Metabolic Research Laboratory, 1 Departments of Radiology and 2 Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
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ABSTRACT |
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This study examines the functional implications of postnatal changes in the expression of the mitochondrial transporter protein, 2-oxoglutarate-malate carrier (OMC). Online 13C nuclear magnetic resonance (13C NMR) measurements of isotope kinetics in hearts from neonate (3-4 days) and adult rabbits provided tricarboxylic acid cycle flux rates and flux rates through OMC. Neonate and adult hearts oxidizing 2.5 mM [2,4-13C2]butyrate were subjected to either normal or high cytosolic redox state (2.5 mM lactate) conditions to evaluate the recruitment of malate-aspartate activity and the resulting OMC flux. During development from neonate (3-4 days) to adult, mitochondrial protein density in the heart increased from 19 ± 3% to 31 ± 2%, whereas OMC expression decreased by 65% per mitochondrial protein content (P < 0.05). Correspondingly, OMC flux was lower in adults hearts than in neonates by 73% (neonate = 7.4 ± 0.4, adult = 2.0 ± 0.1 µmol/min per 100 mg mitochondrial protein; P < 0.05). Despite clear changes in OMC content and flux, the responsiveness of the malate-aspartate shuttle to increased cytosolic NADH was similar in both adults and neonates with an approximate threefold increase in OMC flux (in densitometric units/100 mg mitochondrial protein: neonate = 25.8 ± 2.5, adult = 6.0 ± 0.2; P < 0.05). The 13C NMR data demonstrate that OMC activity is a principal component of the rate of labeling of glutamate.
functional expression; tricarboxylic acid cycle; nuclear magnetic resonance spectroscopy; mitochondria; metabolism
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INTRODUCTION |
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THE INNER MITOCHONDRIAL MEMBRANE is not freely permeable, and transport of a number of tricarboxylic acid (TCA) cycle intermediates is controlled by specific carrier proteins (20). The transport of 2-oxoglutarate across the mitochondrial membrane is specifically limited to the 2-oxoglutarate-malate carrier (OMC) protein (3, 5-6, 8, 25, 29), which is widely distributed in mammalian mitochondria. There is no transport of NADH across the mitochondrial membrane, and reducing equivalents produced in the cytosol from glycolysis are carried into mitochondria as malate through exchange for oxoglutarate. The cytosolic-mitochondrial exchange of 2-oxoglutarate is reversible through this carrier, but net forward flux through the malate-aspartate shuttle during the transport of reducing equivalents into mitochondria results in stimulated exchange of 2-oxoglutarate between these two compartments (6).
Recent work on whole heart preparations in our laboratory has shown that 13C enrichment kinetics of glutamate from a labeled source metabolized via the TCA cycle are dependent on TCA cycle flux (VTCA) and the rate of isotope exchange between mitochondria and the large pool of glutamate in the cytosol (F1) (11, 16, 32, 33). F1 consists of two metabolic processes: 1) transport of 2-oxoglutarate across the membrane and 2) conversion of 2-oxoglutarate to glutamate via glutamate-oxaloacetate transaminase (GOT). However, GOT activity is 20-fold higher than the interconversion rate between 2-oxoglutarate and glutamate (16, 32). As 13C labeling is sensitive to cytosolic glutamate labeling (4, 14, 32), the rate-limiting step in conversion of mitochondrial 2-oxoglutarate to cytosolic glutamate (F1) is the transfer of 2-oxoglutarate across the OMC transporter. Although the exchange of 2-oxoglutarate and malate can occur without a large net forward flux through the malate aspartate shuttle (31), during redox-stimulated malate-aspartate shuttle activity the rate of carbon isotope enrichment of the cytosolic glutamate pool is faster, even under conditions where the TCA cycle flux is constant (31, 32).
