Altered Metabolite Exchange Between Subcellular Compartments in Intact Postischemic Rabbit Hearts
Abstract To examine metabolic regulation in postischemic hearts, we examined oxidative recycling of 13C within the glutamate pool (GLU) of intact rabbit hearts. Isolated hearts oxidized 2.5 mmol/L [2-13C]acetate during normal conditions (n=6) or during reperfusion after 10 minutes of ischemia (n=5). 13C-Nuclear magnetic resonance spectra were acquired every 1 minute. Kinetic analysis of 13C incorporation into GLU provided both tricarboxylic acid (TCA) cycle flux and the interconversion rate (F1) between the TCA cycle intermediate, α-ketoglutarate (α-KG), and the largely cytosolic GLU. The rate-pressure product in postischemic hearts was 46% of normal (P<.05). No difference in substrate utilization occurred between groups, with acetate accounting for 92% of the carbon units entering the TCA cycle at the citrate synthase step. TCA cycle flux in postischemic hearts was normal (normal hearts, 10.7 μmol·min−1·g−1; postischemic hearts, 9.4 μmol·min−1·g−1), whereas F1 was 72% lower at 2.9±0.4 versus 10.2±2.5 μmol·min−1·g−1 (mean±SE) in normal hearts (P<.05). From additional hearts perfused with 2.5 mmol/L [2-13C]acetate plus supplemental 5 mmol/L glucose, any potential differences in endogenous carbohydrate availability were proved not to account for the reduced rate α-KG and GLU exchange, which remained depressed in postischemic hearts. However, specific activities of the transaminase enzyme, catalyzing chemical exchange of α-KG and GLU, were the same, and transaminase flux was 100 μmol·min−1·g−1 in postischemic hearts versus 68 μmol·min−1·g−1 in normal hearts. Normal transaminase activity and the increased flux in postischemic hearts are contrary to the reduced F1. The findings indicate reduced metabolite transport rates across the mitochondrial membranes of stunned myocardium, particularly through the reversible α-KG–malate carrier.
- stunned myocardium
- tricarboxylic acid cycle
- nuclear magnetic resonance spectroscopy
A growing body of literature supports the notion that the intermediary metabolism contributes to the level of functional recovery in the postischemic myocardium.1 2 3 4 5 6 7 8 9 Additionally, although the rates of substrate oxidation in postischemic hearts have been examined previously,10 11 12 13 much less is known regarding the regulation of intermediary metabolism and the coordination of flux rates through different pathways of oxidative metabolism. Of course, postischemic contractile dysfunction in the heart, or myocardial stunning, is known to be a complex process, but the regulatory links between oxidative intermediary metabolism in the mitochondria and the physiochemical state of the cytosol are likely to play an important role in managing the metabolic demands for function in postischemic myocardium. For this reason, we have conducted a study using on-line 13C-NMR spectroscopy of the postischemic myocardium in a dynamic fashion to assess the potential for altered subcellular interactions between the mitochondrial and cytosolic compartments of the intact stunned myocardium. The general implication of the present study is that the pathophysiological state of an organ or intact tissue is translated between subcellular compartments via the exchange of intermediary metabolites. Such exchange, as observed here in the intact heart, may provide a level of metabolic regulation not yet extensively studied.
Previous work from our laboratory on isolated rabbit hearts oxidizing 13C-enriched substrates indicated that the rates of carbon turnover within the glutamate pool were slower in postischemic hearts than in normal hearts.1 14 Because the rate of 13C turnover within glutamate is known to be closely related to the rate of labeled substrate entry and recycling within the oxidative TCA cycle,15 16 17 18 one interpretation of this finding was that the depressed function of the stunned myocardium required less energy production from the oxidation of carbon-based fuels. This notion was consistent with the previous observation of reduced flux across the creatine kinase reaction in the postischemic heart.19 However, in the previous 13C-NMR study, the reduced isotope turnover within the glutamate pool was inconsistent with the relatively normal rates of O2 use by the same postischemic hearts. Subsequently, a study by Weiss et al20 confirmed this reduced rate of 13C turnover within the glutamate pool of postischemic hearts. These investigators used an empirical metabolic model to demonstrate the potential for such reduced 13C recycling to occur if the isotope transfer between the observed glutamate pool and the TCA cycle intermediates was delayed.
Despite the consideration that TCA cycle flux may be normal in postischemic hearts, the reduced interconversion rates between glutamate and the TCA cycle intermediate, α-ketoglutarate, have yet to be directly related to the activity of the GOT enzyme, which catalyzes this reaction. The enzyme is not allosterically regulated and thus does not exist in an active or inactive form. What also remains to be accounted for in this reduced transfer of label between the TCA cycle and glutamate is consideration of the exchange of metabolic intermediates between subcellular compartments.
The TCA cycle enzymes are located in the mitochondrial matrix, whereas >90% of the myocardial glutamate pool, which becomes labeled with 13C, is located in the cytosolic space.17 21 We have already shown that flux through GOT in the normal heart is much too fast, at 20-fold, to account for the observed rates of isotope turnover in glutamate at normal TCA cycle flux rates.17 A more recent study from our laboratory then demonstrated that the rate of 13C incorporation into the glutamate pool of the heart is influenced by the rate of α-ketoglutarate transport from the TCA cycle across the mitochondrial membrane via the malate-aspartate shuttle.22 23 Therefore, the possibility exists for the delayed isotope turnover rates in postischemic hearts to result from altered metabolite transport and not from changes in TCA cycle flux or transaminase activity.
