,³⁸ these findings lend further support to the direct ¹³C observation that glycolysis is not more severely inhibited during ischemia in preconditioned, compared with control, hearts but that slowed glycolysis is primarily due to reduced glycogenolysis.

One mechanism that could be proposed to explain the reduction in glycolytic and glycogenolytic rates during early ischemia in preconditioned hearts is that reduced contractile performance after preconditioning episodes reduces the initial metabolic demand and thereby early flux through glycolysis. To address this directly, nine additional control hearts were studied in which the developed pressure was reduced to match that of the preconditioned hearts just before prolonged ischemia by reducing end-diastolic volume by decreasing the left ventricular balloon volume. Glycolytic and glycogenolytic rates in these control hearts are shown in Fig 6A and were similar to those of prior control hearts and higher than those of preconditioned hearts during early ischemia. Thus, the depression in early glycolytic and glycogenolytic rates observed in preconditioned hearts is not due to the modestly reduced contractile demand at the onset of prolonged ischemia.

View larger version (36K): [in this window] [in a new window]   Figure 6. A, [13C]Glycogen levels (µmol/g wet wt), pHi, and rates of [13C]glycolysis and [13C]glycogenolysis (both µmol/min per gram wet wt) from nine nonpreconditioned hearts (open squares, dashed lines) with ventricular pressures lowered before ischemia to match those of preconditioned hearts. Data from preconditioned (filled inverted triangles, solid line) and original control (open circles, dotted line) hearts are presented from Figs 3 and 4 for comparison. Although left ventricular pressures of these nonpreconditioned hearts matched those of preconditioned hearts, there was no attenuation of ischemic acidosis, glycolysis, or glycogenolysis in these nonpreconditioned hearts, as observed with preconditioned hearts. B, [13C]Glycogen levels (µmol/g wet wt), pHi, and rates of [13C]glycolysis and [13C]glycogenolysis (both µmol/min per gram wet wt) from four nonpreconditioned hearts (open squares, dashed lines) with [13C]glycogen accumulation lowered to levels similar to those of preconditioned hearts. Data from preconditioned hearts (filled inverted triangles, solid line) and original control hearts (open circles, dotted line) are presented again from Figs 3 and 4 for comparison. Although [13C]glycogen levels of these nonpreconditioned hearts matched those of preconditioned hearts, there was no attenuation of ischemic acidosis, glycolysis, or glycogenolysis in these nonpreconditioned hearts, as observed with preconditioned hearts.

Another mechanism that could possibly explain the early reduction in glycolytic and glycogenolytic rates in preconditioned hearts is that the preconditioning episodes reduce glycogen levels and that shorter glycogen chains are more slowly degraded because they are more dependent on the debranching enzymes than on phosphorylase for glycogenolysis. In order to address this possibility, additional control hearts were studied in which less [1-¹³C]glycogen was allowed to accumulate before ischemia, by perfusion with [1-¹³C]glucose for only 20 to 30 minutes, so that the total [1-¹³C]glycogen levels in these hearts matched those of prior hearts after the preconditioning interventions. The results of these experiments are shown in Fig 6B. Although [1-¹³C]glycogen levels in these nonpreconditioned hearts are similar to those of the preconditioned group (Fig 6B, upper left panel), the rate of glycogen breakdown during early ischemia was more rapid than in preconditioned hearts and similar to the rate observed in the prior control hearts. In fact, [1-¹³C]glycogen was nearly completely degraded by 7 minutes of ischemia, which prohibited ¹³C NMR metabolic measures during later ischemia. [¹³C]Glycolytic rates and pH_i were similar to those of the prior control hearts. Thus, reduced [¹³C]glycogen levels resulting from the preconditioning intervals do not alone account for the reduced glycolytic and glycogenolytic rates during early ischemia and, in turn, for the reduced ischemic catabolite accumulation in preconditioned hearts.