During the early postnatal period, the heart undergoes rapid development in terms of both myocyte proliferation and size (18). Subcellular development also occurs inside myocytes with the proportions of mitochondria, myofibrils, and smooth endoplasmic reticulum all increasing (19). During this period of development, the neonatal heart has a reliance on carbohydrate utilization for energy production (12, 24). It has been suggested that the greater dependence on glycolytic metabolism in the neonatal heart is facilitated by the increased expression of OMC protein compared with the adult animal (24).
The functional implications of an enzyme phenotype change in an intact tissue or organ cannot be elucidated reliably from in vitro assays. In consequence, the functional expression of few specific enzymes have been quantitatively investigated in the intact working organ. In this study we have compared the differing expression of OMC in the neonate and adult rabbit heart with kinetic data from the Langendorff perfused heart using dynamic 13C nuclear magnetic resonance (13C NMR) spectroscopy. Kinetic analysis of dynamic 13C NMR spectra allowed direct investigation into the effect of an individual mitochondrial transporter on oxidative metabolism in the whole heart. This study also provides further evidence that OMC flux is a rate limiting processes in the labeling kinetics of glutamate in the heart.
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MATERIALS AND METHODS |
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13C NMR of intact hearts. Hearts from either neonatal rabbits aged 3-4 days or adult rabbits were perfused in retrograde fashion in a NMR magnet, using previously described methods (31-33). Neonate hearts were studied at 3-4 days to ensure that the foramen ovale had fully closed (12). Following heparinization of animals, hearts were excised from anesthetized animals (ketamine, 1,000 U/kg ip; Telozl, 400 U/kg ip) and perfused in retrograde fashion with modified Krebs-Henseleit solution at 100 cm hydrostatic pressure (31).
Hearts contracted spontaneously against a water-filled, intraventricular balloon inflated to an end-diastolic pressure of 5 mmHg and connected to a pressure transducer. Heart rate (HR) and left ventricular developed pressure (LVDP) were continually recorded, and mechanical work was assessed using the rate-pressure product (RPP = HR × LVDP). Experimental groups consisted of hearts perfused with either: 1) 5 mM glucose + 2.5 mM [2,4-13C2]butyrate (Isotec, Miamisburg, OH) (n = 6 for neonates; n = 4 for adults) or 2) 2.5 mM lactate + 2.5 mM [2,4-13C2]butyrate (n = 5 for neonates; n = 5 for adults) to investigate labeling kinetics at two different cytosolic redox potentials. At the start of each protocol, hearts were perfused with unlabeled butyrate and either glucose or lactate for 10 min to reach metabolic equilibrium, during which the natural abundance 13C signals were acquired. The perfusate supply was then switched to labeled medium as listed above, and sequential 13C spectra were acquired every 2.5 min until steady-state enrichment. 31P spectra were acquired before and after perfusion to ensure maintenance of high-energy phosphates during each protocol (31-33). After 40 min, hearts were freeze-clamped for in vitro analysis (31). NMR data were collected on a 9.4-T magnet using a 20-mm NMR probe (Bruker Instruments, Billerica, MA) as previously described (31-33). High-resolution 13C NMR spectra of tissue extracts reconstituted in 0.5 ml of H2O were obtained with a 5-mm probe and were used to determine the fractional enrichment of acetyl-CoA (Fc) (13-14).Kinetic modeling of 13C labeling of glutamate. Kinetic analysis of the 13C enrichment curves for the C-4 and C-2 positions of glutamate was performed by least squares fitting curves to a model described by Yu et al. (31, 32) and developed in this laboratory. The label from [2,4-13C2]butyrate enters the TCA cycle as [2-13C]acetyl-CoA. As indexed in the glutamate pool, 2-oxoglutarate is first labeled at the C-4 position and then with equal probability at the C-2 and C-3 positions on the second turn of the TCA cycle. Kinetic analysis of isotope data provided VTCA and F1 (31, 32).
Biochemical analysis of metabolite concentrations and enzyme activities. Perchloric acid extracts of myocardial tissue freeze clamped at the end of perfusion were assayed for citrate, aspartate, glutamate, and oxoglutarate using ultraviolet and fluorometric techniques (1, 30-31).