By combining a comprehensive kinetic analysis of dynamic-mode 13C-NMR spectra of intact functioning hearts with characterization of the GOT isozyme activities from the mitochondrial and cytosolic compartments, we have previously demonstrated the influence of metabolite transport across the mitochondrial membrane on the rate of isotope turnover within the NMR-observed glutamate pool of the intact heart.17 22 The aim of the present study was to extend such analysis to the stunned myocardium, including evaluation of the GOT isozymes, to determine the source of reduced carbon turnover, which appears to be characteristic of oxidative metabolism in postischemic hearts.1 14 20 The present study examined both flux through the TCA cycle and the rate of label exchange between α-ketoglutarate and glutamate compared with the measured activity of GOT in the postischemic heart. The findings indicate that the exchange of metabolic intermediates between the mitochondria and cytosol is altered in the postischemic stunned heart. Evidence is provided for a link between the pathophysiological state of the myocardium and the metabolic link between the cytosol and mitochondria via exchange of metabolites.
Materials and Methods
Isolated Heart Preparation
Hearts were excised from Dutch belted rabbits (550 to 700 g) that were heparinized (1000 U) and anesthetized with sodium pentobarbital (100 mg/kg IP injection). Immediately upon excision, the heart was immersed in a solution containing 20 mmol/L KCl and 120 mmol/L NaCl for cardioplegia at 0°C. The aorta was cannulated for retrograde perfusion at 100-cm hydrostatic pressure with a modified Krebs-Henseleit buffer equilibrated with 95% O2/5% CO2 at 37°C. The buffer contained (mmol/L) NaCl 116, KCl 4, CaCl2 1.5, MgSO4 1.2, NaH2PO4 1.2, and NaHCO3 25. During preparation, hearts were perfused with buffer containing 5 mmol/L glucose. This perfusate supply was later changed at the start of each 13C-enrichment protocol to a 450-mL reservoir of Krebs-Henseleit buffer containing 2.5 mmol/L [2-13C]acetate (Isotec Inc) with no glucose. A latex balloon was placed in the left ventricle and connected to a pressure transducer line and physiograph (Gould, Inc). The balloon was inflated with water to create a diastolic pressure of 5 to 10 mm Hg. Left ventricular developed pressure (LVDP) and heart rate (HR) were continually measured and recorded from the intraventricular balloon. RPP (HR×LVDP) was used as an index of mechanical work. O2 content of the perfusion medium and coronary effluent was determined with a blood gas analyzer. During either normal perfusion or reperfusion, MV̇o2 was determined for each heart from the difference between the O2 contents of the perfusate at the aortic cannula and the coronary effluent, as described elsewhere.16 24 The temperature of the hearts in the magnet was continuously maintained at 37°C by warm air flow controlled at the NMR system console, and hearts perfused on the bench were maintained at 37°C by a water jacket.
NMR experiments were performed on both normal and postischemic rabbit hearts. Postischemic hearts were subjected to 10 minutes of zero-flow ischemia at 37°C before reperfusion. At the start of each protocol, hearts were perfused (n=6) or reperfused (n=5) with 2.5 mmol/L unlabeled acetate and no glucose for 10 minutes to ensure metabolic equilibrium.25 At this time, a “background” spectrum of naturally abundant (1.1%) 13C was acquired. The substrate supply was then switched from unlabeled acetate to 2.5 mmol/L [2-13C]acetate, and sequential 13C spectra were then acquired every 1.25 minutes. Normal hearts and postischemic hearts were then perfused with 13C-enriched media for an additional 30 minutes. At the end of each experiment, hearts were rapidly frozen for in vitro NMR analysis and tissue chemistry. Acetate was chosen as a substrate in order to compare the results of these experiments with previously published data on TCA cycle flux measurements with 13C-NMR in stunned hearts. By using similar substrate delivery protocols, the hypothesis could then be tested as to whether changes in the transaminase enzyme, which interconverts α-ketoglutarate and glutamate, were the reason that 13C enrichment is delayed in the postischemic heart, that is oxidizing acetate.
In order to control for the potential for differences in endogenous carbohydrate availability, which was due to lower glycogen content in the postischemic group, additional hearts were perfused with 2.5 mmol/L [2-13C]acetate plus supplemental 5 mmol/L unlabeled glucose (normal hearts, n=4; postischemic hearts, n=7). In these groups, hearts received 2.5 mmol/L unlabeled acetate and 5 mmol/L glucose for 10 minutes to ensure metabolic equilibrium before delivering [2-13C]acetate and unlabeled glucose. Normal and postischemic hearts receiving supplemental glucose were subjected to the identical protocol in the NMR magnet and then freeze-clamped, as described above. To further examine whether the supplemental glucose contributed to the energy metabolism of the hearts perfused or reperfused with 2.5 mmol/L acetate, additional hearts were perfused with the reverse labeling scheme. Hearts were either perfused (n=3) or reperfused (n=4) with unlabeled 2.5 mmol/L acetate plus 5 mmol/L [1-13C]glucose and then freeze-clamped after 40 minutes for in vitro NMR analysis and tissue chemistry.
