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(Circulation Research. 1995;77:773-783.)
© 1995 American Heart Association, Inc.

Articles

Nuclear Magnetic Resonance Studies of Cationic and Energetic Alterations With Oxidant Stress in the Perfused Heart

Modulation With Pyruvate and Lactate

Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 1994.

S. Yanagida, C.S. Luo, M. Doyle, G.M. Pohost, M.M. Pike

From the Department of Medicine, Division of Cardiovascular Disease, University of Alabama at Birmingham.

Correspondence to Dr M.M. Pike, Department of Medicine, Division of Cardiovascular Disease, 703 S 19th St, ZRB 308, Birmingham, AL 35294-0007.

	Abstract

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Abstract The postischemic generation of oxygen-derived free radicals may contribute to myocardial reperfusion injury by affecting sarcolemmal ion transport. Recent evidence indicates that exposure to reactive oxygen intermediates induces rapid increases in myocardial cytosolic free Ca²⁺ (Ca²⁺_i). The mechanism is undetermined but may involve disturbances in Na⁺ homeostasis. We tested this hypothesis by interleaving ²³Na and ³¹P nuclear magnetic resonance (NMR) measurements of Na⁺_i and high-energy phosphates in glucose-perfused rat hearts exposed to hydroxyl radicals generated from H₂O₂ and Fe³⁺. In separate experiments, K⁺_i and Ca²⁺_i were measured with ³⁹K and ¹⁹F NMR, respectively. The hearts rapidly exhibited contracture. Threefold Na⁺_i increases and substantial K⁺_i depletion were observed. Glycolytic inhibition was indicated by rapid sugar phosphate accumulation and cellular energy depletion. Notably, however, severe functional and energetic deterioration and substantial elevation of Ca²⁺_i occurred before substantial Na⁺_i accumulation or K⁺_i depletion was observed. Further experiments investigated the ability of pyruvate to scavenge H₂O₂ and to protect the myocardium from oxidant stress. Pyruvate (1 or 2.5 mmol/L) dramatically attenuated functional and energetic alterations and alterations in Na⁺_i and K⁺_i, whereas acetate (2.5 mmol/L) offered no protection. Unlike pyruvate, lactate (5 mmol/L) has little or no capacity to scavenge H₂O₂ but has similar protective effects. In conclusion, pyruvate effectively protects against H₂O₂/Fe³⁺, largely by direct H₂O₂ scavenging. Protection with lactate may involve intracellular pyruvate augmentation. Without exogenous pyruvate or lactate, myocardial Na⁺ homeostasis can be substantially altered by oxidant stress, possibly via cellular energy depletion. Excess Na⁺_i accumulation may, in turn, hasten metabolic and functional deterioration, but a causal link with the initial alterations in function or Ca²⁺_i was not supported.

Key Words: free radicals • intracellular Na⁺ • nuclear magnetic resonance • high-energy phosphates • pyruvate

	Introduction

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Using various free radical–scavenging systems, many studies have shown a link between myocardial ischemic and reperfusion injury and the generation of various toxic free radical species,^{1 2 3 4} although exceptions have been observed.⁵ Free radical generation upon myocardial reperfusion has been demonstrated.^{6 7 8} Mitochondrial respiration is known to generate large quantities of the superoxide radical, · O₂^{- 9} ; it can also be generated via xanthine oxidase activity¹⁰ or from leukocytes.¹¹ Superoxide dismutase scavenges this radical, rapidly converting it to H₂O₂.¹² However, H₂O₂ is itself an oxidant and can also be reduced to the reactive hydroxyl radical, · OH, by Fe²⁺ via the well-known Fenton reaction.^{13 14 15 16} The Fe³⁺ produced can regenerate Fe²⁺ by reaction with either · O₂^{- 13 14 15} or H₂O₂ itself.^{13 16} Many reports have shown that formation of the extremely reactive · OH radical constitutes an important component of both · O₂^-- and H₂O₂-dependent injury and free radical–induced injury in general.^{2 13 14 15 16 17 18 19} Catalase and/or glutathione peroxidase can prevent · OH radical formation by scavenging H₂O₂, converting it to water and oxygen. However, another potentially important scavenging reaction for H₂O₂ that has been largely unexplored is the nonenzymatic decarboxylation of -keto acids such as pyruvate.^{20 21 22 23} This reaction forms a stoichiometric amount of acetate, carbon dioxide, and water from pyruvate.

Increasingly, it is being demonstrated that reactive oxygen intermediates specifically alter certain sensitive ion transport or other systems as opposed to affecting general sarcolemmal membrane integrity or introducing nonspecific leaks.^{24 25} Studies have clearly demonstrated that in myocardial²⁶ as well as other²⁷ cell types, H₂O₂ exposure causes direct inhibition of the glycolytic enzyme GAPDH and results in the accumulation of glycolytic intermediates proximal to that enzyme as well as depletion of high-energy phosphate compounds.^{16 26 27 28 29} Various free radical generation systems have been shown to affect myocardial Ca²⁺ homeostasis. Initial positive inotropy has been observed, followed by triggered activity and increased diastolic tension and/or contracture.^{30 31 32} Recently, cytosolic free Ca²⁺ (Ca²⁺_i) levels have been directly measured and found to increase rapidly in myocardial preparations on exposure to a · OH radical generation system.^{16 28} This system included H₂O₂ and Fe³⁺, a combination that ensures the formation of catalytic quantities of Fe²⁺ and the generation of · OH radicals.^{13 14 15 16} The mechanism for the rapid deterioration of Ca²⁺ homeostasis is undetermined, however. It could involve direct alteration of sarcolemmal and/or sarcoplasmic reticulum Ca²⁺ transport mechanisms. Alternatively, since several studies have reported a decrease in Na⁺,K⁺-ATPase activity with free radical exposure, a common hypothesis has centered around the sarcolemmal Na⁺ gradient and an alteration of both it and Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange.^{18 19 24 33 34} However, little information is available concerning the status of the myocardial sarcolemmal Na⁺ gradient upon controlled exposure to free radicals; no direct measurements of Na⁺_i levels have been reported.