To normalize for differences in mitochondrial density between adult and neonate hearts, flux parameters were expressed in terms of mitochondrial protein content (2, 9). Citrate synthase activity was used to determine the mitochondrial density of myocardium via differential centrifugation (2). Measurement of citrate synthase activity in the total homogenate and mitochondrial fraction allowed determination of mitochondrial protein in the total homogenate. OMC expression in adult and neonate rabbit myocardium was measured by Western blotting of the 31-kDa subunit as described by Scholz and colleagues (28). Myocardial tissue was homogenized with a solution containing 50 mM Tris, 10 mM EDTA, 150 mM NaCl, and 0.1% mercaptoethanol (pH 7.5) with 5 µg/ml soybean trypsin inhibitor, 20 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride and sonicated for 20 s. After centrifugation, total protein was quantified by assay, and then 20-µg samples were separated by SDS-PAGE. SDS-PAGE was performed on the myocardial tissue under reducing conditions on 10% separation gels with a 4% stacking gel in a Miniprotean II cell (Bio-Rad). Proteins were then transferred to a Hybond-ECL nitrocellulose for 2 h and blocked in 5% nonfat milk for 3 h. For immunoreaction, the blot was incubated with 1:2,500 diluted antibodies to OMC (gift from T. Scholz) for 90 min at room temperature. The densities of the bands were detected by a chemiluminescence system and were evaluated using NIH Image. Serial dilution of the membrane preparations revealed a linear relationship between amounts of protein and the densities of the immunoreactive bands (data shown in Fig. 1).
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Statistical comparisons. Comparison of mean values was performed with the Student's unpaired, two-tailed t-test and ANOVA test of variance. Differences in mean values were considered significant at a probability level of <5%. Results are reported as mean ± SE.
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RESULTS |
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As previously reported (19, 24), neonatal hearts had greatly reduced mitochondrial densities (19 ± 3% of the total protein) compared with adult hearts (31 ± 2% of the total protein; P < 0.05). To normalize for this reduction in mitochondrial protein, the flux rates VTCA and F1 obtained by the kinetic fits were expressed in terms of mitochondrial protein mass. This allowed a direct comparison between the adult and neonatal mitochondria despite the differences in mitochondrial density.
To assess OMC protein content in the different groups, quantitative immunoblotting was performed. The intensity of the labeled OMC band was proportional to the amount of cardiac membrane protein electrophoresed in the range of 5-50 µg (Fig. 1, A and B). OMC protein expression in neonatal myocardium was double that found in the adult myocardium (in arbitrary densitometric units: neonate = 119 ± 28; adult = 67 ± 13, P < 0.05; Fig. 1C). Normalizing OMC density to mitochondrial protein content demonstrated that OMC content was threefold higher in the neonate compared with the adult in terms of mitochondrial expression (in arbitrary units per 100 mg of mitochondrial protein: neonate = 630 ± 150; adult = 220 ± 40).
Neonatal hearts displayed physiological and bioenergetic
characteristics typical of that previously reported
(21). Normal contractile function and ATP concentration
were maintained throughout the experimental procedure. No difference in
contractile function between neonatal and adult hearts for a given
substrate was measured (Table 1). There
was little phosphocreatine in the neonatal heart despite the presence
of ATP confirming previously reported low neonatal expression of
phosphocreatine kinase (data not shown) (21).
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Sequential 13C NMR spectra of perfused isolated hearts
provided enrichment kinetics of glutamate over the course of each
experiment (Fig. 2). The relatively high
concentration of cytosolic glutamate acts as a label sink for
13C labeling of 2-oxoglutarate (4, 14). Figure
3 displays enrichment curves and the
least-squares fit of the model for neonatal hearts perfused with
[2,4-13C2]butyrate + lactate. Hearts
perfused with [2,4-13C2]butyrate + lactate showed a more rapid enrichment of the C2 and C4 glutamate pools
than during perfusion with
[2,4-13C2]butyrate + glucose (curves not
shown), as is evident in the values shown in Fig.