At the end point of each NMR experiment, all hearts were rapidly frozen within a liquid nitrogen–cooled clamp. Frozen ventricular tissue was used to determine the tissue concentrations of key metabolites for biochemical assays and in vitro NMR spectroscopy, as described below.
Bench-top experiments were also performed on isolated rabbit hearts to determine the activity of the key enzyme, GOT, in normal (n=5) and postischemic (n=3) myocardium. For characterization of GOT kinetics at the appropriate time point corresponding to the introduction of the carbon isotope, hearts were homogenized fresh after either 10 minutes of normal perfusion or 10 minutes of reperfusion.
Additional sets of hearts were frozen at additional time periods for comparison of glutamate levels in postischemic hearts with levels in heart tissue frozen at the end of the protocol performed in the NMR magnet. Four additional groups of hearts were freeze-clamped (1) after perfusion with 5 mmol/L glucose (n=3), (2) immediately after 10 minutes of ischemia with no reperfusion (n=3), (3) at 10 minutes of reperfusion with 2.5 mmol/L acetate (n=3) to correspond to the introduction of 13C-enriched acetate during the NMR experiments, and (4) at 30 minutes of reperfusion with acetate (n=3). These hearts allowed the metabolite content to be measured after the introduction of acetate immediately after ischemia, at the time point corresponding to the introduction of carbon isotope during the NMR protocol, and at a 30-minute time point corresponding to steady state isotope enrichment. Biochemical assays of tissue metabolites were performed on acid extracts of frozen ventricle as described below. These measurements allowed comparison of tissue metabolite levels between the early and late phases of reperfusion to ensure that the key tissue metabolite compartments for the kinetic analysis were not changing over the course of the reperfusion period.
Over the course of perfusion with 13C-enriched substrate, incorporation of label into the glutamate pool was detected by 13C-NMR. The appearance of resonance peaks that correspond to 13C enrichment at specific carbon positions within the glutamate pool have been described in detail by others.14 15 17 Detection of the 13C-NMR signal from glutamate is based on the relatively high concentration of intracellular glutamate that is in constant exchange with the TCA cycle via α-ketoglutarate. The pattern of pre–steady state isotope enrichment of the carbon positions of the TCA cycle intermediates and glutamate is shown in Fig 1⇓. Initial incorporation of label from the oxidation of [2-13C]acetate into the glutamate pool occurs at the 4-carbon position. The recycling of the label places 13C at either the 2- or 3-carbon of glutamate. The labeling at the 2- and 3-carbon of glutamate is of equal probability because of the symmetry of succinate molecule. Since glutamate is largely located in the cytosol, whereas the TCA cycle occurs in the mitochondria, 13C-enriched α-keto-glutarate is transported out of the mitochondria before chemical exchange occurs between α-ketoglutarate and glutamate.21
Dynamic 13C-NMR Spectroscopy
Perfused hearts were placed within a sample tube in a 20-mm broad-band NMR probe (Bruker Instruments), which was situated in a vertical-bore superconducting NMR magnet operating at a field strength of 9.4 T (Bruker Instruments). NMR data were collected using a Bruker 400-MSL NMR spectrometer. Before each experiment, the magnetic field homogeneity was optimized by shimming on the proton signal of water in the sample to a line width of 15 to 30 Hz.
13C-NMR spectra from intact hearts were acquired at 101 MHz with a 45° pulse angle and 2-second recycle time over 30 scans (1.25 minutes). Bilevel broad-band decoupling at 0.5 W (1.8 seconds) and 7.0 W (17 microseconds) was applied to eliminate carbon-proton coupling and induce nuclear Overhauser enhancement without sample heating. The free induction decay was acquired with an 8 kiloword data set. Changes in relative signal intensities due to nuclear Overhauser enhancement or relaxation effect were negligible under these pulsing conditions.26 27 Natural-abundance 13C signal was digitally subtracted, and the raw signal was processed by exponential filtering with a line broadening of 20 Hz to enhance the signal-to-noise ratio before being converted into frequency domain by Fourier transformation. Peak assignments were referenced to the known resonance of the exogenous 13C-enriched substrate (2-carbon of acetate at 24.1 ppm) and the well-documented glutamate signals.15 28 NMR signal intensities were determined for all spectra by curve-fitting each resonance peak with a lorentzian curve and integrating the area under the fitted curve with NMR-dedicated software (NMR1, Tripos Associates, Inc).
In Vitro 13C-NMR Spectroscopy
High-resolution 13C-NMR spectra of tissue extracts reconstituted in 0.5 mL D2O were obtained with a 5-mm 13C probe (Bruker Instruments). In vitro 13C spectra were collected over 3K data points (45° pulse, 1.8-second recycle time) with broad-band proton decoupling. The free induction decay was acquired over 32K data points and zero-filled to 64K to improve spectral resolution. The signal was processed by 2-Hz exponential filtering, followed by Fourier transformation. The multiplet structure of the glutamate carbon signal allowed Fc and the ratio of anaplerotic flux to citrate synthase (y) to be calculated.29 The amount of glutamate labeled at the 4-carbon position was determined by comparing the signal intensity from glutamate 4-carbon resonance peak within each 13C spectrum to a standard 100 mmol/L (1.1 mmol/L 13C natural abundance) solution of glutamate. Total 13C-labeled glutamate at the 4-carbon position was then divided by total tissue concentration of glutamate from enzymatic assay to obtain the fractional enrichment at the 4-carbon position of glutamate.16 17 27
Perchloric acid extracts were obtained from ventricular muscle of hearts as previously described.16 Glutamate, α-ketoglutarate, aspartate, and citrate contents in myocardium were determined by spectrophotometric or fluorometric techniques.30 31 Extracts were then lyophilized and reconstituted in 0.5 mL D2O. High-resolution 13C spectra of reconstituted extract material were obtained.
