What Products Are Continuously Produced in the Krebs Cycle

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• FASEB J
• PMC2717776

FASEB J. 2009 Aug; 23(8): 2529–2538.

Real-time assessment of Krebs cycle metabolism using hyperpolarized ¹³C magnetic resonance spectroscopy

Marie A. Schroeder

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Helen J. Atherton

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Daniel R. Ball

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Mark A. Cole

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Lisa C. Heather

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Julian L. Griffin

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Kieran Clarke

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

George K. Radda

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Damian J. Tyler

^*Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Received 2009 Jan 8; Accepted 2009 Feb 19.

Abstract

The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2-¹³C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of ¹³C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of ¹³C-labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2-¹³C]pyruvate.—Schroeder, M. A., Atherton, H. J., Ball, D. R., Cole, M. A., Heather, L. C., Griffin, J. L., Clarke, K., Radda, G. K. Tyler, D. J. Real-time assessment of Krebs cycle metabolism using hyperpolarized ¹³C magnetic resonance spectroscopy.

Keywords: dynamic nuclear polarization, ischemia, pyruvate dehydrogenase, TCA cycle

The breakdown of adenosine triphosphate (ATP) is the only immediate source of energy for the heart for contraction, maintenance of active ion gradients, and other vital functions. Cardiac ATP production is controlled largely by the rate at which the Krebs cycle operates (1). The input to the Krebs cycle is the 2-carbon component of acetyl CoA, which is produced from the oxidation of fatty acids, ketone bodies, or glucose via glycolysis and the pyruvate dehydrogenase (PDH) enzyme complex (2, 3). The rates of Krebs cycle metabolism and oxidative phosphorylation are closely coupled to the rate of contractile work and overall ATP demand.

Many pathological states in the heart detrimentally affect cardiac energetics by creating a mismatch between the anaerobic glycolytic pathway and oxidative metabolism via the Krebs cycle. For example, in myocardial ischemia, the glycolytic rate is maintained despite suppressed oxidative metabolism (4, 5). In the hypertrophic heart, fatty acid oxidation is decreased, with an increase in glycolysis (6, 7). However, increased glucose oxidation via PDH does not compensate for reduced fatty acid oxidation, which raises the question as to how Krebs cycle flux and cardiac energetics can be maintained in the state of hypertrophy (8).

The mechanisms that lead to energetic imbalances in heart disease could be better studied by simultaneously monitoring the source and fate of glucose-derived acetyl CoA in the heart (glycolytic and Krebs cycle metabolism). To date, Krebs cycle flux and substrate selection have been measured using carbon-13 MR spectroscopy (¹³C MRS) combined with isotopomer analysis (8,9,10). This technique models the steady-state incorporation of ¹³C label into the glutamate pool and therefore has limited application in non-steady-state situations (11) and in vivo studies of cardiac metabolism (12).

Metabolic imaging with hyperpolarized ¹³C MRS (13, 14) has enabled the measurement of normal and abnormal metabolism in real time in several systems (15,16,17,18,19). In the heart, hyperpolarized [1-¹³C]pyruvate is rapidly converted to [1-¹³C]lactate, [1-¹³C]alanine, and H¹³CO₃ ⁻ (in pH-dependent equilibrium with ¹³CO₂); thus, glycolysis via lactate dehydrogenase (LDH) and flux through the PDH enzyme complex can be assessed (20, 21). However, since PDH-mediated oxidation of hyperpolarized [1-¹³C]pyruvate releases the hyperpolarized ¹³C nucleus as ¹³CO₂, the formation of acetyl CoA and its incorporation into the Krebs cycle cannot be followed.

In this study, we developed the use of hyperpolarized [2-¹³C]pyruvate as a tracer to monitor Krebs cycle metabolism in the isolated perfused heart directly. Hyperpolarized [2-¹³C]pyruvate was infused into healthy hearts, and the metabolic products that had sufficient MR signal to be detected with high temporal resolution were identified. The time courses of the formation of each of these metabolites gave kinetic information describing the relationships among cytosolic metabolism of [2-¹³C]pyruvate, PDH-mediated oxidation of [2-¹³C]pyruvate, and its subsequent incorporation into the Krebs cycle. In addition, hyperpolarized [2-¹³C]pyruvate was infused at the moment of reperfusion into globally ischemic hearts, to identify differences in [2-¹³C]pyruvate metabolism in the reperfused myocardium and to assess the potential of hyperpolarized [2-¹³C]pyruvate as a metabolic tracer to study ischemic heart disease.