The present study has used a unique and diagnostic NMR approach, which on exposure to H₂O₂ and Fe³⁺, rapidly monitored myocardial Na⁺_i with ²³Na NMR spectroscopy while, at the same time, measuring the energetic status with interleaved ³¹P NMR spectra.^{35 36} Energy status is an important factor because of its role in the maintenance of Na⁺,K⁺-ATPase activity.^{37 38} The first objective of the study was to determine what alterations occur in the Na⁺_i and phosphorus metabolites to help assess their role in this model of oxidant injury. To further define changes in ion homeostasis, K⁺_i and Ca²⁺_i changes were also measured in parallel experiments using state-of-the-art ³⁹K and ¹⁹F NMR methodology, respectively. The second goal was to investigate whether pyruvate and/or other metabolic substrates can exert a protective role. The study not only describes the Na⁺_i, K⁺_i, Ca²⁺_i, and energetic alterations that occur in the myocardium with H₂O₂/Fe³⁺ exposure but also documents a powerful protective effect of exogenous pyruvate. The study contains the first report that exogenous lactate also is protective, with an effect comparable to that of pyruvate.

	Materials and Methods

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Heart Preparations
Male Sprague-Dawley rats (270 to 400 g) were heparinized (1000 U IP) and anesthetized with sodium pentobarbital (200 mg/kg IP). The hearts were removed, put in cold perfusate, quickly trimmed, and Langendorff-perfused. The right atrium was removed, atrioventricular node conduction was surgically blocked, and hearts were stimulated to beat at 150 bpm as previously described.³⁶ Heart function was monitored by using a fluid-filled left ventricular balloon in line with a Spectramed p23XL transducer and an Astro-Med MT9500 multichannel recorder; functional data were recorded both on paper and digitally. LVEDP was set to 10 mm Hg. Hearts were initially perfused at 80 mm Hg by using a perfusion system as previously described³⁵ with a modified Krebs-Henseleit bicarbonate perfusate that was equilibrated with 95% O₂/5% CO₂ (32°C), was adjusted to pH 7.4 with NaOH, and contained the following (mmol/L): NaCl 116, KCl 4.7, MgSO₄ 1.2, CaCl₂ 1.5, NaHCO₃ 25, Na₂EDTA 0.5, and glucose 11 (Sigma Chemical Co). After the experiment, all hearts were weighed after drying to a constant weight in a 80°C oven. All animal procedures conformed to University of Alabama at Birmingham Institutional Animal Care and Use Committee guidelines.

Experimental Protocols
For experiments that collected interleaved ²³Na and ³¹P (or ³⁹K) NMR spectra, perfusion was switched to solution containing the ²³Na NMR shift reagent, Tm(DOTP)^5-, after a brief stabilization period with the initial perfusate. This allowed resolution of the intracellular and extracellular ²³Na (or ³⁹K) NMR resonances. The shift-reagent perfusate was similar to the initial perfusate described above except for the inclusion of 4 mmol/L Na₅Tm(DOTP) and additional substrates; NaCl was adjusted to keep the total Na⁺ content unchanged, and the total Ca²⁺ content was adjusted to 5.5 mmol/L to adjust for binding to the shift reagent, resulting in a free ionized Ca²⁺ of 1.07 mmol/L, as previously measured.³⁶ The shift reagent was prepared, purified, and recovered after the experiments, as previously described.³⁵ A total of five different substrate combinations were used in separate heart preparations: (1) glucose at 11 mmol/L (glucose, n=9), (2) glucose at 11 mmol/L and pyruvate at 1 mmol/L [glucose/pyruvate-(1 mmol/L), n=8], (3) glucose at 11 mmol/L and pyruvate at 2.5 mmol/L [glucose/pyruvate-(2.5 mmol/L), n=8], (4) glucose at 11 mmol/L and lactate at 5 mmol/L (glucose/lactate, n=7), and (5) glucose at 11 mmol/L and acetate at 2.5 mmol/L (glucose/acetate, n=4). To allow equilibration, perfusion with one of the above substrate combinations continued for 20 minutes before NMR spectra were acquired. Spectral acquisition was then initiated, and perfusion with the same perfusate continued during a brief control period; after which, infusion of Fe(NTA)₂^3-, the bis complex of ferric chloride and NTA (Sigma), was initiated into the aortic perfusion line just above the heart (final concentration, 10 µmol/L). Two minutes later, Fe(NTA)₂^3- infusion continued while heart perfusion was switched to perfusate which was identical except for the presence of 2 mmol/L H₂O₂ (concentrated stock of H₂O₂ was added 5 minutes earlier). After 4 minutes of perfusion with the H₂O₂-containing perfusate, perfusion was switched back to the perfusate without H₂O₂ that was used immediately before, and Fe(NTA)₂^3- infusion was terminated. The total NMR data collection period for the preparations was <45 minutes; by the end of this period, the hearts had generally been Langendorff-perfused for <80 minutes. The concentration of H₂O₂ in the 10% stock solutions (Sigma) was checked with the absorbance at 230 nm.³⁹

Parallel experiments collected ³⁹K NMR spectra using Tm(DOTP)^5- to resolve intracellular and extracellular K⁺, the same concentration as for the ²³Na NMR experiments. Two experimental groups were studied (n=3 for both), ³⁹K-glucose and ³⁹K-glucose/pyruvate-(2.5 mmol/L), which used perfusion conditions identical to the glucose and glucose/pyruvate-(2.5 mmol/L) groups, respectively, described above.

In another experimental group (n=3), Ca²⁺_i was measured by using ¹⁹F NMR and the fluorinated Ca²⁺ indicator 5F-BAPTA. For these experiments, the hearts were perfused with the initial perfusate (no shift reagent, glucose only) used in the other groups. After 10 minutes of control perfusion, indicator loading was accomplished by switching to a HEPES (10 mmol/L) pH-buffered perfusate solution containing 3% bovine serum albumin (Sigma, No. A-2153, fraction V) and 30 µmol/L of the acetoxymethyl ester of 5F-BAPTA, 5F-BAPTA-AM (Molecular Probes, Inc). A volume of 250 mL of loading solution was perfused/recirculated through the heart for 30 minutes. The inclusion of bovine serum albumin resulted in improved 5F-BAPTA-AM solubilization and tissue loading efficiency compared with previous methods.⁴⁰ After the loading procedure, perfusion was switched back to the initial bicarbonate perfusion solution, and the hearts were subsequently exposed to the H₂O₂/Fe³⁺ intervention.

