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1Department of Biochemistry and Center for Neurosciences and Cell Biology, Faculty of Sciences and Technology, University of Coimbra, Apartado 3126, 3001-401 Coimbra, Portugal; 2Department of Chemistry, University of Texas at Dallas, Richardson 75083-0688; 3Department of Radiology, Mary Nell and Ralph B. Rogers Magnetic Resonance Center, University of Texas Southwestern Medical Center; and 4Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9085; and Department of Veterans Affairs Medical Center, Dallas, Texas 75216
Submitted 14 October 2003 ; accepted in final form 18 March 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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13C nuclear magnetic resonance; tricarboxylic acid cycle; intermediary metabolism; malate-aspartate shuttle; modeling; extracellular calcium concentration
-ketoglutarate (-KG) and glutamate was rapid compared with tricarboxylic acid flux (VTCA), and hence temporal enrichment of glutamate C4 provided a direct readout of cycle activity. Later kinetic studies of isolated hearts demonstrated that two fluxes, VTCA and exchange flux (VX), are required to adequately describe the rates of 13C enrichment at glutamate C4 and C3 (3, 4, 8, 18, 19, 22–26). VX has been ascribed to an exchange process associated with the malate-aspartate shuttle in addition to the transaminase exchange (15). The malate-aspartate shuttle is highly active in cardiac tissue and thought to be the primary process for transporting reducing equivalents (NADH) from cytosol to mitochondria (16). The shuttle consists of the cytosolic and mitochondrial glutamate-oxaloacetate (OAA) transaminase (GOT) and two transporter proteins in the inner mitochondrial membrane: the reversible -KG-malate exchanger and the unidirectional glutamate-aspartate exchanger (10). It has been shown (15) in isolated mitochondria that increased Ca2+ (6, 9, 20, 21) or H+ (decreased pH) concentration increases the affinity of -KG dehydrogenase for -KG and alters mitochondrial levels of -KG. This in turn has been reported to stimulate aspartate formation due to less inhibition of aspartate aminotransferase by -KG and, consequently, overall stimulation of the malate-aspartate shuttle (21). Thus if VX reflects compartmental exchange of glutamate--KG, this working model would predict that increased Ca2+ levels should not only increase contractility and O2 consumption (and hence VTCA) but increase VX as well. A positive correlation between VTCA and VX was indeed observed recently (3) in mouse hearts perfused with various single substrates, although in this case, the variations in VTCA and VX were due to differences in the oxidation state of the substrates presented to the hearts rather than to changes in extracellular Ca2+ concentration ([Ca2+]o). VX also increased significantly in hearts perfused with butyrate upon addition of lactate (25) purportedly due to increased malate-aspartate shuttle activity. More recently, O'Donnell et al. (15) have shown in isolated rabbit heart mitochondria that lowering the pH results in a decrease in the Km of -KG dehydrogenase for -KG and hence a reduced efflux of -KG from mitochondria (reflecting lower malate-aspartate shuttle activity). This was also reflected by a lower VX (F1) value measured by NMR in intact hearts perfused at pH 6.6. These results were consistent with the hypothesis that direct competition between -KG dehydrogenase (Km, 
0.10 mM at pH 6.8) and the -KG-malate transporter (Km, 1.5 mM) for mitochondrial -KG is responsible for the observed decrease in malate-aspartate shuttle activity. Hence, either increased [Ca2+]o or H+ concentration results in a decrease of the Km of -KG dehydrogenase, yet Ca2+ purportedly stimulates malate-aspartate shuttle activity, whereas H+ moderates it. This suggests that the effects of Ca2+ and H+ differ in mechanistic origin. In the present study, the temporal evolution of 13C isotopomers in malate, aspartate, and glutamate in perfused hearts exposed to two different levels of Ca2+ and with either high-lactate/low-pyruvate or high-pyruvate/low-lactate concentrations to alter cytosolic redox was measured in freeze-clamped tissues by 13C NMR with an aim toward better defining the metabolic factors that contribute to VTCA and VX. A kinetic analysis of the data shows that VTCA is influenced more by changes in [Ca2+]o, whereas VX is more sensitive to changes in cytosolic redox. Interestingly, the VX/VTCA ratio was found to be especially sensitive to these physiological variables and could prove useful as an indicator of tissue physiology.