We have demonstrated that Krebs cycle metabolism can be directly and instantaneously monitored in the perfused heart and that new information can be obtained about the coordination of glycolysis, pyruvate oxidation, and Krebs cycle flux in the normal and postischemic myocardium.

MATERIALS AND METHODS

The [2-¹³C]pyruvic acid and all unlabeled compounds were obtained from Sigma (Gillingham, UK). The trityl radical, OX063, was obtained from GE-Healthcare (Amersham, Little Chalfont, UK), and the gadolinium compound 1,3,5-tris-(N- (DO3A-acetamido)-N-methyl-4-amino-2-methylphenyl)-(1,3,5)triazinane-2,4,6-trione, referred to here as 3-Gd, was obtained from Imagnia AB (Malmö, Sweden). All investigations conformed to Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 (HMSO) and to institutional guidelines.

Overview of the experimental protocol

[2-¹³C]Pyruvate was hyperpolarized in a HyperSense system (Oxford Instruments, Abingdon, UK). Six isolated perfused rat hearts were infused with hyperpolarized [2-¹³C]pyruvate in the normal and postischemic metabolic states. Each heart was perfused in the Langendorff mode and placed in the bore of an 11.7 T vertical bore MR scanner. Hyperpolarized ¹³C-labeled metabolic tracer was infused into the heart while it was functioning normally and data were acquired with 1 s temporal resolution. A 10-min period of no-flow global ischemia was then initiated. A second dose of the same ¹³C-labeled hyperpolarized tracer was infused immediately on reperfusion.

Peaks arising from hyperpolarized [2-¹³C]pyruvate were identified using phantom experiments, high-resolution NMR and/or reference to the literature. Differences between metabolism in normal and ischemic hearts, as reported by hyperpolarized [2-¹³C]pyruvate, were quantified, and ¹³C measurements of cardiac metabolism were compared with cardiac function. Details of the heart perfusion, hyperpolarized ¹³C protocol, data processing, analysis, and [2-¹³C]pyruvate peak assignment are described below.

Isolated perfused rat heart

Six male Wistar rats (∼300 g) were anesthetized using a 0.5 ml intraperitoneal injection of pentobarbital sodium (200 mg/ml euthatal). The beating hearts were quickly removed and arrested in ice-cold Krebs-Henseleit perfusion buffer, and the aorta was cannulated for perfusion in a recirculating retrograde Langendorff mode at a constant 85 mmHg pressure and 37°C temperature, as described previously (22, 23). Intraventricular pressure development resulting from thebesian artery drainage was minimized by the insertion of polyethylene tubing through the apex of the heart. A small water-filled balloon was inserted into the left ventricular cavity via the mitral valve, attached via tubing to a pressure transducer, which allowed left ventricular contractile function and heart rate to be measured. End diastolic pressure was set to ∼4 mmHg. Heart rate and left ventricular systolic and diastolic pressure were continuously recorded using a PowerLab/4SP data acquisition system (ADInstruments Ltd., Chalgrove, UK). Myocardial contractile function was assessed by calculating the product of heart rate and left ventricular developed pressure to give the rate pressure product (mmHg/min).

The Krebs-Henseleit perfusion buffer contained 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.75 mM CaCl₂, 0.5 mM Na₂EDTA, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 11 mM glucose, and 2.5 mM pyruvate and was aerated with a mixture of 95% oxygen (O₂) to 5% carbon dioxide (CO₂), to give a final pH of 7.4 at 37°C.

Hyperpolarized ¹³C MR spectroscopy

The perfused heart was placed in a 20 mm NMR sample tube and positioned inside the bore of a 11.7-T Bruker spectrometer (500 MHz for ¹H resonances; Bruker Biospin, Ettlingen, Germany). The function of each heart was allowed to stabilize in the bore of the magnet, while the heart was imaged to localize it in the center of the RF coils, and a slice-selective shim was implemented to reduce ¹H linewidth to ∼50 Hz.

