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Limited transfer of cytosolic NADH into mitochondria at high cardiac workload

¹Program in Integrative Cardiac Metabolism, Department of Physiology and Biophysics, University of Illinois, College of Medicine, Chicago, Illinois 60612; ²Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536; ³Cardiovascular Institute, Department of Cellular and Molecular Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103; and ⁴Department of Molecular and Cellular Physiology, Pennsylvania State University Medical School, Hershey, Pennsylvania 17033

Submitted 30 November 2003 ; accepted in final form 28 January 2004

	ABSTRACT

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Glycolysis supplements energy synthesis at high cardiac workloads, producing not only ATP but also cytosolic NADH and pyruvate for oxidative ATP synthesis. Despite adequate PO₂, speculation exists that not all cytosolic NADH is oxidized by the mitochondria, leading to lactate production. In this study, we elucidate the mechanism for limited cytosolic NADH oxidation and increased lactate production at high workload despite adequate myocardial blood flow and oxygenation. Reducing equivalents from glycolysis enter mitochondria via exchange of mitochondrial -ketoglutarate (-KG) for cytosolic malate. This exchange was monitored at baseline and at high workloads by comparing ¹³C enrichment between the products of -KG oxidation (succinate) and -KG efflux from mitochondria (glutamate). Under general anesthesia, a left thoracotomy was performed on 14 dogs and [2-¹³C]acetate was infused into the left anterior descending artery for 40 min. The rate-pressure product was 9,035 ± 1,972 and 21,659 ± 5,266 mmHg·beats·min^–1 (n = 7) at baseline (n = 7) and with dobutamine, respectively. ¹³C enrichment of succinate was 57 ± 10% at baseline and 45 ± 13% at elevated workload (not significant), confirming oxidation of [2-¹³C]acetate. However, cytosolic glutamate enrichment, a marker of cytosolic NADH transfer to mitochondria, was dramatically reduced at high cardiac workload (11 ± 1%) vs. baseline (50 ± 14%, P < 0.05). This reduced exchange of ¹³C from -KG to cytosolic glutamate at high work indicates reduced shuttling of cytosolic reducing equivalents into the mitochondria. Myocardial tissue lactate increased 78%, countering this reduced oxidation of cytosolic NADH. The findings elucidate a contributing mechanism to glycolysis outpacing glucose oxidation in the absence of myocardial ischemia.

heart; ¹³C nuclear magnetic resonance; malate-aspartate shuttle; metabolism

We hypothesized that the transport of reducing equivalents into the mitochondria becomes limited, resulting in increased lactate production as pyruvate is reduced by cytosolic NADH to restore NAD⁺ reserves. This study examined whether the carrier protein for shuttling reducing equivalents from cytosolic NADH into the mitochondria, namely, the -ketoglutarate (-KG)-malate transporter of the malate-aspartate shuttle, becomes rate limiting at elevated workloads. The activity of the transporter can be monitored by following the exchange of ¹³C-enriched metabolites across the mitochondrial membrane by ¹³C NMR (6, 16, 21, 22, 30). Thus the rate of exchange is an index of cytosolic NADH transfer into the mitochondria for support of oxidative phosphorylation. The findings suggest that oxidation of glycolytically produced NADH in the cytosol is limited at high work states because of relative reductions in malate-aspartate shuttle activity and account for the mechanism of lactate production in the cytosol at high workload despite adequate blood flow and oxygen availability.

	EXPERIMENTAL PROCEDURES

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Animal preparation. Fourteen mongrel dogs of either sex weighing 24–31 kg were sedated with xylazine (1–2 mg/kg im). General anesthesia was induced with thiopental (2–4 mg/kg iv) and, after intubation, was maintained with isoflurane (0.5–1.5%). With the use of a sterile surgical technique, the heart was exposed through a left thoracotomy at the fifth intercostal space. Left ventricle (LV) pressure was measured by implanting a solid-state miniature pressure gauge (model P7, Konigsberg Instruments) in the LV through an apical incision. Tygon catheters (Norton Elastic and Synthetic Division, Akron, OH) were implanted in the descending thoracic aorta and left atrial appendage. A coronary arterial catheter was implanted for infusion of substrate into the left anterior descending artery (LAD) bed. Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, revised 1996).