NMR Methodology
A Bruker AM-360 WB spectrometer was used for all experiments. All ²³Na and ³¹P NMR spectra were obtained with a custom-built, switchable, temperature-controlled NMR probe designed to collect interleaved spectra from both nuclei without retuning.³⁵ Data collection alternated between the collection of ³¹P (2.5 minutes) and ²³Na (0.5 minutes) NMR spectra. ²³Na NMR spectra were obtained at 95.26 MHz with 144 transients; other ²³Na spectral acquisition parameters and the signal area measurement methodology were as previously described.³⁵ Spectral processing parameters were as previously described by Pike et al.³⁶

³¹P NMR spectra were obtained at 145.81 MHz with 72 transients; other ³¹P NMR spectral acquisition and processing parameters and signal area measurement methodology were as previously described.³⁵ For each experiment, a fully relaxed spectrum, using a recycle time of 10 seconds, was acquired immediately before the initial control period. By comparing these with the control spectra, empirical saturation factors for the various resonances were derived: 1.26, 1.08, 1.06, and 1.46 for PCr, ATP, P_i, and SP, respectively. Values for P_i and SP were determined from the glucose group only, because of the low control levels in the other groups.

³⁹K NMR spectra were obtained at 16.81 MHz by using a Bruker 20-mm broadband probe. The pulse sequence (90°_y-acq-180°_-y--90°_-y-acq, where acq is free induction decay acquisition and is a 5-microsecond delay) suppressed the acoustic ringing caused by the short preacquisition delay.⁴¹ The total sequence was rapidly repeated 1400 times (2880 acquisitions, 5 minutes) by using an acquisition time of 0.1 second, a sweep width of 59.5 ppm, a data size of 2048, and a preacquisition delay time of 5 microseconds. A gaussian multiplication was applied to the ³⁹K free induction decays by using NMR1 software (parameters, G1=0, G2=20, G3=0.1; Tripos Associates, Inc). By use of the NMR1 curve-fitting subroutine, lorentzian lineshapes were automatically fit to the spectra.

¹⁹F NMR spectra were obtained by use of a 20-mm Bruker ¹⁹F probe at 338.83 MHz, with 456 transients, a sweep width of 24.2 ppm, a pulse width of 65°, an acquisition time and total recycle time of 0.25 seconds, a preacquisition delay of 78.8 microseconds, and a data size of 2048. The ¹⁹F signal-to-noise ratio was improved by the installation of low-noise GaAsfet ¹⁹F preamplifiers (Advanced Receiver Research). Further improvements in the signal-to-noise ratio were obtained by continuously replacing the effluent surrounding the preparations with a 300 mmol/L mannitol (low dielectric) solution, which increased the Q factor of the ¹⁹F detection circuit. An adequate ¹⁹F signal-to-noise ratio was obtained from the perfused heart in 2 minutes, an improvement over previous studies.⁴⁰ The Ca²⁺_i was calculated according to the following equation: Ca²⁺_i=(K_d)(B)/(F), where B and F are areas under the Ca²⁺-bound and Ca²⁺-free 5F-BAPTA ¹⁹F NMR resonances, respectively; a K_d value of 308 nmol/L was used.⁴⁰

In addition to using exponential or gaussian multiplication of the FIDs, the signal-to-noise ratios of the ²³Na, ³¹P, and ³⁹K NMR spectra were further improved with a recently developed post–Fourier transformation processing technique, called SIFT (for spectral improvement by Fourier thresholding).⁴²

Absolute quantification of the intracellular ³¹P, ²³Na, and ³⁹K NMR resonances was accomplished by comparison with fully relaxed spectra obtained after each experiment from solutions of K₂PO₄, NaCl, and KCl, respectively, placed in heart-sized spheres. Because ²³Na and ³⁹K are quadrapolar nuclei (spin 3/2), the effective NMR visibility of their tissue signals can be <100%, as biexponential transverse relaxation behavior can cause a component of the signal to exhibit a broader spectral line shape.^{43 44} The control values of Na⁺_i measured in the present study agree with the values found in literature reporting the use of ion-selective electrodes in heart homogenates.⁴⁵ Consistent with previous studies,^{44 46} the control K⁺_i levels measured with ³⁹K NMR were lower than the levels measured by other techniques.⁴⁵ However, studies have shown the observation of changes in tissue ³⁹K NMR signals to be a reliable marker for changes in K⁺_i content.^{46 47}

Biochemical Assays
In experiments parallel to the NMR experimental protocols, intracellular pyruvate measurements were made in two groups (n=6 each) of hearts perfused with solutions identical to those used for the glucose and the glucose/lactate groups (without H₂O₂/Fe³⁺). After 20 minutes of perfusion with the shift-reagent perfusates, the hearts were freeze-clamped with Wollenberger clamps at the temperature of liquid N₂. The frozen tissue was pulverized in a mortar under liquid N₂, weighed, and added to 4 mL/g of cold 8% perchloric acid. The mixture was homogenized with a Tissumizer (Tekmar Co) and centrifuged at 4°C and 3000g for 10 minutes. The supernatant was analyzed for pyruvate (Sigma assay kit No. 726).

Pyruvate assays were also conducted on perfusate solutions identical to those used for the glucose/pyruvate-(1 mmol/L) and glucose/pyruvate-(2.5 mmol/L) groups (95% O₂/5% CO₂, 32°C). Assays were also conducted on a solution containing 10 mmol/L acetate, which was otherwise identical to that of the glucose/pyruvate-(2.5 mmol/L) group. Aliquots were taken before and at timed intervals after H₂O₂ addition (2 mmol/L) and combined with 1000 Sigma units/mL of catalase (Sigma No. C-40). This procedure removed H₂O₂ and quenched pyruvate decarboxylation, allowing accurate monitoring of the time course.²³ Lactate assays (Sigma assay kit No. 826) were conducted on samples collected in an analogous fashion from solutions identical to those used in the glucose/lactate groups, both with and without 10 µmol/L Fe(NTA)₂^3-.