Modifications to the Langendorff mode

During image acquisition and shimming, flow rate to the heart was measured from the recirculation line. Buffer delivery to the heart was then switched from the constant pressure system to constant flow through a peristaltic pump (Gilson Minipuls 3; Gilson, Middleton, WI, USA) set to the measured flow rate. Buffer was rerouted from the oxygenation system and 85-mmHg pressure head, via a second water-jacketed umbilical, and delivered to the heart immediately above the cannula. This modification to the Langendorff perfusion setup was introduced to enable the rapid delivery of hyperpolarized buffer to the heart by limiting dead volume in the perfusion line, with a view toward maximizing hyperpolarized signal during ¹³C infusions. Heart function was stabilized at constant flow prior to infusion of hyperpolarized ¹³C-pyruvate.

Hyperpolarized [2-¹³C]pyruvate preparation

[2-¹³C]Pyruvate was prepared and polarized in the HyperSense ¹³C polarizer system with 15 mM OX063 concentration and a trace amount of 3-Gd. On dissolution, 6 ml of effluent hyperpolarized tracer (80 mM sodium pyruvate, 0.27 mM Na₂EDTA, 20 mM TRIS base buffer, 60 mM NaOH, pH ∼7.4, temperature ∼40°C) was infused directly into 190 ml of oxygenated perfusion buffer in a water-jacketed reservoir at 37°C. This diluted the hyperpolarized [2-¹³C]pyruvate concentration to 2.5 mM pyruvate, the concentration used in the initial KH perfusion buffer. The posthyperpolarized buffer contained 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.75 mM CaCl₂, 0.49 mM Na₂EDTA, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 11 mM glucose, and from the hyperpolarized infusion, 2.5 mM hyperpolarized [2-¹³C]pyruvate, and 0.63 mM TRIS base. The hyperpolarized perfusion buffer was identical to the Krebs-Henseleit perfusion buffer initially used to perfuse each heart, except that the 2.5 mM pyruvate was excluded. The primary differences between perfusion and hyperpolarized buffer composition were the slightly decreased Na₂EDTA concentration and the TRIS buffer added to neutralize the pyruvic acid on dissolution in the HyperSense.

After a 2-s delay to allow for mixing, the constant-flow peristaltic pump was switched to draw from the hyperpolarized [2-¹³C]pyruvate reservoir and begin perfusion of the isolated heart with hyperpolarized [2-¹³C]pyruvate. Depending on the exact flow rate measured for each heart, which ranged from 14 to 21 ml/min, hyperpolarized tracer reached the heart in 15–20 s.

¹³C MR data acquisition

Immediately after infusion of hyperpolarized [2-¹³C]pyruvate, MR acquisition of ¹³C-pyruvate and its metabolic derivatives was initiated. Metabolites were detected with a 1-s temporal resolution over the course of 4 min (TR=1 s, excitation flip angle=30°, pulse length=30 μs, 240 acquisitions). Over a bandwidth of 180 ppm, 4096 points were acquired. Spectra were centered at 125 ppm.

Total global ischemia

Global ischemia was initiated in perfused hearts by shutting off the peristaltic pump to stop buffer flow to the heart. Hearts were maintained in this state for 10 min, at which point hyperpolarized [2-¹³C]pyruvate was infused into the buffer reservoir and the pump was switched on (at the same flow rate previously measured) to restart buffer flow. The perfusion line was briefly disconnected immediately above the magnet to wash out nonhyperpolarized buffer from the dead volume, such that each heart was reperfused with nonhyperpolarized buffer for no more than 5 s. By this means, the hyperpolarized pyruvate experiments enabled rapid measurement of changes to the myocardium almost immediately on reperfusion, following no-flow ischemia.

Data analysis

Cardiac ¹³C MR spectra were analyzed using the AMARES algorithm as implemented in the jMRUI software package (24). Spectra were DC-offset corrected based on the last half of acquired points and peaks corresponding with [2-¹³C]pyruvate and its metabolic derivatives (quantifiable at 1-s temporal resolution) were fitted assuming a Gaussian line shape and initial peak frequencies, relative phases and linewidths. Quantified peak areas were plotted against time in Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). The averaged maximum peak area of each metabolite over the 60 s of acquisition was determined for each series of spectra, as described previously (21) along with the area under the metabolic progression curve (AUC). The initial rate of signal production for each metabolite, in arbitrary units per second (s^–1), was measured as the slope of its average metabolite progression plot over the first 5 s following metabolite appearance when the detected signal increased linearly. The time to appearance of signal from each metabolite was defined as the delay between pyruvate appearance in that particular experiment and the first time point that was >10% of the maximum peak area. This corrected for the variation in hyperpolarized pyruvate arrival at the heart due to the variable flow rate at which tracer was delivered. In addition, an exponential signal decay term was fit to each metabolite progression plot from the point of maximum signal over the course of signal decay.