Experimental protocol. After the surgical preparation was completed, hemodynamic variables [aortic pressure, LV pressure, left atrial pressure, rate of change of LV pressure (dP/dt), and heart rate] were monitored continuously throughout the protocol. A control set of dogs (n = 7) stabilized and achieved a basal rate-pressure product (RPP). After baseline measurements, the dogs received 4 mM ¹³C-labeled acetate ([2-¹³C]acetate) with (40 µg·kg^–1·min^–1 iv; n = 4) or without (n = 4) dobutamine or 4 mM unlabeled acetate with (40 mg·kg^–1·min^–1; n = 3) or without (n = 3) dobutamine administered into the LAD at 5 ml/min for 40 min.

Although acetate is clearly not an ideal physiological substrate, it has been a common and affordable substrate for providing ¹³C as used in in vivo NMR studies. Our use of [2-¹³C]acetate was required to explain previously published observations of acetate oxidation in the in vivo dog and pig heart from other laboratories (1, 23). Blood gases and myocardial O₂ consumption (MO₂) were monitored, and respiration rate and tidal volume were adjusted to maintain O₂ levels. MO₂ was determined as milliliters of O₂ per minute with the calculation (A-V difference in volume % O₂) x coronary blood flow/100. At completion of the protocol, a sample of myocardium was excised from the LAD bed of each dog. The sample was rapidly sectioned into subendocardium and subepicardium and immediately freeze clamped at –70°C. We showed previously (15) that this method is rapid enough not to induce changes in metabolic intermediates, such as tissue lactate content.

Myocardial tissue blood flow determination. Regional myocardial blood flow was measured with the radioactive microsphere technique as previously described (9). Three million microspheres (15 ± 1 µm) labeled with ⁹⁵Nb, ⁸⁵Sr, ¹⁴¹Ce, ⁴⁶Sc, ¹¹³Sn, ⁵¹Cr, ¹¹⁴In, or ¹⁰³Ru were suspended in 0.01% Tween 80 solution and placed in an ultrasonic bath for at least 30 min. Before the first injection of microspheres, 1 ml of Tween 80 solution was injected to test for potential adverse cardiovascular effects. Microspheres were injected and flushed with saline via the left atrial catheter. Arterial blood reference samples were withdrawn at a rate of 7.75 ml/min for a total of 120 s. Radioactive microspheres were administered in control animals at baseline and during acetate infusion and in dobutamine animals at baseline, after dobutamine infusion before acetate infusion, and during acetate infusion.

¹³C enrichment of metabolites. The labeling scheme for the oxidation of [2-¹³C]acetate and the incorporation of label into the metabolites of the tricarboxylic acid (TCA) cycle and the cytosolic glutamate pool is depicted in Fig. 1. This labeling scheme has been described in detail elsewhere (13, 16, 21, 22, 31).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Schematic representation of acetate uptake and incorporation into the tricarboxylic acid (TCA) cycle. 13C from labeled acetate enters the first span of the TCA cycle as acetyl-CoA and labels [4-13C]-ketoglutarate (-KG). -KG can be transported from the mitochondria by the -KG-malate transporter and transaminated by glutamate-oxaloacetate transaminase (GOT) to form [4-13C]glutamate, or -KG can be oxidized within the mitochondria by -KG dehydrogenase (-KGDH) and enrich either the C-2- or C-3 positions of succinate. Recycling of 13C within the TCA cycle results in labeling of glutamate C-2 and C-3 positions. The transport of -KG from the mitochondria vs. oxidation within the mitochondria is competitive (21). In this study, we assessed the extent of transport vs. oxidation by measuring glutamate vs. succinate 13C enrichment.

A second set of frozen heart samples were used for chromatographic isolation of succinate and glutamate. After PCA extraction and an initial centrifugation (10 min, 10,000 g, 4°C), extracts were neutralized to pH 6.5 with 3 N potassium hydroxide-0.5 M MOPS-0.1 M EDTA and recentrifuged. The metabolite isolation procedure was adapted from a previously described method for ion-exchange chromatography at room temperature (17). The metabolites of interest were chromatographically separated from the tissue extract with a Dowex chloride column (AG1-X8, 1 x 20 cm) preequilibrated with distilled water. Before the column was loaded, tracer amounts of radiolabeled metabolites ([³H]glutamate and [¹⁴C]succinate) were added to the extract for identification of the eluted fractions containing each metabolite. Metabolites were separated chromatographically over a 2-h period at a rate of 2.0 ml/min as described previously (17). Radiolabeled fractions were identified by scintillation counting (Beckman LS 3801, Beckman Instruments, Columbia, MD) and frozen at –70°C for later NMR analysis. The elution profile of each of the metabolites was previously determined with test samples.