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MATERIALS AND METHODS |
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The [3-13C]pyruvate and [3-13C]lactate (99% BC enrichment) were purchased from Cambridge Isotope Laboratories (Andover, MA). All other materials were of the highest quality available from commercial sources. Male Sprague-Dawley rats (250–300 g) were obtained from Sasco (Houston, TX).
Heart perfusion.
This study was performed under a protocol approved by the University of Texas Southwestern Animal Care and Use Committee. After administration of general anesthesia, hearts were rapidly excised and immersed in cold perfusate. The aorta was immediately cannulated for retrograde Langendorff perfusion at 100 cm of hydrostatic pressure (14) with a modified Krebs-Henseleit bicarbonate buffer maintained at 37°C that contained (in mM) 119.2 NaCl, 4.7 KCl, 0.5 (or 2.5) CaCl2, 1.2 MgSO4, and 25 NaHCO3. The buffer was saturated with 95% O2-5% CO2 and a pH of 7.4 was maintained. A small latex balloon, which was sutured to one end of a polyethylene (PE) tube and attached at the other end to a pressure transducer, was inserted into the left ventricle through the mitral valve. This balloon enabled the continuous measurement of left ventricular developed pressure (LVDP) and heart rate (HR) throughout the experiment. Myocardial O2 consumption (M
O2) was calculated from the difference in O2 tension (measured with a blood gas analyzer) between the perfusion medium in the arterial supply line and the coronary effluent, which was collected via a cannula placed in the pulmonary artery (13). Coronary flow was measured using a stopwatch and a graduated cylinder.
Hearts were initially perfused with substrates that contained natural-abundance levels of 13C to ensure a metabolic steady state (constant pool sizes) before switching to perfusates that contained 13C-enriched substrates at the same concentrations. Four experimental perfusate conditions were chosen: 4 mM [3-13C]pyruvate, 0.4 mM [3-13C]lactate, plus either 0.5 or 2.5 mM Ca2+ (groups 1 and 2) and 0.4 mM [3-13C]pyruvate, 4 mM [3-13C]lactate, plus either 0.5 or 2.5 mM Ca2+ (groups 3 and 4). After perfusion with 13C-enriched substrates for varying periods of time, hearts were freeze-clamped using aluminum tongs precooled in liquid N2. A small portion of frozen tissue was used for determination of the wet-to-dry ratio by weighing the tissue before and after slow oven drying. The remainder of the frozen tissue was pulverized into a fine powder under liquid N2, extracted in 8% perchloric acid, centrifuged at 15,000 g for 15 min at 4°C, neutralized with KOH, and centrifuged a second time. The supernatant was freeze-dried, and the lyophilized heart extract was dissolved in 0.55 ml of 2H2O plus 0.05 ml of a concentrated solution of EDTA in 2H2O. EDTA was added to remove Ca2+ from its citrate complex so that the C2-C4 resonance of citrate was narrow enough to allow analysis of isotopomer populations.
13C NMR.
Proton-decoupled 13C NMR spectra of heart extracts (pH measured in 2H2O solutions, 7.2; 25°C) were acquired at 125.7 MHz on a Varian INOVA spectrometer (Varian Instruments; Palo Alto, CA) using a 5-mm broadband NMR probe, a 45° observed pulse, and a 3-s delay between pulses. Broadband proton decoupling was achieved using Waltz decoupling at two power levels: low during the preacquisition delay and high during acquisition. Relative peak areas for each multiplet component were determined by deconvolution using the personal computer-based NMR program NUTS (Acorn; Freemont, CA).
Tissue measurements.
After acquisition of NMR spectra, samples were freeze-dried, redissolved in H2O, and assayed for total tissue aspartate, glutamate, citrate, and malate using standard enzymatic assays measured spectrophotometrically (1). Appropriate calibration curves prepared from standards were used to derive metabolite tissue contents.
Modeling.