H₂O₂ levels were measured in perfusate solutions identical to those used in all of the various experimental groups, including several assessed both with and without 10 µmol/L Fe(NTA)₂^3-. Assays were also performed on a solution that contained 10 mmol/L acetate, which was otherwise identical to the glucose/pyruvate-(2.5 mmol/L) solution. The H₂O₂ assay used the horseradish peroxidase–mediated oxidation of phenol red and was conducted as described by Pick and Keisari.³⁹ Aliquots were removed from the perfusion solutions at 0, 15, and 30 minutes after H₂O₂ addition (2 mmol/L).

Statistics
Values were expressed as mean±SEM. To minimize multiple comparisons, statistical analysis in the ²³Na/³¹P NMR protocols was restricted to three time points: control data obtained immediately before H₂O₂/Fe³⁺ administration (-1.5 minutes) and at the third and sixth spectroscopic measurements after the start of H₂O₂/Fe³⁺ (7.5 and 16.5 minutes, respectively). One-way ANOVA and, in some cases, paired t tests were used. Results were regarded as significant at P<.05.

	Results

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Functional Measurements
Fig 1 indicates mean LVDP and LVEDP across time for the experimental groups. The data indicate that the glucose and glucose/acetate groups displayed rapid contractile failure and severe increases in LVEDP in response to H₂O₂/Fe³⁺ exposure. In striking contrast, the groups containing pyruvate or lactate were largely protected from such deleterious effects; virtually no increases in LVEDP were observed, and contraction was maintained. However, differences between the two pyruvate groups were observed. The transient positive inotropy observed with the glucose/pyruvate-(1 mmol/L) group was much less than that of the glucose/pyruvate-(2.5 mmol/L) group and was also followed by a decrease in LVDP to levels near 50% of control. Interestingly, the response of the glucose/lactate group was very similar to that of the glucose/pyruvate-(1 mmol/L) group. These data clearly demonstrate a powerful modulation of the functional response to H₂O₂/Fe³⁺ with exogenous pyruvate or lactate.

View larger version (29K): [in this window] [in a new window]   Figure 1. Functional data obtained during the H2O2/Fe3+ protocol are indicated across time for the five experimental groups from which interleaved 23Na and 31P NMR data were obtained. A, Mean LVDP. B, Mean LVEDP. indicates glucose; , glucose/acetate; , glucose/pyruvate-(1 mmol/L); , glucose/pyruvate-(2.5 mmol/L); and , glucose/lactate.

Statistical comparisons of the functional data indicated that compared with control values taken immediately before H₂O₂/Fe³⁺, only the glucose and glucose/acetate groups indicated significant increases in LVEDP at 7 and at 16 minutes after the start of H₂O₂/Fe³⁺. The LVDP was significantly different from control levels for all groups at 7 minutes and for all groups except glucose/pyruvate-(2.5 mmol/L) at 16 minutes. No differences in functional parameters were observed between the glucose and other groups during the control period. However, all groups except the glucose/acetate group differed from the glucose group at the 7- and 16-minute time points in both LVEDP and LVDP.

NMR Measurements
Fig 2 shows typical ³¹P NMR spectra acquired before and 17 minutes after the start of H₂O₂/Fe³⁺. The ³¹P NMR spectra in the glucose group exhibited a complete loss of ATP and PCr with H₂O₂/Fe³⁺ exposure. This energy depletion was concomitant with a large accumulation of SP, whereas the accumulation of P_i (frequency 4.5 ppm) was minimal. Fig 2B demonstrates that with the glucose/pyruvate-(1 mmol/L) group, ATP and PCr depletion was largely prevented and SP accumulation was far less than that observed in the glucose group.

View larger version (11K): [in this window] [in a new window]   Figure 2. Typical 31P NMR spectra that were acquired before (lower spectra) and 17 minutes after H2O2/Fe3+ (upper spectra). Spectra were obtained from a heart within the glucose group (A) and from the glucose/pyruvate-(1 mmol/L) group (B). Resonances from intracellular SP, PCr, and -, ß-, and -ATP, as well as phenylphosphonic acid (PPA) from the left ventricular balloon, are as indicated.

Fig 3 shows mean phosphorus metabolite data from the various experimental groups. Consistent with Fig 2, the glucose group showed SP rapidly increasing to extreme levels (>120 µmol/g dry wt), reaching half-maximal levels only 5 minutes after the start of H₂O₂/Fe³⁺, whereas P_i accumulation did not occur. The data indicate a concomitant decrease in PCr, which rapidly approached near-zero levels. ATP depletion was of similar severity but occurred less rapidly than did PCr depletion. As with the functional response, the glucose/acetate group showed a pattern of phosphorus metabolite changes similar to that of the glucose group, ie, rapid SP accumulation and complete and irreversible depletion of high-energy phosphates. In contrast, inclusion of pyruvate and lactate substantially altered the response. Changes in phosphorus metabolites were entirely prevented in the glucose/pyruvate-(2.5 mmol/L) group. In the glucose/pyruvate-(1 mmol/L) group, alterations occurred but were greatly attenuated compared with those observed in the glucose group: SP accumulation was 50% of that observed in the glucose or glucose/acetate groups, ATP decreased to 70% of control levels, and PCr decreased to 50% of control levels and then largely recovered. Analogous to the trends observed in the functional data, the glucose/lactate group indicated phosphorus metabolite changes that were remarkably similar to those of the glucose/pyruvate-(1 mmol/L) group.

View larger version (28K): [in this window] [in a new window]   Figure 3. Mean phosphorus metabolite data (in micromoles per gram dry weight) obtained during the H2O2/Fe3+ protocol are indicated across time for the same experimental groups as in Fig 1. A, PCr levels. B, ATP levels. C, SP levels. D, Pi levels. indicates glucose; , glucose/acetate; , glucose/pyruvate-(1 mmol/L); , glucose/pyruvate-(2.5 mmol/L); and , glucose/lactate.