The primary reduction in glycogenolysis indicates reduced glucosyl flux through glycogen phosphorylase, which regulates glycogenolysis in normal heart muscle.^{39 40} Phosphorylase occurs in several forms, and its activity is controlled by specific factors, including the interconversion between forms.⁴⁰ Under many conditions,³⁹ including ischemia,⁴¹ glycogenolysis typically occurs via the phosphorylated a or "active" form of phosphorylase, which is independent of ATP concentration. Glycogenolysis can occur by the partially or nonphosphorylated (b) forms of the enzyme, but that activity is inhibited by high ATP concentrations^{39 41} present during early ischemia.⁴ To better understand the mechanisms underlying reduced glycogenolysis during early ischemia in preconditioned hearts, additional studies of phosphorylase a activity were performed in 12 hearts frozen after 2 minutes of ischemia, when the maximum differences in [¹³C]glycolysis and [¹³C]glycogenolysis were observed. Although total (a+b) phosphorylase activity did not differ between control and preconditioned hearts, the fraction in the a or active form at 2 minutes of ischemia was significantly lower in preconditioned than in control hearts (29.1±2.6% versus 41.2±9.8%, P<.05) and similar to that in nonischemic control hearts (27.3±8.3%). Conversion of phosphorylase to the a form can occur after ß-adrenergic stimulation via a cAMP-mediated mechanism.^{36 39} Lower ischemic cAMP levels in preconditioned hearts were recently reported⁴² and may contribute to attenuated conversion of phosphorylase to the a form during early ischemia observed here. Thus reduced conversion of glycogen phosphorylase to the a form during early ischemia in preconditioned hearts occurs and likely contributes to the early attenuation of glycogenolysis before exhaustion of glycogen stores. This, in turn, slows glycolysis from the onset of ischemia and thereby contributes to attenuated glycolytic catabolite accumulation in preconditioned hearts.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Appendix: Evaluation of... References

 
The present study demonstrates that reduced catabolite accumulation during early ischemia in preconditioned hearts is due to decreased glycogenolytic rates and, as a consequence, decreased glycolysis. The fact that the reduction in the glycogenolytic rate is greater than the reduction in the glycolytic rate and that glucose utilization is increased despite the lower glycolytic rate in preconditioned hearts during early ischemia demonstrates that reduced glycogenolysis, and not inhibition of glycolysis, is primary. Reduced glycogenolysis is not due to exhaustion of glycogen stores but occurs while available glycogen remains and is likely due to attenuated or delayed conversion of glycogen phosphorylase to the a form in preconditioned hearts.

Ischemic preconditioning dramatically reduces myocardial infarct size^{1 43} and improves contractile, metabolic, and ionic recovery after severe ischemia.^{4 7} Although the responsible mechanisms are not completely understood, several of the purported signaling pathways limit the severity of intracellular acidosis during the prolonged ischemic period.^{12 13} Interventions that alter pH_i at that time also alter the magnitude of preconditioning's benefit.⁴ The primary source of intracellular acidosis is glycolytic ATP utilization, and glycolytic rates are reduced in preconditioned hearts. Glycogen is the chief glycolytic source during severe or total ischemia, and Murry et al² suggested that anaerobic glycolytic and glycogenolytic rates are reduced in parallel in preconditioned hearts. Wolfe et al³ varied the time interval between the preconditioning episodes and the subsequent prolonged ischemic period. A strong correlation was found between the extent of glycogen repletion and loss of preconditioning's benefit, and the authors concluded that depletion of glycogen stores was responsible for attenuation of intracellular acidosis.

The current ¹³C NMR method allows simultaneous and repetitive quantification of both glycogenolytic and glycolytic rates during total ischemia in the same heart. It provides several advantages over biochemical measures of total glycogen or metabolite levels obtained from different hearts during different ischemic times. The latter may not adequately determine the amount of glycogen available, or being used, for glycolytic metabolism, because glycogen chains composed of alpha-1 to -4 linkages may be more "available" than those from branched or alpha-1 to -6 linkages⁴⁴ and because differences between the low levels present after 15 to 20 minutes of ischemia may be difficult to detect in the absence of serial measures in the same heart. The commonly used method of quantifying glycolysis using perfusion with [³H]glucose and assaying the coronary effluent for tritiated water released in the enolase step of glycolysis cannot be applied to the condition of total ischemia, when coronary flow ceases.

The experiments in which utilization of [2-¹³C]glucose could be distinguished from utilization of [1-¹³C]glycogen clearly demonstrate the unique advantages of ¹³C NMR methodology to follow, nondestructively, cardiac metabolism of the only two glycolytic substrates. These demonstrated that although the glycolytic rate is depressed in preconditioned hearts, the contribution of glucose utilization to that rate is actually increased during early ischemia. Increased glucose utilization and metabolism through the glycolytic pathway in preconditioned hearts strongly indicate that the primary reason for decreased catabolite levels is not inhibition of glycolysis, per se, but rather a decrease in the availability of glycolytic substrate derived from glycogen. A recent study indicated that brief episodes of ischemia cause a substantial translocation of the major glucose transporter, GLUT4, to the plasma membrane of rat cardiac myocytes.⁴⁵ This may account for the increase in glucose utilization during early ischemia in preconditioned hearts in this and a prior study.⁴⁶ It is also possible that the accelerated glycogen synthesis from glucose, which occurs during reperfusion following brief ischemic periods, persists and contributes to reduced ischemic glycogenolysis observed in these experiments.