Metabolite pool sizes and 13C NMR multiplet data were used as input to a kinetic model of the TCA cycle. The program tcaFLUX (8), which is an extension of the model reported by Chance et al. (5), provides a best fit of VTCA, VX, anaplerosis (y), and fractional enrichment of acetyl-CoA (FC2) to time-dependent 13C multiplet data (12). The temporal changes in the glutamate C1 doublet (1D), C2 doublets (D12 and D23), C2 quartet (2Q), C3 doublet and triplet (3D and 3T), and C4 doublet (D34) were used in the fitting procedure for hearts perfused with low-pyruvate/high-lactate concentrations (other metabolites were not detected with these perfusion conditions). In addition to these same glutamate multiplets, temporal data for malate C2 (D23 and Q), malate C3 (D23 and Q), aspartate C2 (D23 and Q), and aspartate C3 (D23 and Q) were also included in the fitting procedure for hearts perfused with high-pyruvate/low-lactate concentrations. Dilution of [3-13C]pyruvate/[3-13C]lactate by other endogenous substrates, expressed as the fraction of acetyl-CoA that is unenriched (FC0 = 1 – FC2), was also a variable fitted by the kinetic analysis. All results are presented as means ± SD. Differences in mean values were considered statistically significant at a probability level of <5% (P < 0.05). Student's t-test was used to compare means.
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RESULTS |
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HR, LVDP, and MO2 were measured for each heart. The rate pressure product (RPP) was not sensitive to the pyruvate-lactate ratio but was significantly higher (P < 0.0001) at increased [Ca2+]o. At 0.5 mM Ca2+, the RPP values were (12 ± 4) x 103 and (10 ± 4) x 103 mmHg/min at low- and high-pyruvate concentrations, respectively, and at 2.5 mM Ca2+, RPP values were (42 ± 3) x 103 and (43 ± 5) x 103 mmHg/min at low- and high-pyruvate concentrations, respectively. MO2 values also increased significantly between the low- and high-[Ca2+]o groups as expected. Interestingly, MO2 was also higher between the low- vs. high-pyruvate groups with either 0.5 or 2.5 mM Ca2+ in the perfusate.
Tissue levels of TCA cycle intermediates.
The most abundant metabolite in hearts perfused with a mixture of 0.4 mM [3-13C]pyruvate and 4.0 mM [3-13C]lactate was glutamate (Table 1). The tissue contents of other cycle intermediates and aspartate were small and could therefore be neglected in the kinetic analysis (22). Tissue glutamate content tended to decrease at higher [Ca2+]o (20.4 ± 5.3 vs. 17.5 ± 4.2 µmol/g dry wt at 0.5 vs. 2.5 mM Ca2+, respectively) but did not reach statistical significance. The metabolite content of hearts perfused with 4.0 mM [3-13C]pyruvate and 0.4 mM [3-13C]lactate was, however, quite different; glutamate content was lower, whereas citrate, malate, and aspartate were all present in significant amounts (Table 1). With this substrate mix, a increase in [Ca2+]o from 0.5 to 2.5 mM did produce significant increases in glutamate content (4.8 ± 1.6 vs. 6.6 ± 0.9 µmol/g dry wt), aspartate (3.6 ± 0.5 vs. 8.1 ± 1.3 µmol/g dry wt), and malate (2.7 ± 0.7 vs. 4.6 ± 0.9 µmol/g dry wt). Thus an increase in the pyruvate-lactate ratio from 0.1 (typical of physiological conditions) to 10 resulted in an approximately fourfold reduction in tissue glutamate level at low [Ca2+]o (from 20.4 ± 5.3 to 4.8 ± 1.6 µmol/g dry wt) and an approximately threefold reduction at high [Ca2+]o (from 17.5 ± 4.2 to 6.6 ± 0.9 µmol/g dry wt) and concomitant increases in aspartate, malate, and citrate (Table 1).
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30 min in hearts perfused with 2.5 mM [Ca2+]o and after 45 min in hearts perfused with 0.5 mM [Ca2+]o.