4. No difference was found between the
fractional enrichment of acetyl-CoA between the neonate and adult for
the two groups or between substrate mixtures (for butyrate + glucose, neonate Fc = 0.88 ± 0.04 and adult
Fc = 0.91 ± 0.02; for butyrate + lactate,
neonate Fc = 0.88 ± 0.04 and adult
Fc = 0.96 ± 0.09). However, the total
pool of aspartate was increased in the neonate compared with the adult
for glucose and butyrate perfusion (Table 2) (P < 0.05).
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VTCA and F1 values for both the neonatal and adult heart groups are shown in Fig. 4. VTCA values are constant between hearts receiving [2,4-13C2]butyrate + glucose and [2,4-13C2]butyrate + lactate for a given age group. VTCA was unchanged and remained largely determined by butyrate oxidation. Increasing the cytosolic reducing potential by switching from glucose to lactate stimulated flux through the malate-aspartate shuttle (32), thereby increasing the rate of interconversion between 2-oxoglutarate and glutamate in both age groups (P < 0.05 for the neonate; P < 0.005 for the adult).
Comparing the two age groups, the OMC-facilitated interconversion of 2-oxoglutarate and glutamate (F1) in neonates was increased relative to adults for the same substrate mixture (in µmol/min per 100 mg of mitochondrial protein: for +glucose, neonate = 7.4 ± 0.4 and adult = 2.0 ± 0.1, P < 0.05; for +lactate, neonate = 25.8 ± 2.5 and adult = 6.0 ± 0.2, P < 0.05). For a given substrate mixture the increase in F1 detected in neonates was 3.6- to 4.3-fold higher than that found in adults under similar redox conditions. OMC expression and F1 rate measurements correlated well for both redox loads for the two age groups.
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DISCUSSION |
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Our finding that relates the expression of the OMC protein to glutamate labeling kinetics contributes to the notion of OMC providing a measurable, rate-determining step in the labeling of glutamate. The detection of glutamate labeling from intact systems, organs, and whole animals has often been interpreted directly as TCA cycle flux in isotope studies. However, this study demonstrates the molecular basis for 2-oxoglutarate efflux from mitochondria via OMC as being a significant factor in the measurement of metabolic flux from glutamate labeling.
Although labeling of cytosolic glutamate does occur when there is little transport of NADH across the mitochondrial membrane, because of the reversible nature of OMC, efflux of 2-oxoglutarate from mitochondria via OMC is stimulated during the recruitment of net forward flux through the malate/aspartate shuttle (32, 33). The increased exchange of 2-oxoglutarate across the mitochondrial membrane accelerates carbon isotope enrichment of the observed glutamate pool (32), as was observed in this study for both neonatal and adult hearts following an increase in redox load in the cytosol. The malate-aspartate shuttle is comprised of two distinct carrier proteins, the irreversible, electrogenic aspartate-glutamate transporter (AGT) protein and the reversible OMC protein (10, 26). With decreasing reliance of the developing myocardium on glycolytic metabolism, the postnatal production of cardiac OMC is reduced, yet present in adult hearts (27, 28). In the neonatal myocyte, as well as the malate-aspartate shuttle, the glycerophosphate shuttle also facilitates indirect exchange of NADH/NAD+ across the mitochondrial membrane (27). The enzymes associated with this shuttle are minimally expressed in adult myocardium. However, the presence of the glycerophosphate shuttle in the neonate hearts did not affect our measurements, because the 13C labeling process is specific to the interconversion of 2-oxoglutarate and glutamate (16, 31). Thus the method used is dependent on the influx/efflux of 2-oxoglutarate through OMC and is therefore specific to the activity of OMC while being insensitive to the glycerophosphate shuttle and flux through AGT.