The conclusions of the present study are not dependent on the "last on, first off" hypothesis of glycogen metabolism as the sole mechanism for glucosyl addition/deletion, which has been recently questioned.⁴⁷ Although the ¹³C NMR method does not detect unlabeled glycogen metabolism, the ¹H NMR data and biochemical measures of total glycogen independently demonstrate that unlabeled glycogen utilization is very small under these conditions and is similar in control and preconditioned hearts. The ¹H NMR observations also show that during ischemia in the presence of insulin, the preference of glycogenolysis for newly synthesized glucosyl moieties is predominant, but not absolute. Partial preference for ordered glycogen degradation was also observed during glucagon stimulation,⁴⁸ although to a lesser extent than that observed under these ischemic conditions in the presence of insulin using direct measures in each heart. The recent recognition that glycogen can exist in different forms (macroglycogen and proglycogen)⁴⁹ does not alter these conclusions either. Evidence indicates that all ¹³C NMR glycogen is detected or "visible,"^{22 23} presumably independent of form. Importantly, glycogenolysis data generated from the appearance of separately labeled end products (Fig 5) are independent of glycogen NMR "visibility" and independent of the form of glycogen. These too confirm the significant early depression in glycogenolysis in preconditioned hearts.

Although glucose was the sole exogenous substrate in these experiments, this approach does not prevent the addition of other nonenriched substrates such as lactate, fatty acids, or ketone bodies in future studies. Since the oxidation of nonglucose substrates is rapidly inhibited during total ischemia, it is very unlikely that the observed effects of preconditioning on glycolytic metabolism are dependent on the use of glucose as the sole substrate. In future studies, this NMR approach can be modified to improve the time resolution of the measurements by using [1,6-¹³C]glucose instead of [1-¹³C]glucose or by using ¹H-detect, ¹³C-decouple, or ¹H NMR methods with their higher sensitivity than direct ¹³C NMR spectroscopy. If the current ¹³C NMR methods are combined with simultaneous measurements of ATP by ³¹P NMR, the rates of glycolytic ATP utilization could be determined directly, and issues such as the quantitative relationship between proton generation and glycolytic ATP hydrolysis during ischemia could be experimentally addressed.⁵⁰ The isolated globally ischemic heart model does not allow conventional studies of infarct size reduction; therefore, the postischemic benefits of ischemic preconditioning are typically demonstrated by improved metabolic and contractile recovery in this model.^{7 14} Postischemic metabolic and contractile recovery were not studied in these hearts in order that the destructive measures of unlabeled glycogen use could be obtained at the end of ischemia. Nonetheless, we feel that these findings are relevant to ischemic preconditioning because this method of preconditioning in this model was previously shown to improve metabolic and contractile recovery after prolonged ischemia.⁴ Thus, although a single model of preconditioning was studied, the above observations suggest, but cannot prove, that these findings are not an artifact of the substrate composition or perfusion protocol.

View this table: [in this window] [in a new window]   Table 1. Fractional [13C]Alanine Enrichment of Control and Preconditioned Hearts After 2.5, 12.5, and 22.5 Minutes of Total Ischemia Determined From 1H Nuclear Magnetic Resonance Spectra of Tissue Extracts

In summary, prior ischemic preconditioning significantly slows glycolytic rates during total ischemia, and this occurs with a profound decrease in the rate of glycogenolysis from the onset of ischemia and before exhaustion of available glycogen stores. This occurs despite an increase in glucose utilization and is associated with subsequent attenuation of proton and lactate accumulation. Since the attenuation of glycolytic catabolite accumulation during ischemia in ischemia-preconditioned hearts can contribute to the postischemic contractile and metabolic recovery,⁴ it would be interesting to determine the coupling between proposed triggering events in preconditioning (eg, adenosine receptor stimulation, -adrenergic stimulation, PKC activation, and ATP-dependent K⁺ channel activation) and attenuated ischemic glycogenolysis and catabolite accumulation as well as to reexplore whether manipulation of glycogenolytic rates,^{51 52} with or without glycogen depletion,^{14 53} during ischemia affects ischemic pH and lactate and can improve postischemic recovery in a manner comparable to that of ischemic preconditioning.