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1.4-fold), whereas VX decreased four to fivefold. Similar trends in VTCA and VX were obtained by analysis of the temporal changes in glutamate C4D34 alone (Table 3).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Exposure of hearts to different ratios of pyruvate and lactate (with total concentrations held constant at 4.4 mM) alters the cytosolic redox state and subsequently the steady-state levels of several tissue metabolites (see Table 1). One should note that these changes do not result from a change in tissue pH, because even the highest level of lactate used here (4 mM) does not exceed the buffering capacity of the tissue. Rather, these changes must originate from changes in the redox state of the tissue (11).
A 10-fold increase in pyruvate from 0.4 to 4 mM would predictably decrease cytosolic NADH and increase cytosolic NAD+. This in turn should favor formation of OAA from malate (via cytosolic malate dehydrogenase) and subsequent conversion of OAA to aspartate and -KG to glutamate (via the transaminase GOT). This would result in a net shift in the tissue content of many TCA cycle and related intermediates (aspartate, malate, and citrate). This was observed experimentally at both high and low [Ca2+]o, although it is interesting to note that there was a net decrease in total tissue metabolites in the low-[Ca2+]o group as pyruvate was increased from 0.4 to 4 mM (20.4 ± 5.3 vs. 14.7 ± 3.0 µmol/g dry wt) and a net increase in total tissue metabolites in the high-[Ca2+]o group as pyruvate was increased from 0.4 to 4 mM (17.5 ± 4.2 vs. 22.9 ± 3.3 µmol/g dry wt). This demonstrates that [Ca2+]o does indeed have a significant effect on total tissue metabolite levels, although the effect is more evident when the metabolites are more evenly distributed among glutamate, aspartate, malate, and citrate (high-pyruvate group).
An increase in [Ca2+]o from 0.5 to 2.5 mM had a more subtle effect on tissue metabolites (see Table 1). With an increase in [Ca2+]o, tissue metabolites (mostly glutamate) tended to be lower in the low-pyruvate group but clearly increased in the high-pyruvate group. Here the observed changes in metabolite levels are not as easily predicted based on shifts in known equilibria, but studies of isolated mitochondria have demonstrated that mitochondrial Ca2+ content, which is known to increase with an increase in [Ca2+]o, significantly decreases the Km of -KG dehydrogenase and thereby lowers mitochondrial -KG (21). This in turn should increase mitochondrial aspartate and decrease mitochondrial OAA and glutamate contents.
Influence of altered Ca2+ on TCA cycle kinetics.
An increase in [Ca2+]o is known to increase both contractility (2) and MO2 (22). In these Langendorff-perfused hearts, an increase in [Ca2+]o from 0.5 to 2.5 mM resulted in an approximately twofold increase in MO2. This was also detected in our kinetic analyses as an increase in VTCA (approximately twofold) between the low- vs. high-Ca2+ groups for both low-pyruvate and high-pyruvate groups. It was found that VX was also stimulated at higher Ca2+ levels, and, in fact, VX was about twice as sensitive to the fivefold increase in [Ca2+]o as was VTCA. The origin of this effect is unknown. It has been reported that increases in [Ca2+]o stimulate both glycolysis and glucose oxidation by direct stimulation of phosphofructokinase-1 and pyruvate dehydrogenase by Ca2+ (17), but glucose was not present in these experiments, so the increase in VX must have a different origin. Perhaps it is related to the increase in contractility and O2 demand that is produced by increased [Ca2+]o. It has been proposed that VX reflects an exchange between -KG and glutamate (15), which in turn could reflect an increase in the malate-aspartate shuttle activity (21). Hence, higher [Ca2+]o may stimulate the shuttling of more reducing equivalents from the cytosol into the mitochondria to contribute to the increased O2 demand. In the present experiments, these reducing equivalents would most likely be contributed by cytosolic lactate, but unfortunately, we cannot differentiate between oxidation of pyruvate vs. oxidation of lactate because both substrates were labeled with 13C equivalently. It is interesting to note, however, that the change in VX with increased O2 demand (low- vs. high-[Ca2+]o groups) was much higher when the cytosol was more reduced (pyruvate-lactate ratio, 0.1).