Statistical analysis

Data are reported as means ± se. Statistical significance between healthy and ischemic groups was assessed using a paired Student's t test. Statistical significance was considered at P < 0.05.

Peak assignment

[2-¹³C]pyruvate phantom

Infusion of hyperpolarized [2-¹³C]pyruvate into an empty perfusion rig was performed according to the same protocol as infusion into the isolated perfused heart. ¹³C data were acquired and processed identically for comparison to perfused heart spectra.

Metabolite extraction

One heart was perfused further for ∼30 min with the Krebs-Henseleit perfusion buffer described above containing both 80 mM [1-¹³C]pyruvate and [2-¹³C]pyruvate, after which time the heart was immediately excised and frozen in liquid nitrogen before being stored at −80°C until analysis. Metabolites were extracted from heart tissue using methanol/chloroform/water as described previously (25). Briefly, frozen tissue (∼100 mg) was placed in methanol/chloroform (2:1, 600 μl) and homogenized. Samples were then sonicated for 5 min before chloroform/water (1:1) was added (200 μl of each). Samples were centrifuged (13,500 rpm, 20 min), and the aqueous layer was removed and dried overnight in an evacuated centrifuge (Eppendorf, Hamburg, Germany).

NMR spectroscopy

Dried extracts were rehydrated in 600 μl of D₂O and buffered in 0.24 M sodium phosphate (pH 7.0) containing 1 mM sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropionate (TSP; Cambridge Isotope Laboratories, Andover, MA, USA) as an internal standard. The samples were analyzed using an Avance II+ spectrometer operating at 500 MHz for the ¹H frequency (Bruker) equipped with a 5-mm broadband TXI automatic tuning and matching (ATMA) probe. One-dimensional spectra were collected using a solvent suppression pulse sequence based on a 1-D nuclear Overhauser effect spectroscopy pulse sequence to saturate the residual [¹H] water proton signal (relaxation delay=2 s, t ₁= 3 μs, mixing time=150 ms, solvent presaturation applied during the relaxation time and the mixing time, NS=128, SW=12 ppm, T=37°C). Two-dimensional heteronuclear multiple bond correlations (HMBCs; ref. 26) were also performed to correlate ¹H and ¹³C resonances [hmbcgplpndqf Bruker pulse program, TD=4096 (F1) and 256 (F2), NS=256, D1=2.5 s, SW(¹H)=12 ppm, SW(¹³C)=250 ppm, AQ=0.31 s). Peak assignments were based on their ¹³C chemical shifts relative to the known 1-D ¹H peaks using the 2-D NMR spectrum.

RESULTS

Polarization of [2-¹³C]pyruvate

[2-¹³C]Pyruvic acid polarized similarly to the [1-¹³C]pyruvic acid used previously (20). [2-¹³C]Pyruvate polarization levels were slightly below the ∼30% polarization reached by [1-¹³C]pyruvate and were estimated by comparison of solid-state polarization data to be ∼27%. In both compounds, the rate of polarization buildup had a time constant of ∼850 s.

Cardiac function throughout the protocol

Figure 1 shows cardiac function throughout the protocol, in terms of developed pressure, heart rate, and rate pressure product. The perfused rat hearts (n=6) had a rate pressure product of 34,000 ± 5000 mmHg/min. During constant pressure perfusion, buffer flow to the heart was 19 ± 3 ml/min and was set at this rate for the remainder of the perfusion protocol. Infusion of hyperpolarized pyruvate tended to decrease transiently developed pressure and, therefore, the rate pressure product, without affecting heart rate. Global ischemia decreased heart function to negligible levels within 5 min. At 45 s after reperfusion with hyperpolarized pyruvate, the rate pressure product recovered to 10,000 ± 4000 mmHg/min. At 5 min following reperfusion, the rate pressure product recovered to 56% of its original value.