NMR spectroscopy. PCA extracts from heart tissue and isolated succinate and glutamate fractions were lyophilized. The powder was reconstituted in 0.5 ml of deuterium oxide for NMR analysis. NMR data were collected on a Bruker MSL400 series spectrometer interfaced to a 9.4-T vertical-bore superconducting magnet (Bruker Instrument, Billerica, MA). Details of high-resolution data collection and postprocessing have been described elsewhere (13, 16, 18, 21, 22, 31).

Tissue chemistry. Fractional enrichments were determined from NMR spectra and enzymatic assays (18, 29). The concentrations of ¹³C-labeled metabolites were calculated from the area of the ¹³C resonance that was referenced to the spectra of known succinate and glutamate standards. Total metabolite concentrations were determined by enzymatic analysis of the same NMR samples (succinate test kit, Boehringer Mannheim). Tissue lactate, glutamate, and succinate were assayed as previously described (2, 13, 14). Tissue alanine content was determined by cross-referencing the NMR proton resonance of lactate to alanine (2, 14).

Statistical analysis. Data are reported as means ± SD unless otherwise stated. Data set comparisons were performed with Student's unpaired, two-tailed t-test unless otherwise stated. Differences in mean values were considered statistically significant at a probability level of <5% (P < 0.05).

	RESULTS

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Functional data and workload. Values of contractile function and coronary flow for baseline and elevated workload groups are shown in Tables 1 and 2, respectively. The RPP of the baseline group was 9,035 ± 1,972 mmHg·beats·min^–1, whereas the dobutamine group reached 21,659 ± 5,266 mmHg·beats·min^–1. LV dP/dt was fivefold greater for the dobutamine group relative to baseline (dobutamine group 7,568 ± 1,863 mmHg/s, baseline 1,540 ± 414 mmHg/s). Thirty-five minutes into the protocol MO₂ was 2.23 ± 0.06 ml/min in the baseline group and 6.98 ± 2.94 ml/min in the high-workload group.

Isotopic enrichment of oxidative intermediates. Representative high-resolution ¹³C NMR spectra obtained from myocardial extracts are shown in Fig. 2 for baseline and elevated workload conditions. The isotopic enrichment displayed occurs in the relatively large pool of glutamate in the cytosol (10, 21, 31). Consistent with previous observations (1, 23), baseline workloads produced obvious ¹³C enrichment of glutamate at the C-2, C-4, and C-3 positions (55.8, 34.6, and 28.3 ppm, respectively). In contrast, glutamate enrichment was extremely limited at the elevated workloads. Importantly, high-resolution ¹³C NMR of succinate samples (Fig. 3) showed ¹³C enrichment of succinate at both basal and elevated workloads in all eight hearts provided [2-¹³C]acetate. The incorporation of ¹³C into the succinate pool confirms the oxidation of [2-¹³C]acetate, disproving previous speculation that acetate oxidation is out-competed at high metabolic demand (23). Consequently, the lack of isotopic enrichment in cytosolic glutamate that occurred at elevated RPP is a function of transport activity rather than substrate oxidation.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Representative high-resolution 13C NMR spectra from heart extracts after [2-13C]acetate infusion in the in vivo canine myocardium. In agreement with previous reports (1, 23), glutamate carbons C-2, C-3, and C-4 reveal labeling at baseline workloads (B) but not at elevated workloads (A). The spectrum acquired at elevated workload shows only natural abundance enrichment of endogenous metabolites for this heart.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Representative high-resolution 13C NMR spectra from the TCA cycle intermediate succinate at baseline (B) and elevated (A) workload. Succinate was chromatographically isolated from the in vivo canine heart oxidizing [2-13C]acetate. At both workloads, succinate carbons C-2 and C-3 revealed significant enrichment relative to succinate carbons C-1 and C-4. This is consistent with oxidation of [2-13C]acetate. In these spectra the area of the C-2, C-3 resonance, relative to the C-1, C-4 area, is 30:1.