Statistical comparison of the phosphorus metabolite data during the control period (obtained 1 minute before the start of H₂O₂/Fe³⁺) with values obtained at 8 and 17 minutes after the start of H₂O₂/Fe³⁺ indicated that at those latter times, SP levels were significantly elevated and both PCr and ATP were significantly decreased within all groups except the glucose/pyruvate-(2.5 mmol/L) group. Comparisons between the glucose and the other groups across time indicated that during the control period, there were no significant differences in ATP. Consistent with previous observations, the glucose group indicated slightly but significantly higher P_i levels during the control period than the other substrate groups and statistically lower PCr than observed in one other group, glucose/pyruvate-(1 mmol/L).^{48 49 50} At the 8- and 17-minute time points, SP (and P_i at 17 minutes only) was significantly higher and ATP and PCr were lower in the glucose group than in all other groups, except for the glucose/acetate group.

Table 1 shows pH_i values calculated from the chemical shift of the P_i peak.³⁵ The values indicate that pH_i changes occurred only in the glucose and glucose/acetate groups, decreasing moderately from 7.2 to 6.8. At the 8-minute time point, the pH_i in the glucose group was significantly below the control level; at 17 minutes, both glucose and glucose/acetate were below control levels. None of the groups differed significantly in pH_i from the glucose group during the control period. However, at 8 and at 17 minutes, the glucose group indicated pH_i levels that were significantly lower than those for all groups except the glucose/acetate group.

View this table: [in this window] [in a new window]   Table 1. Mean pHi for Five Experimental Groups at Three Time Points Relative to the Start of H2O2/Fe3+

Fig 4 displays ²³Na NMR spectra, which were obtained in an interleaved fashion with the ³¹P NMR spectra shown in Fig 2, on the same preparations. Excellent resolution of the unshifted Na⁺_i resonance was obtained, allowing accurate estimations of Na⁺_i changes. Fig 4A indicates that by 19 minutes from the start of H₂O₂/Fe³⁺, extensive Na⁺_i accumulation had occurred in the glucose group. In contrast, Fig 4B indicates that in the glucose/pyruvate-(1 mmol/L) group, virtually no Na⁺_i accumulation had occurred; the spectra actually indicate a slight decrease. Fig 4C shows the mean Na⁺_i data for the various experimental groups. The graph shows that by 19 minutes after the start of H₂O₂, threefold increases in Na⁺_i occurred in the glucose and glucose/acetate groups. In contrast, Na⁺_i increases were entirely prevented in both of the pyruvate groups as well as in the glucose/lactate group.

View larger version (23K): [in this window] [in a new window]   Figure 4. A and B, 23Na NMR spectra were obtained from the same preparations as used for the 31P NMR spectra in Fig 2 before (lower spectra) and 19 minutes after the start of H2O2/Fe3+ (upper spectra). Spectra are from a heart within the glucose group (A) and from the glucose/pyruvate-(1 mmol/L) group (B). The unshifted resonance at 0 ppm is from Na+i. That at 3 ppm is from Na+o, shifted downfield with 4 mmol/L Tm(DOTP)5-. The resonance at 7 ppm is from Na+ within the left ventricular balloon, also shifted with Tm(DOTP)5- (larger shift due to absence of Ca2+). C, Mean Na+i data (in micromoles per gram dry weight) obtained during the H2O2/Fe3+ protocol are indicated across time for the same experimental groups as shown in Figs 1 and 3. indicates glucose; , glucose/acetate; , glucose/pyruvate-(1 mmol/L); , glucose/pyruvate-(2.5 mmol/L); and , glucose/lactate.

Statistical comparisons (by ANOVA) of the control Na⁺_i levels (obtained 2 minutes before H₂O₂/Fe³⁺) with data obtained at 7 and 16 minutes after the start of H₂O₂/Fe³⁺ indicated that only the glucose group showed a significant increase at 7 minutes, with both glucose and glucose/acetate groups showing significantly elevated levels at 16 minutes. Interestingly, further analysis by paired t test not only confirmed this result but also indicated that slight but significant decreases in Na⁺_i levels were evident in the glucose/pyruvate-(1 mmol/L) and glucose/pyruvate-(2.5 mmol/L) groups at the 7-minute time point, 10% below their respective control levels. During the control period, slight differences in Na⁺_i were noted between the experimental groups, with the glucose/lactate (18.1±0.7 µmol/g dry wt) and glucose/acetate (20.3±1.8 µmol/g dry wt) groups indicating statistically higher Na⁺_i levels than the glucose group (14.9±0.8 µmol/g dry wt). At 7 minutes, only the glucose/pyruvate-(1 mmol/L) group showed significantly different Na⁺_i (lower) than the glucose group. However, at 16 minutes, all groups except the glucose/acetate group showed significantly lower levels than the glucose group.

The pattern of Na⁺_i changes noted in these experiments in some respects suggests an insensitivity of Na⁺ homeostasis to oxidant stress compared with the other parameters monitored; little or no change in Na⁺_i was observed in the glucose/pyruvate-(1 mmol/L) and glucose/lactate groups, whereas clearly observable changes were noted in function and in phosphorus metabolites. In the groups in which alterations in Na⁺ homeostasis did occur, the alterations were severe and associated with irreversible oxidant stress conditions. However, even in these groups (glucose and glucose/acetate), the large increases in Na⁺_i occurred with a delayed time course. The functional changes with H₂O₂ were extremely rapid: at 7 minutes from the start of H₂O₂ administration, the LVDP was near zero and LVEDP was 40 mm Hg for the glucose and glucose/acetate groups and increasing rapidly. Fig 3 indicates that at that time, the precipitous decrease in PCr had reached 10% and 20% of the control value for the glucose and glucose/acetate groups, respectively (at the 8-minute time point). SP was in the range of 100 µmol/g dry wt and approaching maximal values. ATP had decreased to 20% and 40% of the control value for the glucose and glucose/acetate groups, respectively, and was still falling rapidly, However, at 7 minutes, the Na⁺_i increase had only just started and reached levels only 30% and 10% above control levels for the glucose and glucose/acetate groups, respectively, a small fraction of the 300% increases observed later in the protocol.