A representative recording of cardiac function throughout the perfusion protocol, showing developed pressure, heart rate and rate pressure product. Left panel: effects of hyperpolarized pyruvate infusion during the first 2 min of MR data acquisition. Right panel: ischemia and 5 min of postischemic recovery.

[2-¹³C]pyruvate and metabolite peak assignment

Figure 2 shows an example of stacked spectra acquired from a healthy heart over the first 60 s of data acquisition. The signal from [2-¹³C]pyruvate had the highest intensity at 207.8 ppm. Other identifiable peaks are shown inset (averaged over 10 acquisitions) and annotated. All chemical shifts were referenced to the doublet of [2-¹³C]lactate (peak 7) at 71.2 ppm. Based on previous work with [1-¹³C]pyruvate and literature values, peaks 4, 5, and 8 were assigned to natural abundance [1-¹³C]pyruvate at 172.8 ppm, [2-¹³C]pyruvate hydrate at 96.5 ppm, and [2-¹³C]alanine at 53.3 ppm, respectively. Two impurities in the [2-¹³C]pyruvate preparation, located at ∼149 and 89 ppm (peak 6), were assigned by comparison of the spectra acquired from a healthy heart with that acquired from an infusion into an empty perfusion rig. A very small acetyl CoA peak was also detected at ∼202 ppm (only visible when all data were summed). In addition, 3 singlet peaks with sufficiently high SNR to be quantified in single scans were detected at 183.7 ppm (peak 1), 181.0 ppm (peak 2), and 175.2 ppm (peak 3).

Example stacked spectra acquired in the first 60 s following [2-¹³C]pyruvate infusion into a perfused rat heart. [2-¹³C]Pyruvate was observed at 207.8 ppm. Peaks 1, 2, and 3 represent the metabolic products [5-¹³C]glutamate (183.7 ppm), [1-¹³C]citrate (181.0 ppm), and [1-¹³C]acetylcarnitine (175.2 ppm), respectively. [1-¹³C]Pyruvate derived from natural abundance ¹³C was seen as a quartet at 172.8 ppm (peak 4, left inset). [2-¹³C]Pyruvate hydrate, which is in chemical equilibrium with pyruvate, was detected at 96.5 ppm (peak 5, right inset). Impurities in the [2-¹³C]pyruvic acid preparation were observed at ∼149 and 89 ppm (peak 6, right inset). [2-¹³C]Lactate and [2-¹³C]alanine could also be observed (peaks 7 and 8, respectively).

To assign these peaks, 2-D HMBCs were also performed on one additional heart, which had been infused with [1,2-¹³C]pyruvate for 30 min to correlate ¹H and ¹³C resonances. The annotated results are shown in Fig. 3 , which confirmed that the ¹³C label was taken up into the Krebs cycle, based on the coordinated appearance of Krebs cycle intermediates in both ¹H and ¹³C spectra. Inspection of the expanded ¹H/¹³C spectral region of interest, shown inset, confirmed that myocardial perfusion with [2-¹³C]pyruvate produced [5-¹³C]glutamate at ∼183.5 ppm (peak 1), [1-¹³C]citrate at ∼181.2 ppm (peak 2) and [1-¹³C]acetyl carnitine at ∼175.0 ppm (peak 3). To further the assignment of peak 3 (Fig. 2) confirmation, previous work regarding peak assignment following infusion of hyperpolarized [1-¹³C]acetate was referenced (27). When infused in vivo, hyperpolarized [1-¹³C]acetate generates a low-amplitude acetyl CoA peak and a higher-magnitude [1-¹³C]acetyl carnitine peak at ∼175 ppm.

Annotated 2-D heteronuclear multiple bond correlations. The y axis shows ¹³C chemical shifts, and the x axis shows ¹H chemical shifts. Shaded regions within the plot indicate nuclei for which coupling between ¹H and ¹³C resonances had been detected. Peaks were assigned based on both ¹H and ¹³C resonances. Inset: spectral region corresponding with previously unassigned hyperpolarized ¹³C peaks detected in the isolated perfused heart.

These findings confirm the utility of hyperpolarized [2-¹³C]pyruvate as a metabolic tracer to follow Krebs cycle metabolism with high temporal resolution.