The exchange of carbon isotope from the TCA cycle to glutamate occurs through a transamination reaction catalyzed by the GOT enzyme. Two isoforms of GOT exist, mitochondrial and cytosolic.32 The Km values for both α-ketoglutarate and aspartate of both GOT isozymes have been determined in a previous study from our laboratory.17 In the present study, the specific activities of the GOT in normal versus postischemic hearts were compared. From the measured tissue concentrations of the substrates for this enzyme, α-ketoglutarate and aspartate, flux through GOT could then be determined for both normal and postischemic hearts. Tissue was harvested at the 10-minute mark of normal perfusion or reperfusion, with the time point corresponding to the introduction of label in the 13C-enrichment protocol.
Isolated rabbit hearts were perfused or reperfused, as described in the protocols above, with MSEE buffer (mmol/L: mannitol 225, sucrose 75, MOPS 5, EDTA 0.1, and EGTA 0.2 at 37°C and pH 7.0). From homogenized tissue, the cytosolic and mitochondrial fractions were separated by differential centrifugation.33 Measurement of protein content per milliliter in the total homogenate and in the supernatant after high-speed centrifugation (14 000g) yielded the percent of supernatant (cytosolic) present in the total rabbit heart homogenate. Measurements of citrate synthase activity34 in the total homogenate and the isolated mitochondrial fraction allowed determination of the percent mitochondrial protein in the total homogenate. The fraction of mitochondrial protein was determined to be 31%. The contamination of the supernatant fraction with mitochondrial matrix enzymes was determined by measuring citrate synthase in the supernatant. This value was used to correct the GOT activity in the supernatant for any contaminating mitochondrial GOT isozyme.
The specific activity of GOT for the synthesis of glutamate and oxaloacetate was measured at 37°C in (mmol/L) KCl 150, MOPS 25, Tris 10, NaCl 5, and NADH 0.15, along with 2 U/mL malate dehydrogenase at pH 7.2. The reaction was executed in the presence of 10 mmol/L aspartate and 10 mmol/L α-ketoglutarate. Enzymatic activity in the forward and reverse directions was determined by the change in absorbance at 340 or 280 nm, respectively. Mitochondrial GOT activity was determined at 37°C and at pH 7.7 to mimic the mitochondrial matrix.35 Aliquots of mitochondria were preincubated with 0.05% Triton X-100 to permeabilize the mitochondria. The activity of citrate synthase was measured at 412 nm in 50 mmol/L Tris-HCl, 0.1 mmol/L 5,5′-dithiobis-(2-nitrobenzoic acid), 0.5 mmol/L acetyl-CoA, and 0.05% Triton X-100, pH 8.0 at 37°C. The reaction was started by the addition of 1 mmol/L oxaloacetate.
Kinetic Model and Data Analysis
Analysis of metabolic activity was based on observations of pre–steady state 13C-NMR spectra of glutamate, reflecting the dynamic labeling pattern described above. A kinetic model was used to investigate 13C labeling within key metabolic compartments, as described elsewhere in greater detail.17 The original model is derived from the simplified metabolic compartment model, which includes key rate-limiting tricarboxylate pools as well as the major contributing amino acid pools, glutamate and aspartate. Analysis was performed at steady state flux through the TCA cycle and with constant metabolite pool sizes. Effects of both the exogenous 13C and endogenous substrate entry into the TCA cycle at the citrate synthase reaction along with unlabeled anaplerosis are accounted for by incorporating data measured from high-resolution 13C-NMR as parameters into the model. This model can be used with or without MV̇o2 as a input parameter to improve the accuracy of the output variables. Previous analysis has demonstrated that only a 10% difference was generated in the VTCA determined from this model when MV̇o2 was not used.
As previously described, the kinetic model is composed of nine differential equations and was derived by the principle of mass conservation among the metabolic compartments or pools to characterize both VTCA and the interconversion rates between metabolite pools.17 18 22 The entire compartment model could be simplified by focusing on the 13C enrichment at the 4-carbon and 2-carbon positions of glutamate and eliminating equations that describe 13C enrichment at the 3-carbon site due to the symmetry of 2- and 3-carbon labeling. The resulting equations that were applied for this analysis are as follows: CIT, αKG, GLU, MAL, OAA, and ASP denote the metabolites citrate, α-ketoglutarate, glutamate, malate, oxaloacetate, and aspartate, respectively. F1 and F2, the fluxes of interconversion between α-ketoglutarate and glutamate and between aspartate and oxaloacetate, respectively, include both transamination and membrane transport. Pool sizes are expressed as μmol/g dry wt; flux values, as μmol·min−1·g dry wt−1.