	Appendix: Evaluation of Assumptions of the 13C NMR Approach

Top Abstract Introduction Materials and Methods Results Discussion Appendix: Evaluation of... References

 
Tricarboxylic Acid Cycle Inhibition During Total Ischemia
An important assumption of the above method of calculating glycolytic flux is that tricarboxylic acid flux is rapidly inhibited during total ischemia, such that pyruvate is converted to lactate and alanine and not to acetyl-coenzyme A for tricarboxylic acid cycle oxidation. To determine whether tricarboxylic acid cycle inhibition occurs rapidly after total ischemia, other hearts were perfused with [1-¹³C]glucose and unlabeled acetate, which resulted in accumulation of [¹³C]glycogen but no ¹³C enrichment of the tricarboxylic acid cycle–derived intermediates.²⁴ When the buffer was switched to one containing [1-¹³C]glucose without acetate, pyruvate dehydrogenase was no longer inhibited, and [4-¹³C]glutamate rapidly appeared as ¹³C moved from pyruvate through the first span of the Krebs cycle to 2-oxoglutarate and through the transaminase reaction to glutamate.^{24 26} Three minutes after the switch, total ischemia was induced, and the enrichment of the C4 of glutamate was observed with ¹³C NMR spectra acquired every 1.25 minutes. The results from three such hearts are shown in Fig 7 along with data obtained in the absence of ischemia and demonstrate that the ¹³C enrichment of glutamate C4 stops rapidly after the onset of total ischemia. Since total glutamate levels do not change significantly in the first 10 minutes after total ischemia in glucose-perfused rat hearts,²⁰ these data are consistent with rapid inhibition of flux from pyruvate into the tricarboxylic acid cycle after the onset of total ischemia.

View larger version (14K): [in this window] [in a new window]   Figure 7. 13C enrichment of glutamate C4 by the tricarboxylic acid cycle versus time. Hearts were initially perfused with [1-13C]glucose and unlabeled acetate, and at time 0 the acetate was removed. 13C flux into the tricarboxylic acid cycle can be followed by the 13C enrichment of glutamate C4.26 27 The dashed line shows data acquired in the absence of ischemia and demonstrates the rapid monotonic 13C enrichment of glutamate C4 by tricarboxylic acid cycle flux. The other data (filled circles connected by a solid line) are from three hearts subjected to total ischemia 3 to 4 minutes after removal of the unlabeled acetate (onset of ischemia denoted by vertical dotted line). The observation that ischemia rapidly halts further 13C enrichment of glutamate C4 is consistent with rapid inhibition of carbon flux from pyruvate through the tricarboxylic acid cycle by total ischemia in this model.

Quantification of Glycolytic End Products by ¹³C NMR Spectroscopy
The quantification of glycolytic rates during total ischemia by ¹³C NMR spectroscopy also depends on rapid and reliable quantification of the ¹³C-enriched glycolytic end products, alanine and lactate. These ¹³C NMR methods can reliably quantify tricarboxylic acid cycle–derived labeling of glutamate and aspartate in intact hearts.^{26 27} In addition, ¹³C NMR data acquired after [1-¹³C]glucose loading in this model show no detectable formation of [3-¹³C]lactate or [3-¹³C]alanine during total ischemia, when glycolysis is totally inhibited by prior administration of 1.0 mmol/L iodoacetic acid (data not shown). Total lactate levels measured biochemically and by NMR were also compared in extracts from four control hearts not exposed to iodoacetic acid and frozen after 22 minutes of ischemia. The mean concentration of [3-¹³C]lactate determined from ¹³C NMR intact heart spectra was 10.9±0.7 µmol/g wet wt, the fractional ¹³C enrichment of lactate C3 by ¹H NMR spectroscopy of those extracts was 39±1%, and the NMR estimate of total tissue lactate therefore was 10.9/0.39, or 27.5±1.6 µmol/g wet wt. This agrees with the total tissue lactate concentration, 28.5±1.2 µmol/g wet wt, determined fluorometrically in the same extracts. In addition, samples from perfusate obtained at the end of 22 minutes of ischemia from the top of the NMR tube just outside the sensitive volume of the NMR coil showed relatively little lactate (0.38±0.06 µmol/g wet wt, n=3). Therefore, we conclude from all of these findings that these methods provide a valid method for quantifying the accumulation of the principal glycolytic end product, lactate, during total ischemia under these conditions.