Influence of changes in cytosolic redox on TCA cycle kinetics.
One would anticipate that altering cytosolic redox by changing the pyruvate-lactate ratio would have predictable effects on both VTCA and VX. As a first approximation, one would anticipate that high-lactate (low-pyruvate) concentration would stimulate the malate-aspartate shuttle (25), and hence, more reducing equivalents would be transported from the cytosol into the mitochondria. Indeed, VX was about four- to fivefold higher in hearts perfused with low-pyruvate/high-lactate concentrations at both levels of [Ca2+]o. Furthermore, import of more reducing equivalents from the cytosol into the mitochondria should also decrease the requirement of the TCA cycle to produce NADH; hence, one would expect that VTCA would be lower. This was also detected by the kinetic analysis and by a direct measure of MO2. As shown in Table 2, both VTCA and MO2 increased significantly between the low-pyruvate/high-lactate group compared with the high-pyruvate/low-lactate group (at both low and high [Ca2+]o), although contractile function as measured by RPP did not change at either [Ca2+]o level. The percent increases in MO2 between the low- and high-pyruvate groups were similar for hearts perfused with 0.5 mM (23% increase) vs. 2.5 mM (21% increase) [Ca2+]o. This indicates that the increase in MO2 is not related to increased demand brought about by the increased contractility promoted by Ca2+ but is rather due to the substrates available to the heart. It has been observed in a perfused, working heart preparation that switching the perfusate substrate available to hearts from glucose to pyruvate can result in an increase in MO2 without a concomitant increase in RPP (7). Yu et al. (25) also found that VX (equivalent to F1 in Ref. 25) increased by four- to fivefold in hearts perfused with butyrate plus lactate compared with butyrate alone, whereas VTCA was no different in these two groups. They also found no net oxidation of lactate; however, we observed net oxidation of lactate (FC2 values, 0.9 for all groups). These differences likely reflect the expected differences in mitochondrial redox state between hearts perfused with pyruvate vs. butyrate.
Comparisons to previous studies.
The primary role of the TCA cycle in heart tissue is to provide reducing equivalents for electron transport and subsequent ATP synthesis. The values reported here for VTCA are significantly smaller than the values reported previously for acetate-perfused hearts but are similar to the values reported for acetate- plus propionate-perfused hearts (4). Although acetate has a single metabolic fate in the heart (to contribute to acetyl-CoA and generate reducing equivalents in the TCA cycle), both lactate and pyruvate are capable of contributing extra reducing equivalents and thus reducing VTCA. Perfusion of hearts with any substrate or substrate mixture that contributes reducing equivalents external to the TCA cycle results in lower cycle flux relative to hearts perfused with acetate alone. This trend was observed by Burgess et al. (3) in mouse hearts, where it was found that VTCA was significantly higher in hearts perfused with acetate alone vs. octanoate alone and vs. lactate alone (27.1 vs. 17.1 vs. 13.1 µmol·min–1·g dry wt–1, respectively). Jeffrey et al. (8) also observed a similar trend in rat hearts perfused with glucose plus acetate vs. glucose plus pyruvate, where VTCA decreased from 11.3 to 6.1 µmol·min–1·g dry wt–1.
The observations that a more highly oxidized cytosol results in an increase in VTCA and a decrease in VX whereas an increase in [Ca2+]o results in an increase in both VTCA and VX are at least consistent with a parallel between VX and malate-aspartate shuttle activity. However, it is clear from these and other experiments that the process represented by VX does not require net transport of reducing equivalents from the cytosol to the mitochondria under all conditions, so VX may be more accurately described as a metabolite-exchange flux across the inner mitochondrial membrane (15). The VX/VTCA ratio (see Tables 2 and 3) was found to vary 10-fold among the four groups of hearts reported here (0.3 for hearts perfused with low-[Ca2+]o and high-pyruvate concentrations to 2.7 for hearts perfused with high-[Ca2+]o and low-pyruvate concentrations), so the ability of the heart to alter both VTCA and VX reflects an intricate coordination of energy demand and exchange kinetics.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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