The detected signals in this model are the 2- and 4-carbon–enrichment sites of glutamate. Acetyl-CoA enrichment (Fc) and the ratio of anaplerosis to citrate synthase (y) were determined from the high-resolution NMR spectrum of tissue extracts. Glutamate, aspartate, α-ketoglutarate, and citrate concentrations were measured by enzymatic assays. Other metabolite concentrations whose values are relatively stable to the changes in VTCA were taken from literature on hearts perfused under similar substrate conditions: malate, 0.60 μmol·g dry wt−1; oxaloacetate, 0.04 μmol·g dry wt−1.36 37 As shown by extensive sensitivity analysis, the model is relatively insensitive to changes in these metabolite pools values taken from the literature.17 18
Since alanine and pyruvate concentrations in all four experimental groups were low, alanine aminotransferase activity was minimal. Therefore, the interconversion rate between α-ketoglutarate and glutamate and between oxaloacetate and aspartate were set equal, ie, F1=F2. As performed in an earlier application of the model, the effect of possible variations or errors in these pool sizes was evaluated by sensitivity analysis with a 3-fold change in pool sizes.17 18 The TCA cycle flux, given as VTCA, and the interconversion rate between TCA cycle intermediates and glutamate, given as F1, were determined by nonlinear least-squares fitting of the model to 13C-enrichment data from NMR spectra using the Levenberg-Marquardt method (MATLAB, The MathWorks Inc) on a computer workstation (Sun Microsystems).
Comparison of intragroup data sets was performed with Student’s paired two-tailed t test. Testing for differences between multiple groups or data sets was performed using ANOVA. Analysis of multiple data sets obtained from the same group over time was performed using repeated-measures ANOVA. Differences in mean values were considered statistically significant at P<.05.
Contractile Function and MV̇o2
RPP values indicated a level of contractile dysfunction in the postischemic group that was consistent with previous observations of postischemic dysfunction in isolated hearts.38 39 40 Fig 2A⇓ displays the values of RPP over the course of perfusion and illustrates the reduced capacity for mechanical function in postischemic hearts oxidizing acetate compared with control hearts oxidizing acetate. Before the 10-minute period of ischemia in the postischemic group, RPP (mean±SD) was 20 389±2490 bpm·mm Hg, a value similar to the RPP value of 19 274±6660 bpm·mm Hg observed before the start of the control perfusion with acetate (initial perfusion with glucose). Also consistent with previous observations,14 MV̇o2 was not statistically different between normal and postischemic hearts (19.4±3.7 μmol·min−1·g dry wt−1 for normal hearts and 17.4±3.6 μmol·min−1·g dry wt−1 for postischemic hearts) despite the large sustained differences in mechanical function between both groups.
Fig 2B⇑ displays the values of RPP over the course of perfusion in hearts receiving both [2-13C]acetate and supplemental glucose (5 mmol/L) to control for differences in carbohydrate availability between normal and postischemic hearts. Again, mean RPP values were consistently depressed in the postischemic group (P<.05). RPP values were not affected by the presence of glucose in this protocol and were similar to those for hearts oxidizing acetate alone, as shown in Fig 2A⇑. MV̇o2 was also not significantly affected by the addition of supplemental glucose (normal hearts, 17.5±0.67 μmol·min−1·g dry wt−1; postischemic hearts, 17.0±1.3 μmol·min−1·g dry wt−1) compared with MV̇o2 in hearts oxidizing acetate alone.
Metabolite Content, In Vitro NMR, and GOT Kinetics
Steady state metabolite contents for all experimental groups are tabulated in Table 1⇓. Of note is the lower level of glutamate in postischemic hearts compared with normal hearts that were oxidizing acetate (P<.05). Fig 3⇓ displays the results of glutamate assays performed on myocardium sampled at various times during the ischemia/reperfusion protocol with acetate alone. As observed in a previous study, glutamate levels did not drop during the brief (10-minute) period of ischemia compared with preischemic perfusion with glucose.7 (All hearts in this study received glucose as sole substrate before either normal perfusion or reperfusion with 13C-enriched acetate.) During reperfusion, the mean glutamate levels (11.1 to 15.2 μmol/g dry wt) were not as elevated as in the normal hearts that were perfused with acetate, which had a mean glutamate content of 29.1 μmol/g dry wt (Table 1⇓). Instead, the relatively low glutamate level that was present in the heart during perfusion with glucose remained unchanged throughout 10 minutes of ischemia and 40 minutes of reperfusion. Therefore, the glutamate pool did not change over time, enabling kinetic analysis of the 13C turnover in glutamate. Reduced glutamate content in postischemic hearts did not result from glutamate loss during ischemia but resulted from a lack of increased glutamate in response to reperfusion with acetate. Postischemic hearts did not respond to acetate by elevating tissue glutamate content, as did the normal hearts in this study (reported as control in Fig 3⇓) and those reported elsewhere for earlier periods of perfusion.17 20 In view of our results, this apparent discrepancy suggests that altered transporter function within the malate-aspartate shuttle has induced a difference balance of carbon mass among the intermediates of oxidative metabolism in postischemic hearts.