Unlabeled Glycogen Utilization
These ¹³C NMR methods do not detect the utilization of unlabeled glycogen or its contribution to glycolysis and glycolytic ATP production. The design of the experiments was such that all experiments were terminated when [1-¹³C]glycogen was still present; thus, the vast majority of glycogen used was labeled rather than unlabeled.^{17 18} To directly evaluate unlabeled glycogen utilization under these conditions, so as to exclude the possibility that reduced ¹³C lactate production and calculated glycolytic rates during prolonged ischemia in preconditioned hearts were accompanied by higher unlabeled glycogen use, ¹H NMR spectroscopy was performed on tissue extracts from four control and six of the original preconditioned hearts frozen after 22 minutes of ischemia as well as in control and preconditioned hearts studied in parallel fashion and frozen after only 2.5 and 12.5 minutes of ischemia (n=4 each, 16 total). The fractional ¹³C enrichments of alanine were comparable in control and ischemia-preconditioned hearts, indicating a similar proportion of unlabeled glycogen utilization between groups and over time (Table). These data also demonstrate that [¹³C]glucosyl moieties were the predominant carbon source for glycolysis (80% to 85%), even at the longest ischemic period studied under these conditions, and that contributions from unlabeled glycogen were relatively small (15% to 20%, at most). The fractional ¹³C enrichments of lactate also did not differ in control and preconditioned hearts (data not shown). Total glycogen levels were measured biochemically in hearts from parallel experiments at the onset of ischemia and after 12.5 minutes of ischemia, the longest time studied in all NMR hearts, and also provide additional evidence that minimal unlabeled glycogen degradation occurred. Net total glycogen breakdown during the first 12.5 minutes of total ischemia was less in preconditioned hearts (11.0±1.2-10.8±2.0=0.2 µmol/g wet wt) than in control hearts (21.4±1.2-17.3±2.5=4.1 µmol/g wet wt). The fact that total glycogen breakdown was similar to, or less than, ¹³C-labeled glycogen breakdown also suggests that minimal unlabeled glycogen degradation occurred, although these biochemical measures were associated with considerable interanimal variability as previously observed.^{14 48}

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-17655-20 and HL-52315-01 and an American Heart Association Grant-in-Aid. The research was performed during the tenure of Dr Weiss as an Established Investigator of the American Heart Association and Scientist for the Samuel J. Katcef Memorial. The authors wish to thank Olive Stebbing for assistance preparing the manuscript, and Dr E. David Mellits and Shirley Quaskey for statistical expertise.

Received April 24, 1995; accepted June 11, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion Appendix: Evaluation of... References

 
1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124-1136.[Abstract/Free Full Text]

2. Murry CE, Richard VJ, Reimer KA, Jennings RB. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ Res. 1990;66:913-931.[Abstract/Free Full Text]

3. Wolfe CL, Sievers RE, Visseren FLJ, Donnelly TJ. Loss of myocardial protection after preconditioning correlates with the time course of glycogen recovery within the preconditioned segment. Circulation. 1993;87:881-892.[Abstract/Free Full Text]

4. de Albuquerque CP, Gerstenblith G, Weiss RG. Importance of metabolic inhibition and cellular pH in mediating preconditioning contractile and metabolic effects in rat hearts. Circ Res. 1994;74:139-150.[Abstract/Free Full Text]

5. Kida M, Fujiwara H, Ishida M, Kawai C, Ohura M, Miura I, Yabuuchi Y. Ischemic preconditioning preserves creatine phosphate and intracellular pH. Circulation. 1991;84:2495-2503.[Abstract/Free Full Text]

6. Liu GS, Thornton J, Van Winkle DM, Stanley AWH, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1-adenosine receptors in rabbit heart. Circulation. 1991;84:350-356.[Abstract/Free Full Text]

7. Steenbergen C, Perlman ME, London RE, Murphy E. Mechanism of preconditioning: ionic alterations. Circ Res. 1993;72:112-125.[Abstract/Free Full Text]

8. Gross GJ, Auchampach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res. 1992;70:223-233.[Abstract/Free Full Text]

9. Speechly-Dick ME, Mocanu MM, Yellon DM. Protein kinase C: its role in ischemic preconditioning in the rat. Circ Res. 1994;75:586-590.[Abstract/Free Full Text]

10. Bankwala A, Hale SL, Kloner RA. -Adrenoceptor stimulation with exogenous norepinephrine or release of endogenous catecholamines mimics ischemic preconditioning. Circulation. 1994;90:1023-1028.[Abstract/Free Full Text]

11. Mitchell MB, Meng X, Ao L, Brown JM, Harken AH, Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res. 1995;76:73-81.[Abstract/Free Full Text]

12. Capogrossi MC, Kachadorian WA, Gambassi G, Spurgeon HA, Lakatta EG. Ca²⁺ dependence of -adrenergic effects on the contractile properties and Ca²⁺ homeostasis of cardiac myocytes. Circ Res. 1991;69:540-550.[Abstract/Free Full Text]

13. Iwakura K, Hori M, Watanabe Y, Kitabatake A, Cragoe EJ, Yoshida H, Kamada T. 1-Adrenoceptor stimulation increases intracellular pH and Ca²⁺ in cardiomyocytes through Na⁺/H⁺ and Na⁺/Ca²⁺ exchange. Eur J Pharmacol. 1990;186:29-40.[Medline] [Order article via Infotrieve]

14. Schaefer S, Carr LJ, Prussel E, Ramasamy R. Effects of glycogen depletion on ischemic injury in isolated rat hearts: insights into preconditioning. Am J Physiol. 1995;268:H935-H944.[Abstract/Free Full Text]