In Fig 5, ³⁹K NMR spectra, obtained from hearts in the ³⁹K-glucose and ³⁹K-glucose/pyruvate-(2.5 mmol/L) experimental groups, are shown before and after H₂O₂/Fe³⁺. The Tm(DOTP)^5- shifted the extracellular ³⁹K resonance sufficiently to obtain adequate resolution from the K⁺_i resonance, and signal areas were reliably integrated by using curve-resolution techniques. This allowed direct measurement of the K⁺_i without having to use strategies based on differences in intracellular and extracellular T₁ (spin-lattice relaxation time) values.⁴⁷ The spectrum from the ³⁹K-glucose group indicates a reduction in the K⁺_i signal with H₂O₂/Fe³⁺. In contrast, that from the ³⁹K-glucose/pyruvate-(2.5 mmol/L) group does not show any such depletion. Fig 5C indicates the mean K⁺_i data. Loss of K⁺_i was clearly evident in the ³⁹K-glucose group. At the 16.5-minute measurement and beyond (relative to the start of H₂O₂/Fe³⁺), the ³⁹K-glucose group indicated significantly lower K⁺_i values compared with control values and with values in the ³⁹K-glucose/pyruvate-(2.5 mmol/L) group at equivalent times. The accuracy of the amount of intracellular K⁺_i depletion in fractional or absolute terms may be affected by issues of intracellular compartmentation and NMR visibility peculiar to ³⁹K NMR.^{43 44 46} However, the time course of the changes is informative and indicates that like that of Na⁺_i, the deterioration of K⁺ homeostasis in the ³⁹K-glucose group lags behind the other metabolic and functional alterations. No change was evident at 7 minutes, and little is evident even at 12 minutes after the start of H₂O₂/Fe³⁺.

View larger version (20K): [in this window] [in a new window]   Figure 5. A and B, Typical 39K NMR spectra that were acquired from hearts before (lower spectra) and 26.5 minutes after the start of H2O2/Fe3+ (upper spectra). Spectra were obtained from a heart within the 39K-glucose group (A) and from the 39K-glucose/pyruvate-(2.5 mmol/L) group (B). C, Mean K+i data (in micromoles per gram dry weight) obtained during the H2O2/Fe3+ protocol are indicated across time for the 39K-glucose () and 39K-glucose/pyruvate-(2.5 mmol/L) () experimental groups.

Previous reports have indicated rapid and extensive changes in Ca²⁺_i in cardiac preparations upon exposure to H₂O₂/Fe³⁺.^{16 28} This was confirmed in the present study by the measurement of Ca²⁺_i in several heart preparations perfused with glucose (only). Fig 6A shows ¹⁹F NMR spectra obtained before and after H₂O₂/Fe³⁺ administration in a 5F-BAPTA–loaded rat heart. The spectrum obtained after H₂O₂ shows an increased Ca²⁺-bound–to–Ca²⁺-free ratio, indicating that Ca²⁺_i increased with the H₂O₂/Fe³⁺ intervention. Fig 6B shows the mean Ca²⁺_i concentrations across time with 2-minute time resolution and confirms that Ca²⁺_i did increase to very high levels, >1 µmol/L. The Ca²⁺_i became noticeably higher at 6 minutes after the start of the H₂O₂ intervention. A marked increase in LVEDP also began at that time, increasing by 40 mm Hg by the 10th minute.

View larger version (15K): [in this window] [in a new window]   Figure 6. A, Typical 19F NMR spectra (2-minute acquisitions) were obtained before (lower spectrum) and 14 minutes after the start of H2O2/Fe3+ administration (upper spectrum) in a 5F-BAPTA–loaded rat heart. B, Mean Ca2+i concentrations obtained during the H2O2/Fe3+ protocol were calculated from the 19F NMR spectral data and are indicated across time.

Biochemical Measurements
Changes in H₂O₂ concentrations over time are shown for the different perfusate solutions in Table 2. To more accurately detect differences within and between substrate groups, the entire assay was repeated five times in each group, and the calculated means of each group were normalized to a zero time value of 2.0 mmol/L H₂O₂ (the quantity of H₂O₂ added at that time). The table indicates that in the solutions containing pyruvate, H₂O₂ decreased markedly over time. At 15 and 30 minutes after H₂O₂ addition, all pyruvate groups indicated H₂O₂ levels that were significantly below the control level (2 mmol/L) and below levels measured in the glucose group at those times. To within the limits of the experimental variability of the H₂O₂ assay, such differences were not detected in the other groups at 15 minutes. However, at 30 minutes, the glucose/lactate group with Fe(NTA)₂^3- indicated significant differences from both control and the glucose group, although the differences were not nearly as large as those observed in the pyruvate groups. The group containing 10 mmol/L acetate, which was otherwise identical to that of the glucose/pyruvate-(2.5 mmol/L) group, indicated a H₂O₂ decrease that was indistinguishable from that of the latter group, indicating that acetate had no effect.

View this table: [in this window] [in a new window]   Table 2. Concentrations of H2O2 in Different Perfusate Solutions at 15 and 30 Minutes After Addition of 2 mmol/L H2O2

Fig 7 shows the change in pyruvate with time in the glucose/pyruvate-(1 mmol/L) and glucose/pyruvate-(2.5 mmol/L) perfusion solutions after the addition of 2 mmol/L H₂O₂. In both solutions, pyruvate steadily decreased with time. In the glucose/pyruvate-(2.5 mmol/L) solution that also contained 10 mmol/L acetate, the decrease in H₂O₂ observed at 5 minutes was indistinguishable from that of the analogous solution without acetate.

View larger version (17K): [in this window] [in a new window]   Figure 7. Measured pyruvate concentrations are shown at various time intervals after H2O2 addition for glucose/pyruvate-(2.5 mmol/L) () and glucose/pyruvate-(1.0 mmol/L) () perfusion solutions, as well as for glucose/pyruvate-(2.5 mmol/L) with 10 mmol/L acetate (). Sample aliquots were combined with catalase to remove H2O2.

Lactate levels measured in the glucose/lactate perfusate solution indicated no change from the initial level (5 mmol/L) 30 minutes after the addition of 2 mmol/L H₂O₂, with or without Fe(NTA)₂^3-. However, when H₂O₂ and Fe(NTA)₂^3- were increased by 25-fold (50 mmol/L and 250 µmol/L, respectively), lactate was found to decrease slightly, from 5 to 4.6 mmol/L after 30 minutes (and to 4.3 mmol/L at 60 minutes). No decrease in lactate was detected in 50 mmol/L H₂O₂ solutions without Fe(NTA)₂^3-.