In vitro analysis provided data on the fractional enrichments of key metabolites for the model. No significant differences in the fractional 13C enrichment of metabolites occurred between normal and postischemic hearts. From in vitro 13C-NMR spectroscopy of tissue extracts, the end-point enrichment of glutamate in both groups was not different, ranging between 95% and 100%. Fc and the ratio of anaplerotic flux to citrate synthase activity (y) calculated from high resolution 13C-NMR spectra were determined. For normal hearts, values (mean±SD) were Fc=0.92±0.07 and y=9±4%. Values in the postischemic group were similar at Fc=0.92±0.09 and y=10±6%. Values from hearts oxidizing acetate in the presence of supplemental glucose were not significantly different for normal hearts (Fc=0.86±0.07, y=6±2%) and postischemic hearts (Fc=0.87±0.09, y=10±6%), owing to the expected inhibition of glucose oxidation by acetate or short chain fatty acids.21 However, values of Fc are not of significance and y exerts very little influence in determining the flux rates via kinetic analysis of these 13C-NMR data.17 18
Reversing the labeling of the substrate combination to use unlabeled acetate and 13C-enriched glucose demonstrated no differences in glucose metabolism between normal and postischemic hearts. 13C-NMR spectra of extracts of hearts perfused or reperfused with unlabeled acetate and the 13C-enriched glucose generally showed no NMR signal from the metabolites of glucose. This lack of signals indicates that little, if any, glucose metabolism was occurring in the presence of acetate. Thus, irrespective of substrate availability, no differences in substrate competition or utilization occurred between normal and postischemic hearts, whereas metabolic flux rates were different, as shown below.
Specific activity levels of GOT isozymes from both the cytosol and mitochondria of normal and postischemic hearts oxidizing acetate are presented in Table 2⇓. As shown, the specific activity of either isozyme of GOT, which catalyzes the interconversion between α-ketoglutarate and glutamate, was essentially the same in normal as in postischemic myocardium. With the same amount and activity of the GOT enzyme, which is not allosterically regulated, the actual reaction rate was then determined using the measured Vmax of the cytosolic isozyme (Table 2⇓) and previously determined Km values for the cardiac GOT17 in the double-displacement reaction equation.17 From these parameters, the calculated rates for the GOT reaction in the cytosol, where 90% of the glutamate resides, of normal and postischemic hearts were found to be 68 μmol·min−1·g dry wt−1 in normal hearts and 100 μmol·min−1·g dry wt−1 in postischemic hearts. These values are also presented in Fig 6⇓ for comparison with the observed rate of interconversion between α-ketoglutarate and glutamate (F1), which, in contrast to flux through the GOT enzyme, was much lower in the postischemic myocardium. Note that because of differences in the aspartate content (Table 1⇑), the postischemic heart actually demonstrates a faster reaction rate than that of the normal heart. Thus, delayed 13C turnover in the glutamate pool of postischemic hearts is not due to reduced transaminase activity.
13C-NMR Spectroscopy, Isotope Kinetics, and Metabolic Flux
Fig 4⇓ shows representative sequential 13C-NMR spectra acquired from a normal (Fig 4A⇓) and a postischemic (Fig 4B⇓) heart oxidizing [2-13C]acetate. Isotopic enrichment of the glutamate pool was significantly delayed in reaching steady state in postischemic hearts compared with normal hearts. This delay in the 13C enrichment of glutamate in postischemic hearts is evident in the spectra shown in Fig 4⇓. For illustrative purposes, Fig 5⇓ graphically displays the time course of 13C enrichment of glutamate (mean±SD) at 2- and 4-carbons from all acquired spectra of hearts in each group shown along with the least-squares fitting of the kinetic model. Note the closeness of fit, which corresponded to a correlation coefficient of at least .98 between the model and the experimental data from either group. Values of VTCA and F1 were the same for each experimental group when least-squares fitting was performed either to the mean enrichment curves for all hearts, as shown in Fig 5⇓, or on each of the curves from individual hearts to provide statistical mean and standard deviations to allow comparison between the two groups. Output from this model provided measures of TCA cycle flux and the interconversion rate between α-ketoglutarate and glutamate, which are graphically displayed in Fig 6⇓.
As shown in Fig 6⇑, mean VTCA was not significantly different in the postischemic group of hearts compared with normal hearts. However, VTCA in postischemic hearts was not proportional to the large reduction in RPP, as is consistent with previous observations of respiratory rate in stunned myocardium.1 16 38 39 40 The rate of interconversion between α-ketoglutarate and glutamate (F1) was 72% lower in postischemic hearts than in normal hearts, despite the much faster chemical exchange via GOT flux in postischemic hearts, as described above. Therefore, in postischemic hearts, the reduced 13C labeling kinetics of glutamate and the 3-fold-lower rate of interconversion between α-ketoglutarate and glutamate were not due to reduced flux across the GOT reaction (nor VTCA). Therefore, the rate of the cytosolic GOT enzyme was not reduced in postischemic hearts and could not account for the reduced isotope exchange between α-ketoglutarate and glutamate.
The difference in F1 between normal and reperfused hearts was not due to differences in carbohydrate availability due to glycogen depletion during ischemia. Values for VTCA and F1 were not affected by the presence of glucose to control for glycogen depletion in postischemic groups. For comparison with values shown in Fig 6⇑, values from normal hearts perfused with acetate plus glucose were VTCA=8.9±0.8 μmol·min–1·g–1 and F1=9.8±3.4 μmol·min–1·g–1, and values from postischemic hearts reperfused with acetate plus glucose were VTCA=8.3±0.9 μmol·min–1·g–1 and F1=2.1± 1.1 μmol·min–1·g–1. Again, in the second protocol, in which hearts received supplemental glucose to control for glycogen loss during ischemia, F1 was significantly depressed in the postischemic group (P<.05). A significant rate-determining step in carbon isotope turnover between the citric acid cycle intermediates and glutamate has been shown to be the transport of metabolic intermediates between the mitochondria (TCA cycle intermediates) and the cytosolic space (glutamate) via the malate-aspartate shuttle.41 In the presence of normal GOT activity, the lower F1 value reported here for postischemic hearts must result from reduced rates of metabolite transport via this shuttle.