15. de Albuquerque CP, Gerstenblith G, Weiss RG. Myocardial buffering capacity in ischemia preconditioned rat hearts. J Mol Cell Cardiol. 1995;27:777-781.[Medline] [Order article via Infotrieve]

16. Opie LH. Substrate utilization and glycolysis in the heart. Cardiology. 1972;56:2-21.

17. Brainard JR, Hutson JY, Hoekenga DE, Lenhoff R. Ordered synthesis and mobilization of glycogen in the perfused heart. Biochemistry. 1989;28:9766-9772.[Medline] [Order article via Infotrieve]

18. Laughlin MR, Petit WAJ, Dizon JM, Shulman RG, Barrett EJ. NMR measurements of in vivo myocardial glycogen metabolism. J Biol Chem. 1988;263:2285-2291.[Abstract/Free Full Text]

19. Rovetto MJ, Lamberton WF, Neely JR. Mechanisms of glycolytic inhibition in ischemic rat hearts. Circ Res. 1975;37:742-751.[Abstract/Free Full Text]

20. Peuhkurinen KJ, Takala TES, Nuutinen EM, Hassinen IE. Tricarboxylic acid cycle metabolites during ischemia in isolated perfused rat heart. Am J Physiol. 1983;244:H281-H288.[Abstract/Free Full Text]

21. Kalil-Filho R, Gerstenblith G, Hansford RG, Chacko VP, Vandegaer KM, Weiss RG. Regulation of myocardial glycogenolysis during post-ischemic reperfusion. J Mol Cell Cardiol. 1991;23:1467-1479.[Medline] [Order article via Infotrieve]

22. Gruetter R, Prolla TA, Shulman RG. ¹³C NMR visibility of rabbit muscle glycogen in vivo. Magn Reson Med. 1991;20:327-332.[Medline] [Order article via Infotrieve]

23. Garlick PB, Pritchard RD. Absolute quantification and NMR visibility of glycogen in the isolated, perfused rat heart using ¹³C NMR spectroscopy. NMR Biomed. 1993;6:84-88.[Medline] [Order article via Infotrieve]

24. Weiss RG, Chacko VP, Gerstenblith G. Fatty acid regulation of glucose metabolism in the intact beating rat heart assessed by carbon-13 NMR spectroscopy: the critical role of pyruvate dehydrogenase. J Mol Cell Cardiol. 1989;21:469-478.[Medline] [Order article via Infotrieve]

25. Weiss RG, Chacko VP, Glickson JD, Gerstenblith G. Comparative ¹³C and ³¹P NMR assessment of altered metabolism during graded reductions in coronary flow in intact rat hearts. Proc Natl Acad Sci U S A. 1989;86:6426-6430.[Abstract/Free Full Text]

26. Weiss RG, Gloth ST, Kalil-Filho R, Chacko VP, Stern MD, Gerstenblith G. Indexing tricarboxylic acid cycle flux in intact hearts by carbon-13 nuclear magnetic resonance. Circ Res. 1992;70:392-408.[Abstract/Free Full Text]

27. Weiss RG, Kalil-Filho R, Herskowitz A, Chacko VP, Litt M, Stern MD, Gerstenblith G. Tricarboxylic acid cycle activity in postischemic rat hearts. Circulation. 1993;87:270-282.[Abstract/Free Full Text]

28. Chacko VP, Weiss RG. Intracellular pH determination by ¹³C NMR. Am J Physiol. 1993;2640:C755-C760.

29. Jacobus WE, Moreadith RW, Vandegaer KM. Mitochondrial respiratory control: evidence against the regulation of respiration by extramitochondrial phosphorylation potentials or by [ATP]/[ADP] ratios. J Biol Chem. 1982;257:2397-2402.[Abstract/Free Full Text]

30. Walaas O, Walaas E. Effect of epinephrine on rat diaphragm. J Biol Chem. 1950;187:769-776.[Free Full Text]

31. Wexler LF, Lorell BH, Momomura S, Weinberg EO, Ingwall JS, Apstein CS. Enhanced sensitivity to hypoxia-induced diastolic dysfunction in pressure-overload left ventricular hypertrophy in the rat: role of high-energy phosphate depletion. Circ Res. 1988;62,4:766-775.