Characterization of spectra derived from [2-¹³C]pyruvate

In the healthy perfused hearts examined, many peaks derived from [2-¹³C]pyruvate were of sufficiently high magnitude to be quantified with 1-s temporal resolution, which thereby allowed kinetic analyses. Figure 4 shows the metabolic progression plots of [2-¹³C]lactate, [1-¹³C]acetyl carnitine, [1-¹³C]citrate, [2-¹³C]alanine, and [5-¹³C]glutamate; Fig. 5 details the fitted parameters for each metabolite.

Progression of the metabolic products of [2-¹³C]pyruvate, for healthy perfused hearts (n=6). Data were acquired with a 1-s temporal resolution, using a 30° RF pulse.

Summary of the metabolic fate of infused [2-¹³C]pyruvate along with the measured parameters of the observed metabolites.

The [2-¹³C]lactate peak was first detected 4 ± 1 s after pyruvate arrival and was the highest amplitude peak (maximum peak area=0.05±0.01, relative to maximum [2-¹³C]pyruvate peak area). The [1-¹³C]acetyl carnitine and [1-¹³C]citrate peaks were first detected at 5 ± 1 s and 4 ± 1 s after pyruvate arrival and with maximum normalized peak areas of 0.029 ± 0.004 and 0.014 ± 0.002 respectively. The [5-¹³C]glutamate peak appeared significantly later than the other metabolites, at 7 ± 1 s following pyruvate arrival (P<0.001 vs. citrate arrival) and had the lowest maximum normalized peak area (0.009±0.002).

The time constant of [2-¹³C]lactate MR signal decay was calculated to be 24 ± 1 s, whereas the decay time constant of hyperpolarized [1-¹³C]acetyl carnitine was the longest of all [2-¹³C]pyruvate-derived metabolites at 30 ± 1 s. The decay time constants of [5-¹³C]glutamate and [1-¹³C]citrate were calculated to be 17 ± 1 and 26 ± 5 s, respectively.

Hyperpolarized [2-¹³C]pyruvate in the ischemic myocardium

Figure 6 shows the averaged metabolic time courses for [2-¹³C]lactate, [1-¹³C]acetyl carnitine, [1-¹³C]citrate, and [5-¹³C]glutamate for all hearts in the preischemic and postischemic states. On reperfusion with [2-¹³C]pyruvate, the ischemic myocardium produced significantly more [2-¹³C]lactate than the healthy myocardium (area under curve (AUC) = 3.8±0.6 postischemia vs. 1.6±0.3 in the healthy heart, P=0.001). Also, the decay time constant of [2-¹³C]lactate MR signal decreased by 23% in the postischemic myocardium.

Comparison of the average metabolic time courses for lactate, acetylcarnitine, citrate, and glutamate in both the healthy and postischemic hearts. Image shows a significant elevation in the lactate signal following a 10 min ischemic period, along with a significant reduction in the citrate and glutamate signals.

No significant difference was found in the level of [1-¹³C]acetyl carnitine production following ischemia (AUC=0.7±0.1 postischemia vs. 1.0±0.1 in the healthy heart, P=0.1). However, the initial rate of [1-¹³C]acetyl carnitine signal production was significantly reduced on reperfusion by 50% (P=0.005). In contrast to lactate, the signal production of [1-¹³C]citrate and [5-¹³C]glutamate were both significantly reduced following ischemia (AUC=0.23±0.04 vs. 0.42±0.06, P=0.02 and AUC=0.15±0.05 vs. 0.27±0.06, P<0.01, respectively).

DISCUSSION

This work has demonstrated the use of hyperpolarized [2-¹³C]pyruvate as a metabolic tracer. Observation of [2-¹³C]pyruvate metabolism in real time in the perfused heart has provided simultaneous information on glycolysis, via [2-¹³C]lactate appearance; PDH flux and energy demand, via [1-¹³C]acetyl carnitine; and a direct assessment of Krebs cycle metabolism. Detection of [1-¹³C]citrate and [5-¹³C]glutamate has enabled the first instantaneous measurements of oxidative metabolism in whole organs. The simultaneous appearance of [1-¹³C]citrate, [2-¹³C]lactate, and [1-¹³C]acetyl carnitine revealed that pyruvate-derived acetyl CoA was incorporated into the Krebs cycle within 1 s of both its production by PDH and the cytosolic conversion of pyruvate to lactate. All of these enzymatic conversions occurred within 5 s of [2-¹³C]pyruvate arrival at the coronary arteries.