In the present study, concurrent measurements of TCA cycle flux and MV̇o2 were mismatched to the impaired contractile performance of postischemic hearts. TCA cycle flux and O2 belied the reduction in the RPP in postischemic hearts. These metabolic observations reported in the present study are consistent with previous findings from both our own laboratory1 14 and those of other investigators.13 20 More important, these experiments demonstrated that the carbon isotope turnover in the intramyocardial glutamate pool, as detected with 13C-NMR, was significantly reduced and that chemical exchange between metabolite pools alone did not account for this reduction. Indeed, the present study indicates that the specific activity of the cardiac GOT enzyme, which catalyzes this interconversion, is normal in the postischemic heart. Therefore, the reduced turnover of 13C in the glutamate pool must instead be due to other factors that influence the exchange of isotope between glutamate and α-ketoglutarate. A principal factor, recently shown to directly influence 13C turnover within the glutamate of the heart, is the rate of metabolite transport between the TCA cycle in the mitochondria and the glutamate pool in the cytosol.17 22 In documenting normal transaminase activity despite reduced interconversion between mitochondrial and cytosolic metabolite pools in postischemic hearts, the results of the present study indicate altered exchange of metabolic intermediates across the mitochondrial membrane of the stunned myocardium.
A series of studies has led to the present understanding that three principal factors predominate the rate of 13C enrichment of glutamate in the heart. These factors are as follows: (1) TCA cycle flux16 17 20 42 43 ; (2) the rate of the equilibrium reaction between α-ketoglutarate and glutamate, which is catalyzed by the GOT enzyme17 44 ; and (3) physical exchange of metabolic intermediates between the mitochondria, where the TCA cycle enzymes are located, and the cytosol, where >90% of the glutamate pool resides.17 22 42 Each of these factors must then be considered in examining the reduced 13C kinetics observed in the postischemic heart. The present set of experiments was designed to address each factor with the benefit of the understanding that has been gained from previous 13C-NMR investigations.
After an initial report of reduced 13C turnover rates within the glutamate pool of postischemic hearts from our laboratory,14 a subsequent study by Weiss et al20 offered the important finding that the observed changes need not be due to reduced TCA cycle flux alone. However, in the present study, the analysis of GOT clearly shows normal levels of the enzyme activity in the postischemic heart. Thus, the delay in isotope turnover cannot be attributed to impaired enzyme action in catalyzing the chemical interconversion between the NMR-detected glutamate pool and the TCA cycle intermediates. In contrast, a recent study has demonstrated that the 13C-enrichment rates of glutamate are strongly influenced by the activity of the malate-aspartate shuttle,22 a mechanism by which the labeled α-ketoglutarate from the mitochondria gains access to the cytosol for exchange with the bulk of the glutamate pool via the cytosolic GOT.17 21 41 In that study, the rate of 13C turnover within glutamate was accelerated simply by stimulating metabolite transport across the mitochondrial membrane with increases in cytosolic redox state and without changes in TCA cycle flux. In view of this previous result and the present data showing normal GOT activity in postischemic hearts, the pronounced delay in 13C enrichment of glutamate in postischemic hearts reflects neither TCA cycle flux nor flux through GOT. Therefore, the delay in isotope kinetics indicates a reduced rate of metabolite transport across the mitochondrial membrane of postischemic myocytes.
Net forward flux through the malate-aspartate shuttle involves the concerted activity of a unidirectional glutamate-aspartate exchanger and a reversible α-ketoglutarate–malate exchanger, both on the mitochondrial membrane.21 23 However, the exchange of isotope labeled α-ketoglutarate from the mitochondria with the cytosolic glutamate pool does not necessarily require net forward flux through the malate-aspartate shuttle. Instead, of the two transporters that constitute this shuttle, the reversible α-ketoglutarate–malate exchanger need be available only to enable the interconversion of labeled α-ketoglutarate with cytosolic glutamate. Therefore, the transport of metabolites across the mitochondrial membrane may play a significant role in coordinating TCA cycle flux with cytosolic metabolism in the absence of significant activity through the malate-aspartate shuttle.
Experiments with 13C-enriched acetate and supplemental glucose controlled for potential differences in the availability of endogenous carbohydrates due to the depletion of glycogen during ischemia. These experiments demonstrated that any differences in carbohydrate availability for cytosolic NADH production did not account for the differences in transport rates (F1) between normal and postischemic hearts, because supplemental glucose had no effect on F1. Also, negligible glycolytic activity for cytosolic NADH production was in evidence from experiments with [1-13C]glucose plus acetate, which suggests that net forward flux through the entire malate-aspartate shuttle is not likely to have produced the observed changes in metabolite transport. The data suggest very little activity through the malate-aspartate shuttle. In contrast, only the reversible, α-ketoglutarate–malate exchanger is likely to have facilitated the measured exchange of α-ketoglutarate with glutamate. Thus, the efflux of labeled α-ketoglutarate from the mitochondria was delayed in the postischemic hearts, most likely without involvement of the unidirectional glutamate-aspartate exchanger and net transport of reducing equivalents into the mitochondria.