Intracellular pyruvate concentrations were determined in hearts perfused with either the glucose or glucose/lactate perfusion solutions. The pyruvate levels in the glucose group were 0.074±0.019 mmol/L when an intracellular water–to–gram wet weight ratio of 0.42 was used.⁴⁵ The intracellular pyruvate levels in the glucose/lactate group (0.197±0.018 mmol/L) were significantly augmented (P=.0009).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many studies have documented an increase in ³¹P NMR phosphomonoester resonance intensity (6 ppm relative to PCr) in myocardium exposed to H₂O₂.^{16 26 28 29} Chatham et al²⁶ demonstrated this to result from glycolytic inhibition and the accumulation of various SP glycolytic intermediates; analogous to a previous study with P388D₁ cells,²⁷ the glycolytic enzyme GAPDH was effectively inhibited, while glycolytic intermediates proximal to GAPDH increased, particularly fructose 1,6-bisphosphate. This metabolite was also identified as the major phosphomonoester constituent in ferret myocardium exposed to H₂O₂/Fe³⁺.²⁸ The GAPDH enzyme has long been known to have extreme in vitro sensitivity to H₂O₂ and/or oxidant stress, which causes inactivation by sulfhydryl modification.⁵¹ This is the mechanism by which the glycolytic inhibitor iodoacetate works; analogous to H₂O₂, iodoacetate also induces excess fructose 1,6-bisphosphate accumulation.^{52 53}

The present study provides clear evidence that myocardial Na⁺ and K⁺ homeostasis can be seriously altered under conditions of acute oxidant stress. This is consistent with reports of decreased Na⁺,K⁺-ATPase activity after exposure to reactive oxygen intermediates.^{18 19 24 33 34} Kukreja et al¹⁹ found the Na⁺,K⁺-ATPase activity of myocardial preparations to be sensitive to the · OH radical. Various free radical scavengers can protect the Na⁺-K⁺ activity of ventricular muscle reperfused after 2 hours of ischemia.³⁴ Recently, Shattock and Matsuura²⁴ observed a decrease in Na⁺-K⁺ pump current with rose bengal photoactivation (singlet oxygen and · O₂^- generator); the current fell by 50% over 10 minutes and was attributed to direct enzyme alteration. However, because Na⁺,K⁺-ATPase activity also depends on cellular energy status, such changes require careful interpretation.^{37 38}

In terms of the identification of potential causal factors for alterations in Na⁺_i homeostasis, the comprehensive NMR approach used in the present study has certain advantages.^{35 36} It was shown that the H₂O₂/Fe³⁺ intervention induced rapid depletion of ATP and PCr in the glucose and glucose/acetate groups that preceded substantial elevations in Na⁺_i. Hence, energy depletion could have been an important causal factor. This would also be consistent with the absence of Na⁺_i increases in the glucose/pyruvate-(1 mmol/L) and glucose/lactate groups, which suffered only mild high-energy phosphate depletion. Direct alteration of the Na⁺,K⁺-ATPase would appear unlikely in those groups, although potentially, such an effect could have been masked by reserve enzyme capacity. It is also possible that compensating decreases in sarcolemmal Na⁺ current occurred,²⁵ perhaps consistent with the slight Na⁺_i decreases that were detected in the glucose/pyruvate-(1 mmol/L) and glucose/pyruvate-(2.5 mmol/L) groups. Ultimately, however, the perturbation of Na⁺ homeostasis via direct enzyme alteration of the enzyme is not entirely consistent with the observation that when Na⁺_i increases do occur after the termination of acute H₂O₂/Fe³⁺ administration, they lag behind changes in energetic and functional parameters and accelerate gradually. Importantly, K⁺_i depletion was also delayed well beyond the energetic and functional changes; substantial changes were not observed until 16 minutes after the start of H₂O₂/Fe³⁺. Like Na⁺_i, K⁺_i is an indicator for changes in Na⁺,K⁺-ATPase activity.^{37 38} Hence, as alterations in Na⁺_i and K⁺_i were invariably preceded by a severely depleted energy condition, that condition must be considered as playing a potentially important role.

Recent evidence has indicated that Ca²⁺_i accumulation is likely to be an important player in free radical–induced injury. Studies have reported that in myocardium exposed to oxidant stress, arrhythmogenic activity occurs which is likely to be related to Ca²⁺_i overload.^{16 30 31 32} Severe Ca²⁺_i overload can induce irreversible injury via mechanisms such as phospholipase and/or protease activation⁵⁴ or mitochondrial Ca²⁺ sequestration.⁵⁵ An electron microscopy study by Vandeplassche et al⁵⁶ using photoactivation of rose bengal indicated contraction band necrosis and severe ultrastructural damage to mitochondria, including the appearance of calcium precipitates. Direct Ca²⁺_i measurements in rat myocytes using the fluorescent Ca²⁺ indicator indo 1 have indicated that Ca²⁺_i increases substantially upon exposure to · OH radicals generated via H₂O₂/Fe³⁺.¹⁶ The increases correlated temporally with increased twitch amplitude and automaticity, followed shortly by contracture and inexcitability. Corretti et al²⁸ used ¹⁹F NMR spectroscopy to measure Ca²⁺_i in the 5F-BAPTA–loaded ferret hearts and found that upon brief exposure of the heart to H₂O₂/Fe³⁺, Ca²⁺_i increased rapidly and extensively, in parallel with diastolic tension and contracture. The ¹⁹F NMR data obtained in the present study with glucose-perfused hearts confirmed this, indicating a rapid increase in Ca²⁺_i, from a control level of 230 nmol/L to levels threefold higher at 630 nmol/L, at only 6 minutes from the start of H₂O₂/Fe³⁺. At 6 minutes, a sharp increase in LVEDP began in the 5F-BAPTA–loaded hearts, as had occurred in the analogous glucose group shown in Fig 1. This is consistent with the previous report indicating that 5F-BAPTA loading attenuates LVDP but does not substantially alter the time course of H₂O₂/Fe³⁺-induced changes in mechanical function and phosphorus metabolites.²⁸ Hence, the data in this and in previous studies provide convincing evidence that Ca²⁺_i changes are on the leading edge of the functional and metabolic abnormalities in this model of oxidant stress, rising rapidly in tandem with LVEDP.