32. Zimmer SD, Ugurbil K, Michurski SP, Mohanakrishnan P, Ulstad VK, Foker JE, From AHL. Alterations in oxidative function and respiratory regulation in the post-ischemic myocardium. J Biol Chem. 1989;264:12402-12411.[Abstract/Free Full Text]

33. Schjott J, Jynge P, Holten T, Brurok H. Ischaemic episodes of less than 5 minutes produce preconditioning but not stunning in the isolated rat heart. Acta Physiol Scand. 1994;150:281-291.[Medline] [Order article via Infotrieve]

34. Asimakis GK, Inners-McBride K, Conti VR. Attenuation of postischaemic dysfunction by ischaemic preconditioning is not mediated by adenosine in the isolated rat heart. Cardiovasc Res. 1993;27:1522-1530.[Abstract/Free Full Text]

35. Asimakis GK, Inners-McBride K, Medellin G, Conti VR. Ischemic preconditioning attenuates acidosis and postischemic dysfunction in isolated rat heart. Am J Physiol. 1992;263:H887-H894.[Abstract/Free Full Text]

36. Neely JR, Morgan HE. Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Annu Rev Physiol. 1974;36:413-459.[Medline] [Order article via Infotrieve]

37. Neely JR, Whitmer JT, Rovetto MJ. Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts. Circ Res. 1975;37:733-741.[Abstract/Free Full Text]

38. Williamson JR. Glycolytic control mechanisms: inhibition of glycolysis by acetate and pyruvate in the isolated, perfused rat heart. J Biol Chem. 1965;240,6:2308-2321.

39. Morgan HE, Parmeggiani A. Regulation of glycogenolysis in muscle: control of glycogen phosphorylase reaction in isolated perfused heart. J Biol Chem. 1964;239:2435-2439.[Free Full Text]

40. Neely JR, Whitfield CF, Morgan HE. Regulation of glycogenolysis in hearts: effects of pressure development, glucose, and FFA. Am J Physiol. 1970;219:1083-1088.

41. Wollenberger A, Krause EG, Heier G. Stimulation of 3', 5'-cyclic amp formation in dog myocardium following arrest of blood flow. Biochem Biophys Res Commun. 1969;36:664-670.[Medline] [Order article via Infotrieve]

42. Sandhu R, Thomas U, Diaz RJ, Wilson GJ. Effect of ischemic preconditioning of the myocardium on cAMP. Circ Res. 1996;78:137-147.[Abstract/Free Full Text]

43. Li Y, Whittaker P, Kloner RA. The transient nature of the effect of ischemic preconditioning on myocardial infarct size and ventricular arrhythmia. Am Heart J. 1992;123:346-353.[Medline] [Order article via Infotrieve]

44. Bailey IA, Radda GK, Seymour AL, Williams SR. The effects of insulin on myocardial metabolism and acidosis in normoxia and ischaemia. Biochim Biophys Acta. 1982;720:17-27.[Medline] [Order article via Infotrieve]

45. Sun D, Nguyen N, Degrado TR, Schwaiger M, Brosius FC III. Ischemia induces translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane of cardiac myocytes. Circulation. 1994;89:793-798.[Abstract/Free Full Text]

46. Janier MF, Vanoverschelde JJ, Bergmann SR. Ischemic preconditioning stimulates anaerobic glycolysis in the isolated rabbit heart. Am J Physiol. 1994;267:H1353-H1360.[Abstract/Free Full Text]

47. Henning SL, Wambolt RB, Schonekess BO, Lopaschuk GD, Allard MF. Contribution of glycogen to aerobic myocardial glucose utilization. Circulation. 1996;93:1549-1555.[Abstract/Free Full Text]

48. Goodwin GW, Arteaga JR, Taegtmeyer H. Glycogen turnover in the isolated working rat heart. J Biol Chem. 1995;270:9234-9240.[Abstract/Free Full Text]

49. Alonso MD, Lomako J, Lomako WM, Whelan WJ. A new look at the biogenesis of glycogen. FASEB J. 1995;9:1126-1137.[Abstract]

50. Dennis SC, Gevers W, Opie LH. Protons in ischemia: where do they come from; where do they go? J Mol Cell Cardiol. 1991;23:1077-1086.[Medline] [Order article via Infotrieve]

51. Neely JR, Grotyohann LW. Role of glycolytic products in damage to ischemic myocardium: dissociation of adenosine triphosphate levels and recovery of function of reperfused ischemic hearts. Circ Res. 1984;55:816-824.[Abstract/Free Full Text]

52. Lagerstrom CF, Walker WE, Taegtmeyer H. Failure of glycogen depletion to improve left ventricular function of the rabbit heart after hypothermic ischemic arrest. Circ Res. 1988;63:81-86.[Abstract/Free Full Text]

53. Cross HR, Opie LH, Radda GK, Clarke K. Is a high glycogen content beneficial or detrimental to the ischemic rat heart?: a controversy resolved. Circ Res. 1996;78:482-491.[Abstract/Free Full Text]

This article has been cited by other articles:

				J.-H. Yang, L. Yang, Z. Qu, and J. N. Weiss Glycolytic Oscillations in Isolated Rabbit Ventricular Myocytes J. Biol. Chem., December 26, 2008; 283(52): 36321 - 36327. [Abstract] [Full Text] [PDF]