The normal activity of the GOT enzyme that is documented here is not surprising, given that the duration of global ischemia was relatively short and that the myocardium retained viability. Without much cell lysis, significant loss of the transaminase was unlikely to occur. In fact, this was the case upon reperfusion, when the myocardium was harvested at a time that would otherwise have allowed for ample washout of extracellular enzymes that may have leached out of the myocyte. In addition, the GOT enzyme is not allosterically regulated and would not be expected to display altered kinetics in the reperfused myocardium. Rather, the GOT activity is more dependent on the concentration of substrate for the reaction, given that the amount of the enzyme present has not changed, which was the case in the present study. Therefore, the evidence points to normal chemical interconversion rates between α-ketoglutarate and glutamate in postischemic hearts with altered metabolite transport to account for the discrepancy between the observed exchange of label between the two metabolites and calculated flux through the GOT enzyme.
Such altered metabolite exchange is reflected by the shift in the glutamate pool sizes that were observed in postischemic hearts compared with normal hearts. In normal hearts, oxidation of acetate induces relatively high concentrations of glutamate in the myocardium.16 27 The significantly lower glutamate pool in postischemic hearts did have an impact on the net flux of the isotope through the rate-determining metabolite compartments. This finding of lower glutamate content in postischemic hearts is consistent with normal flux through the TCA cycle enzyme, α-ketoglutarate dehydrogenase, and our observation of reduced exchange with the cytosolic glutamate pool. However, if either the substrate affinity (Km) or level of activity of α-ketoglutarate dehydrogenase is increased, as may occur at altered pH or elevated calcium concentration,45 the mitochondrial α-ketoglutarate pool becomes less available for efflux to the cytosol.21 The reduced exchange of metabolites during reperfusion that have been observed in the present study may then result from either changes in the exchange proteins or changes that are intrinsic to the TCA cycle enzymes.
In the present study, the lack of an increase in glutamate content during reperfusion after 10 minutes of ischemia is also consistent with the previous observation of reduced glutamate levels in rat hearts that were reperfused with acetate. This previous study20 in rat hearts was performed with a concentration of acetate that was double the concentration used in the present study. However, in that previous investigation, hearts were also perfused with 5 mmol/L acetate before ischemia, which elevates the level of glutamate well above the level observed in the hearts that were perfused with glucose before ischemia, as performed in this present set of experiments. Additionally, values for glutamate are reported here per gram of dry weight tissue to eliminate the potential confounding effects of increased wet weight that occurs during reperfusion of the isolated heart that is reperfused with crystalloid medium that does not contain colloidal material. Therefore, although glutamate values were observed to be lower in the reperfused hearts that were examined in both the present study and the previous study by Weiss et al,20 the mechanisms for lower glutamate content may also differ because of the distinctions between the two different protocols. Despite the reduced glutamate concentration in postischemic hearts, which would tend to increase the turnover of label, a consistent feature of such studies on postischemic hearts is the significant reduction in 13C kinetics.
In summary, the findings of this investigation again document the respiratory inefficiency of contractile performance in postischemic hearts, as shown by the apparent mismatch between mechanical work and MV̇o2. TCA cycle flux rates were normal in postischemic hearts, confirming this inefficiency at the level of intermediary metabolism. The reduced rates of isotope turnover in the glutamate pools of postischemic hearts have been shown here to occur despite normal activity of the associated transaminase enzyme, GOT, in both mitochondria and cytosol. Reduced efflux of α-ketoglutarate across the mitochondrial membrane is then left to account for the slower exchange of label between the mitochondrial metabolites and cytosolic glutamate in postischemic myocardium. The findings of the present study indicate that the interconversion between α-ketoglutarate and glutamate is reduced in postischemic myocardium, as a result of reduced α-ketoglutarate efflux rates from the mitochondria. The results also indicate that the altered exchange of labeled α-ketoglutarate with cytosolic glutamate is most likely to have been mediated by reduced flux across the reversible α-ketoglutarate–malate exchanger without involvement of the entire malate-aspartate shuttle system. In either case, the altered metabolite exchange between subcellular compartments in the stunned myocardium that is reported in the present study may prove to be a factor in the metabolic inefficiency of postischemic myocardium. As a consequence, the role of metabolite exchange across the mitochondrial membrane in coordinating the metabolic support of physiological function warrants further investigation in normal and diseased tissues.
Selected Abbreviations and Acronyms
|acetyl-CoA||=||acetyl coenzyme A|
|F1, F2||=||fluxes of interconversion|
|Fc||=||fraction of 2-carbon–labeled acetyl-CoA entering TCA cycle|
|MV̇o2||=||myocardial O2 consumption|
|NMR||=||nuclear magnetic resonance|
|VTCA||=||TCA cycle flux|
This study was supported by National Heart, Lung, and Blood Institute grants RO1 HL-49244 and RO1 HL-56178 (Dr Lewandowski) and by Grant-in-Aid 13-522-912 from the American Heart Association, Massachusetts Affiliate, Inc (Dr Lewandowski) and was performed during the tenure of an Established Investigator Award from the American Heart Association to Dr Lewandowski.
This manuscript was sent to Ketty Schwartz, Associate Editor, for review by expert referees, editorial decision, and final disposition.
Presented in part in abstract form at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995.
- Received September 3, 1996.
- Accepted May 6, 1997.
- © 1997 American Heart Association, Inc.
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