View larger version (32K): [in this window] [in a new window]   Fig. 4. The 13C fractional enrichments of 2 intermediates, succinate and glutamate, from in vivo canine myocardium oxidizing [2-13C]acetate at baseline and elevated workloads. Fractional enrichment is calculated as 13C-labeled intermediate concentration ([13C-labeled intermediate])/total intermediate concentration ([total intermediate]). [13C-labeled intermediate] values were determined from the 13C NMR spectra, and [total intermediate] values were determined by enzymatic assay of the same NMR sample. Given that succinate is clearly labeled at both workloads, limited labeling of cytosolic glutamate at elevated workloads indicates reduced transport of labeled intermediates from the mitochondria. *Significant difference between glutamate at high workload and all other groups (P < 0.05).

	DISCUSSION

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Previous observations and NMR studies. Our results demonstrate a mechanism for a phenomenon of increased cytosolic NADH at high workload that was the subject of speculation in a report by Kobayashi and Neely (8). They measured whole tissue NADH and NAD⁺ content as a function of workload in the isolated heart and calculated cytosolic and mitochondrial fractions of NADH and NAD⁺. They found that the cytosolic NADH-to-NAD⁺ ratio increased immediately after a jump in workload. They speculated that the rise in cytosolic NADH/NAD⁺ indicated that the rate of transport into the mitochondria could not keep pace with NADH production by glycolysis. Our data demonstrate that the -KG-malate transporter is in fact reduced at high workload, thus indicating that malate-aspartate activity does not increase with workload or glycolytic rates when exogenous sources of pyruvate or lactate are not provided.

Indeed, a seemingly unrelated report by Robitaille et al. (23) describes a similar lack of ¹³C enrichment of glutamate during [2-¹³C]acetate infusion at elevated workloads in the in vivo canine heart, which at the time was speculated to result from a lack of acetate oxidation. The mechanisms elucidated by our data resolve a longstanding question on this poorly understood metabolic response to workload, first reported by Robitaille and colleagues (1, 23). In their earlier work, as in the present study, the oxidation of ¹³C-enriched acetate in the myocardium was monitored in vivo. Under baseline workload conditions (RPP < 10,800 mmHg·beats·min^–1), the myocardium oxidized the infused [2-¹³C]acetate and incorporated the labeled carbon into the NMR-detectable glutamate pool as expected. At the high workloads induced by Robitaille et al. (23) (RPP 24,400 mmHg·beats·min^–1), they originally reported that enrichment of glutamate was significantly reduced. In explanation, and contrary to the known unregulated entry of acetate into mitochondria, the suggested mechanism was that acetate was not oxidized at the higher workloads. However, the absence of glutamate labeling does not necessarily indicate that TCA cycle intermediates are not enriched by the oxidation of ¹³C-labeled substrate, because of compartmentation of the observed cytosolic glutamate pool from the TCA cycle (13).

Indeed, the significant level of isotopic enrichment of succinate that is in evidence in our data from both experimental groups demonstrates that a similar proportion of [2-¹³C]acetate contributed to oxidative metabolism at both baseline and high workloads. The oxidation of [2-¹³C]acetate was clearly evident from the labeling of the TCA cycle intermediate succinate (13). Therefore, similar levels of isotopic enrichment of succinate demonstrate similar contributions of acetate oxidation, despite the lack of enrichment of the cytosolic glutamate pool, contrary to previously published speculation (1, 23). Furthermore, any mobilization of endogenous lipid by dobutamine infusion did not alter the extent of the oxidation of [2-¹³C]acetate, which bypasses the highly regulated entry of long-chain free fatty acids into the mitochondria. In view of these additional considerations, a number of previous, and seemingly unrelated, studies on the metabolic state of the in vivo heart at high workload are now linked by a common mechanism that is elucidated by the current protocol.

Mechanism for reduced transport. As shown in Fig. 1, mitochondrial -KG is transported from the mitochondria by the -KG-malate transporter and subsequently transaminated to form glutamate by glutamate-oxaloacetate transaminase (GOT). In earlier studies (30, 31), we showed that the rate of GOT is much too fast to be a rate-determining component of the observed interconversion rate between -KG and glutamate. Thus our results suggest that the shuttle is significantly downregulated under the condition of parallel dobutamine and acetate infusion. This may be due to a competition for substrate between the -KG dehydrogenase of the TCA cycle and the -KG-malate transporter of the shuttle.