Direct assessment of Krebs cycle flux

The detection of [5-¹³C]glutamate 3 s later than [1-¹³C]citrate appearance has followed the oxidative metabolic fate of [2-¹³C]pyruvate carbon atoms across a defined part of the Krebs cycle. Within these 3 s, [1-¹³C]citrate was converted reversibly to isocitrate by aconitase and converted irreversibly to α-ketoglutarate by isocitrate dehydrogenase. This irreversible reaction would have also generated a molecule of NADH, released a carbon nucleus as CO₂ (Fig. 5), and served as a potential point of Krebs cycle control (30). In addition, prior to [5-¹³C]glutamate detection, the labeled carbon must have accumulated to a detectable level in the glutamate pool, via glutamate dehydrogenase-mediated exchange between α-ketoglutarate and glutamate. Integration of hyperpolarized MR measurements of [5-¹³C]glutamate production with steady-state Krebs cycle data, acquired with thermal equilibrium ¹³C MRS, may enable validated modeling of these steps of mitochondrial metabolism with unique levels of temporal detail.

Investigation of PDH flux

These experiments have provided new insight into the nature of both [2-¹³C]pyruvate and [1-¹³C]pyruvate metabolism, as assessed in numerous hyperpolarized MR studies. Previous work from this group (20, 21) and others (15, 37) have used the conversion of hyperpolarized [1-¹³C]pyruvate into H¹³CO₃ ⁻ to measure flux through the PDH enzyme complex. While this application enables assessment of an important indicator of cardiac substrate selection, cardiac H¹³CO₃ ⁻ production should not be used as an absolute marker of glucose oxidation, or pyruvate incorporation into the Krebs cycle. The maximal [1-¹³C]acetyl carnitine MR signal produced following [2-¹³C]pyruvate infusion was ∼2-fold greater than the [1-¹³C]citrate signal produced. Though MR signal production is affected by both substrate concentration and hyperpolarized signal decay, the high magnitude of the [1-¹³C]acetyl carnitine peak has qualitatively indicated that even in healthy hearts, the majority of pyruvate-derived acetyl CoA may not be immediately taken up into the Krebs cycle (29).

Acetyl carnitine production is mediated by the carnitine acetyl transferase (CAT) system, which is stimulated by abundant mitochondrial acetyl CoA and carnitine (4). When available acetyl CoA exceeds the Krebs cycle capacity for ATP production, the acetyl carnitine pathway enables acetyl group storage for later utilization when fuel supply is reduced (28). Further, Lysiak et al. (29) have reported that in heart mitochondria, acetyl CoA derived from pyruvate is particularly accessible to CAT, and that a greater proportion of acetyl carnitine is produced from pyruvate-derived acetyl CoA than from fatty acid-derived acetyl CoA.

Metabolism in the ischemic myocardium

Hyperpolarized [2-¹³C]pyruvate can also be used to study the metabolic consequences of myocardial ischemia. Global ischemia is characterized by accumulation of the anaerobic metabolic products lactate, NADH, and H⁺, as glycolysis is maintained while oxidative phosphorylation is decreased (5, 30, 31). In the postischemic myocardium, the 2.4-fold increase in [2-¹³C]lactate most likely indicates lactate accumulation, as hyperpolarized ¹³C-lactate production has been demonstrated to reflect LDH-mediated exchange of the ¹³C-label (16, 32). In addition, the 23% decrease in the time constant of [2-¹³C]lactate MR signal decay provided information about lactate washout on reperfusion (33).

The reduced rate of [1-¹³C]acetyl carnitine production observed in postischemic hearts indicated a reduced rate of acetyl CoA production from [2-¹³C]pyruvate during the early reperfusion period. This reduction may have resulted from residual PDH inhibition, due to accumulation of intracellular NADH during ischemia (5, 30, 34). The magnitude of the maximal [1-¹³C]acetyl carnitine signal was not significantly changed, but it was delayed by ∼10 s, such that the peak [1-¹³C]acetyl carnitine MR signal was not observed until ∼25–30 s after [2-¹³C]pyruvate arrival. Therefore, it seemed that PDH flux was normalized within ∼30 s of reperfusion.