				J. Sun, M. Morgan, R.-F. Shen, C. Steenbergen, and E. Murphy Preconditioning Results in S-Nitrosylation of Proteins Involved in Regulation of Mitochondrial Energetics and Calcium Transport Circ. Res., November 26, 2007; 101(11): 1155 - 1163. [Abstract] [Full Text] [PDF]

				W. R. Tracey, J. L. Treadway, W. P. Magee, J. C. Sutt, R. K. McPherson, C. B. Levy, D. E. Wilder, L. J. Yu, Y. Chen, R. M. Shanker, et al. Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H1177 - H1184. [Abstract] [Full Text] [PDF]

				M. Singh and H. K. Saini Resident Cardiac Mast Cells and Ischemia-Reperfusion Injury Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2003; 8(2): 135 - 148. [Abstract] [PDF]

				C. Weinbrenner, M. Nelles, N. Herzog, L. Sarvary, and R. H Strasser Remote preconditioning by infrarenal occlusion of the aorta protects the heart from infarction: a newly identified non-neuronal but PKC-dependent pathway Cardiovasc Res, August 15, 2002; 55(3): 590 - 601. [Abstract] [Full Text] [PDF]

				R. A. Kloner and R. B. Jennings Consequences of Brief Ischemia: Stunning, Preconditioning, and Their Clinical Implications: Part 2 Circulation, December 18, 2001; 104(25): 3158 - 3167. [Abstract] [Full Text] [PDF]

				R. Schulz, M. V Cohen, M. Behrends, J. M Downey, and G. Heusch Signal transduction of ischemic preconditioning Cardiovasc Res, November 1, 2001; 52(2): 181 - 198. [Full Text] [PDF]

				S. Schneider, W. Chen, J. Hou, C. Steenbergen, and E. Murphy Inhibition of p38 MAPK {alpha}/{beta} reduces ischemic injury and does not block protective effects of preconditioning Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H499 - H508. [Abstract] [Full Text] [PDF]

				H. Tong, W. Chen, C. Steenbergen, and E. Murphy Ischemic Preconditioning Activates Phosphatidylinositol-3-Kinase Upstream of Protein Kinase C Circ. Res., August 18, 2000; 87(4): 309 - 315. [Abstract] [Full Text] [PDF]

				H. Tong, W. Chen, R. E. London, E. Murphy, and C. Steenbergen Preconditioning Enhanced Glucose Uptake Is Mediated by p38 MAP Kinase Not by Phosphatidylinositol 3-Kinase J. Biol. Chem., April 14, 2000; 275(16): 11981 - 11986. [Abstract] [Full Text] [PDF]

				C. E. Ganote and S. C. Armstrong Adenosine and preconditioning in the rat heart Cardiovasc Res, January 1, 2000; 45(1): 134 - 140. [Full Text] [PDF]

				R. de Jonge and J. W. de Jong Ischemic preconditioning and glucose metabolism during low-flow ischemia: Role of the adenosine A1 receptor Cardiovasc Res, September 1, 1999; 43(4): 909 - 918. [Abstract] [Full Text] [PDF]

				C. Depre, J.-L. J. Vanoverschelde, and H. Taegtmeyer Glucose for the Heart Circulation, February 2, 1999; 99(4): 578 - 588. [Full Text] [PDF]

				S. A. Gabel, H. R. Cross, R. E. London, C. Steenbergen, and E. Murphy Decreased intracellular pH is not due to increased H+ extrusion in preconditioned rat hearts Am J Physiol Heart Circ Physiol, November 1, 1997; 273(5): H2257 - H2262. [Abstract] [Full Text] [PDF]

				S. Schaefer and R. Ramasamy Glycogen utilization and ischemic injury in the isolated rat heart Cardiovasc Res, July 1, 1997; 35(1): 90 - 98. [Abstract] [Full Text] [PDF]

				D. Pucar, P. P. Dzeja, P. Bast, N. Juranic, S. Macura, and A. Terzic Cellular Energetics in the Preconditioned State. PROTECTIVE ROLE FOR PHOSPHOTRANSFER REACTIONS CAPTURED BY 18O-ASSISTED 31P NMR J. Biol. Chem., November 21, 2001; 276(48): 44812 - 44819. [Abstract] [Full Text] [PDF]

				R. A. Forbes, C. Steenbergen, and E. Murphy Diazoxide-Induced Cardioprotection Requires Signaling Through a Redox-Sensitive Mechanism Circ. Res., April 27, 2001; 88(8): 802 - 809. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Weiss, R. G. Articles by Gerstenblith, G. Search for Related Content PubMed PubMed Citation Articles by Weiss, R. G. Articles by Gerstenblith, G.