There was no difference in the fractional enrichment of acetyl-CoA, demonstrating that substrate selection was similar for both substrate mixtures and between age groups. A similar effect has previously been observed in the postischemic heart where substrate selection between acetate and glucose was identical with that of controls, despite a large difference in flux through OMC (17). Despite differences in mitochondrial density, apart from aspartate concentration during perfusion of neonatal hearts with butyrate and glucose, the metabolite pool sizes were indistinguishable within the experimental error between the two age groups for a given substrate mixture. Interestingly, the labeling kinetics of glutamate are relatively insensitive to large intermediate metabolite pool concentrations (~20%) in the heart (32, 33).
Immunoassay analysis of the expression of the OMC protein in the neonate and adult heart demonstrated a greater OMC content per mitochondrial protein content by threefold in the neonate. Scholz and colleagues (28) noted that the developmental decline in malate/aspartate flux as measured in isolated mitochondria is accompanied by a decrease in production of mRNA for OMC protein in the porcine heart. Conversely, they also noted that the mRNA levels for aspartate-glutamate carrier, mitochondrial malate dehydrogenase, and mitochondrial aspartate aminotransferase increased during development, suggesting the rate of flux through the malate/aspartate shuttle is determined by OMC. This was confirmed by 13C NMR spectroscopy measurements of the labeling kinetics of glutamate, with OMC flux increasing 3.6- to 4.3-fold in response to an increase in OMC expression of 2.8-fold in terms of mitochondrial protein. Although the increase in flux is greater than the increase in OMC expression, it is well established that other factors beyond enzyme content affect total activity, such as substrate and cofactor levels. This suggests that the increased rate of glutamate labeling detected in these experiments arises from an increase in OMC activity in situ as well as its level of expression. Only by directly observing transport across OMC were we able to determine the actual effect that OMC expression had on metabolism during development. This demonstrates the importance of functional measurements and how they correlate to the level of enzyme expression.
Immunoblot assays determined protein expression, but 13C NMR enabled measurement of enzyme activity relative to that of competing enzymes in the heart. The greater value of F1 in the neonatal heart compared with the adult may in part reflect reduced affinity of 2-oxoglutarate dehydrogenase for 2-oxoglutarate. The TCA cycle can be conceptualized as two spans, the first from acetyl-CoA to 2-oxoglutarate and the second from 2-oxoglutarate to oxaloacetate (15, 22). The enzymes 2-oxoglutarate dehydrogenase and the OMC transporter occur at a pivotal point to link these spans through competition between oxidation of 2-oxoglutarate and the efflux of 2-oxoglutarate from the mitochondrial matrix. The balance between 2-oxoglutarate oxidation/efflux can serve as a mechanism for metabolic flux homeostasis for maintaining TCA cycle flux (23). The balance between OMC and 2-oxoglutarate dehydrogenase activities may in turn be caused by changes in 2-oxoglutarate dehydrogenase expression or an alteration in matrix free calcium concentration, a known regulator of several TCA cycle enzymes including 2-oxoglutarate dehydrogenase (7), during development.
In conclusion, we have used direct observe 13C NMR to measure the activity of the OMC carrier protein and assess the functional responses to oxidative flux regulation during differential expression of this protein in the developing myocardium. The data document the sensitivity of 13C NMR spectroscopy to rates of metabolite exchange between subcellular compartments in the myocytes of the whole heart, on the basis of molecular changes in OMC expression from the neonate to the adult heart. Such kinetics have previously been restricted to isolated mitochondria preparations, whereas the expression, but not the net activity, of the OMC proteins could only be determined from immunoblot assay. Thus we have demonstrated the use of 13C enrichment kinetics to evaluate the functional consequences of metabolite exchange protein expression and activity in the intact, beating heart.
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ACKNOWLEDGEMENTS |
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We are grateful to Thomas Scholz, University of Iowa, for providing antibodies to OMC.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-49244, RO1-HL-56178, and RO1-HL-62702 (to E. D. Lewandowski).
Address for reprint requests and other correspondence: E. D. Lewandowski, Dept. Physiology and Biophysics, MC 901, Univ. of Illinois at Chicago, College of Medicine, 835 S.Wolcott Ave., Rm. 240, Chicago, IL 60612 (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.
Received 14 April 2000; accepted in final form 26 June 2000.
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