The decrease in oxidative metabolism compared with glycolysis, characteristic of ischemia, has also been linked to substrate depletion of the Krebs cycle intermediates and the glutamate pool (35, 36). To promote metabolic and functional recovery of the reversibly ischemic heart, pyruvate may be consumed as an anaplerotic substrate to maintain mitochondrial levels of malate and oxaloacetate (36). Taegtmeyer's findings are consistent with our observations of Krebs cycle metabolism in the postischemic heart. The delay in [1-¹³C]acetyl carnitine production that we observed on reperfusion may have reflected an initial diversion of hyperpolarized [2-¹³C]pyruvate from PDH-mediated oxidation to anaplerotic pathways. In addition, the reduction of the [5-¹³C]glutamate and [1-¹³C]citrate peak areas indicated a decline in hyperpolarized [2-¹³C]pyruvate incorporation into Krebs cycle and depletion of the glutamate pool.

General discussion

Our study has led to several conclusions on the ischemic mismatch between glycolysis and Krebs cycle metabolism and the utility of hyperpolarized [2-¹³C]pyruvate as a metabolic tracer. However, our perfusion protocol omitted fatty acids as a source of fuel to the heart, to limit competition for [2-¹³C]pyruvate metabolism and thus maximize MR signal from [2-¹³C]pyruvate-derived metabolites. Acetyl CoA produced from β-oxidation may reduce PDH activity, thereby affecting [1-¹³C]acetyl carnitine production from hyperpolarized [2-¹³C]pyruvate (29). Also, fatty acid metabolism contributes to Krebs cycle recovery following ischemia (28, 37). Therefore, these relationships should be explored in a more physiological perfusion system and in in vivo models of cardiac dysfunction.

Hyperpolarized [2-¹³C]pyruvate remains a challenging metabolic tracer to use. Only those metabolites that accumulate to a sufficiently high concentration and retain enough polarization can be visualized with 1-s temporal resolution. For example, [5-¹³C]glutamate and [1-¹³C]citrate, the most valuable metabolic products of [2-¹³C]pyruvate, were detected with low signal amplitudes in this study. Future work will determine the temporal resolution and length of data acquisition possible to enhance the study of these metabolites.

Work will also focus on the development of a suitable protocol for in vivo [2-¹³C]pyruvate experiments. The higher cardiac workload observed in vivo, compared with the Langendorff perfused heart, may compensate for the increased sensitivity of the perfused heart system, enabling [2-¹³C]pyruvate hyperpolarized MR studies in living animals. Alternately, systems in which cardiac workload is elevated, for example after infusion of a β-adrenergic agonist, may be useful for in vivo studies with [2-¹³C]pyruvate.

Finally, several cardiac metabolic states exhibit elevated citrate levels, including fasting and experimental diabetes (38), and modulation of citrate levels has also been observed in tumor cells (39, 40). These metabolic states may also warrant investigation with hyperpolarized [2-¹³C]pyruvate.

In summary, initial investigations in the isolated perfused heart with hyperpolarized [2-¹³C]pyruvate have supplied the first direct, whole-organ measurements of instantaneous Krebs cycle metabolism. Simultaneous measurement of [1-¹³C]citrate and [1-¹³C]acetyl carnitine metabolism have provided an indication of the coupling between glycolytic and oxidative metabolism in normal hearts and after a period of global ischemia. Visualization of ¹³C incorporation into the glutamate pool has enabled real-time assessment of flux through a defined portion of the Krebs cycle and may enable validation of hyperpolarized MR measurements based on thermal equilibrium ¹³C MRS and isotopomer analysis. Further investigations with [2-¹³C]pyruvate in a variety of physiological and pathological situations, both in the perfused heart and in vivo, may yield a sensitive diagnostic test for impaired heart function and enhance basic understanding of the mechanisms regulating Krebs cycle metabolism.

Acknowledgments

The authors acknowledge previous work from scientists at Imagnia AB (Malmö, Sweden), which aided peak assignment in this study. We also thank Dr. Matthew Merritt and Professor Craig Malloy for helpful discussions regarding perfused heart techniques. M. S. gratefully acknowledges the Newton Abraham Scholarship Foundation for her D.Phil. studentship, and NIH grant 1-F31-EB006692-01A1. This work was funded by research grants from the Medical Research Council (MRC grant G0601490) and the British Heart Foundation (BHF grant PG/07/070/23365) and through equipment support from GE-Healthcare and Oxford Instruments Molecular Biotools.

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