As described previously, the two enzymatic reactions compete for the substrate -KG by virtue of their apparent K_m values (10, 21). The -KG-malate transporter of the mitochondrial membrane has an apparent K_m of 1.5 mM for -KG on the matrix side of the carrier (25), whereas the K_m of -KG dehydrogenase (-KGDH) for -KG was reported as 0.67 mM (10). This makes both oxidation and efflux very sensitive to regulation by the -KG concentration in the mitochondrial matrix. The Ca²⁺-sensitive -KGDH increases its affinity for substrate, and the K_m is lowered at elevated Ca²⁺ levels, whereas the transporter K_m does not change (27). Thus, as mitochondrial Ca²⁺ content increases with -adrenergic activation (3), both -KGDH activity and TCA cycle turnover increases to meet increased energy demands. It is postulated that increased activity through the dehydrogenase reduces the availability of substrate for transport (21). Importantly, the observed drop in the cytosolic glutamate pool is consistent with reduced transport of the substrate from the mitochondria.

We have observed other conditions in which flux through the -KG-malate transporter is similarly substrate limited. In particular, in the reperfused heart and in hearts exposed to elevated Ca²⁺ and H⁺ levels, the -KG-malate transporter flux was shown to decrease from control levels (16, 21, 22). In addition, we showed (6, 22, 30) that the transport flux increases in isolated hearts when exogenous lactate is provided to boost cytosolic NADH/NAD⁺ state.

The maintenance of the largely unlabeled glutamate pool may reflect the contributions of either alanine aminotransferase activity or conversion of cytosolic glutamine to glutamate via the glutaminase enzyme. However, the 55% drop in glutamate indicates that glutaminolysis does not increase enough to account for the significant drop in glutamate ¹³C enrichment observed at high workload. Also, alanine is an unlikely source because of a shift in the chemical equilibrium toward lactate production, which then limits the alanine pool (2, 14, 26).

Tissue lactate and alanine. The rise in tissue lactate and alanine levels observed with increased workload is consistent with elevated glycolysis in response to the reduced -KG-malate exchange and coupled transfer of cytosolic reducing equivalents. Normally, cytosolic NADH passes its reducing equivalents onto malate, which is transported into the mitochondria for exchange with -KG. The NAD⁺ regenerated in the cytosol is available to fuel glycolysis. Without the transporter, the only other major mechanism to restore NAD⁺ for glycolytic demands is the reduction of pyruvate to lactate. Although we did not measure glycolytic activity directly, increased tissue alanine content at high workloads indicates glycolytic activation (2, 11, 26). The parallel increase in lactate content signifies that not all glycolytic pyruvate is oxidized. Rather, pyruvate is reduced to form lactate, thus providing a mechanism for the disposal of glycolytic NADH and the regeneration of NAD⁺ to fuel glycolysis. However, elevated tissue lactate under conditions of normoxia is not an index of reduced tissue pH, because the lactate will not necessarily exceed the buffering capacity of the cytosol (12).

We found that the ratio of lactate to alanine, an index of cytosolic redox state, was similar between baseline and high workload groups (Table 3). The earlier Kobayashi and Neely work (8) reported increased redox state immediately after inotropic stimulation. However, their work was done under dynamic conditions. In our study, redox state was estimated at the end of the protocol, long after the heart has reached a new steady state. Under such conditions metabolic processes have responded and then adjusted to the increased demands, restoring the redox potential as indexed by lactate/alanine.

In this study, we explored a mechanism of cytosolic reducing equivalent use for energy production in functioning myocardium that was little understood, in part because of previous limitations in evaluating the extent of malate-aspartate activity in the intact or in vivo heart. In elucidating the mechanism for limited transfer of cytosolic reducing equivalents, this study explains the previous observations of elevated cytosolic redox state despite adequate tissue oxygenation at high workloads (8). Therefore, the mechanism elucidated in this study may have an impact on future investigations of the hypoperfused myocardium resulting from coronary artery constriction. The near-complete lack of a contribution of cytosolic NADH to oxidative energy production in the mitochondria of the well-perfused heart at high workload is a surprising new finding. Furthermore, the data resolve a misunderstood phenomenon of limited ¹³C transfer from oxidative intermediates to glutamate at high cardiac workload (23).

	GRANTS

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This work was supported in part by National Institutes of Health Grants HL-49244, HL-56178, HL-59139, HL-33107, HL-33065, HL-69020, HL-37404, and RR-16592.

	FOOTNOTES

 

Address for reprint requests and other correspondence: E. D. Lewandowski, Program in Integrative Cardiac Metabolism (MC 901), Dept. of Physiology and Biophysics, Univ. of Illinois, College of Medicine, 835 S. Wolcott Ave., Rm. 240 CMW, Chicago, IL 60612-7342 (E-mail: dougl{at}uic.edu).

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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