Because the primary sarcolemmal Ca²⁺ extrusion mechanism is Na⁺-Ca²⁺ exchange, measurements of Na⁺ homeostasis are critical in investigating potential mechanisms leading to free radical–induced Ca²⁺_i overload. In this regard, the observed threefold changes in Na⁺_i are certain to bias Na⁺-Ca²⁺ exchange toward reduced Ca²⁺ extrusion and increased Ca²⁺ entry.⁵⁷ Hence, Na⁺_i accumulation clearly is a marker for severe oxidant stress in the present study and supports a role for Na⁺_i accumulation in oxidant stress–related Ca²⁺_i accumulation and injury. Importantly, however, examination of the early period after the H₂O₂/Fe³⁺ intervention reveals that at that time, the involvement of Na⁺_i accumulation in the functional and metabolic deterioration of the myocardium is an open question. The Ca²⁺_i increased threefold by 6 minutes after H₂O₂/Fe³⁺, yet at that point Na⁺_i increases of at most 30% had occurred in the glucose group. It is unlikely that such changes could induce a Ca²⁺_i overload state.⁵⁷ At a later point, however, the progression of Na⁺_i accumulation would most likely accelerate Ca²⁺ entry. This is consistent with the observed Ca²⁺_i increase, which continued unabated until the 15-minute time point.

Mechanisms that might explain the more immediate functional and metabolic responses to H₂O₂/Fe³⁺ include Ca²⁺_i overload occurring via increased L-type Ca²⁺ current, previously shown to increase in response to a xanthine oxidase/hypoxanthine free radical generation system.³¹ This would be consistent with the observation that the Ca²⁺ channel blocker nitrendipine inhibits the contractile dysfunction induced with H₂O₂/Fe³⁺.¹⁶ An activation of Ca²⁺ leak channels is also possible,⁵⁸ as well as inhibition of the sarcolemmal Ca²⁺ ATPase.⁵⁹ Strong evidence also exists for effects localized to the sarcoplasmic reticulum, which could induce arrhythmias and transient positive inotropy; however, effects on the sarcoplasmic reticulum alone are not likely to induce sustained Ca²⁺_i overload.^{17 30 32} Finally, an interesting recent report has suggested that the accumulation of glycolytic byproducts may have some direct (but unspecified) effect on Ca²⁺ homeostasis that is separate from that of energy depletion.⁵³

An important observation of the present study is that exogenous pyruvate and lactate provided a high degree of protection against the H₂O₂/Fe³⁺ radical generation system. It is unlikely that this effect is simply due to provision of a nonglycolytic energy substrate that circumvents glycolytic block. Perfused rat hearts have ample nonglycolytic substrate (triglyceride) reserves, and exogenous acetate provides no protection from functional and metabolic deterioration. This is consistent with a previous study that found little protection against H₂O₂ toxicity with butyrate.²⁶ In the context of ischemia, pyruvate has been reported to improve reperfusion functional recovery because of an enhanced ability to quickly reenergize the phosphorylation potential of the myocardium,^{49 50} related in part to pyruvate dehydrogenase activation.⁶⁰ It is unclear to what extent this effect would play a role in terms of protection from oxidant stress under continuously aerobic conditions. However, the present study provides clear evidence that the protection from pyruvate could be largely related to the direct reaction it undergoes with H₂O₂. Fig 7 demonstrates that pyruvate decreases steadily in the presence of H₂O₂ under the experimental conditions of the present study. Similarly, Table 2 indicates that H₂O₂ also decreases in the pyruvate perfusion solutions. With the possible exception of the lactate/Fe(NTA)₂^3- solution, H₂O₂ was stable in all the other solutions. The time course of the H₂O₂ and pyruvate changes was very consistent with that previously reported, and to within the error of the measurements, the changes in H₂O₂ and pyruvate were consistent with the reported 1:1 stoichiometry of the decarboxylation reaction; the H₂O₂ decrease in glucose/pyruvate-(1 mmol/L) solution was limited to 1 mmol/L.^{20 22 23} As would be expected from a reaction that breaks a carbon-carbon bond and evolves CO₂, product inhibition from acetate was not observed.²⁰ Consistent with the mechanism of the decarboxylation reaction, iron catalysis, ie, exposure to the · OH radical, was not required.²⁰ In the context of previous studies, these findings indicate conclusively that the nonenzymatic decarboxylation reaction decreased exogenous H₂O₂ in our experimental system. The time course in Fig 7 indicates that during the 5 minutes that lapsed between H₂O₂ addition and tissue perfusion, the H₂O₂ concentration in the pyruvate perfusion solutions would have decreased substantially. At 5 minutes, according to 1:1 pyruvate/H₂O₂ reaction stoichiometry, the H₂O₂ would be at 30% and 60% of the initial (2 mmol/L) level for the glucose/pyruvate-(2.5 mmol/L) and glucose/pyruvate-(1 mmol/L) solutions, respectively. Hence, exogenous H₂O₂ scavenging is likely to play an important role in the extraordinary protection observed with pyruvate.

It is an interesting observation that like pyruvate, lactate also provides potent protection from the H₂O₂/Fe³⁺ intervention. Lactate is not an -keto acid and cannot participate in a decarboxylation reaction; previous reports have clearly demonstrated that it does not decrease in the presence of H₂O₂ alone, and this was confirmed in the present study.²³ However, an Fe²⁺-catalyzed H₂O₂ oxidation of lactate (to pyruvate) was reported as early as 1900 by Fenton and Jones.⁶¹ Upon investigation of this reaction in glucose/lactate perfusion solution containing Fe(NTA)₂^3-, we found it to be either nonexistent or extremely slow. No significant change in H₂O₂ (from 2 mmol/L) had occurred by 15 minutes of incubation, but a slight decrease was measured after 30 minutes. Lactate depletion was not observed, possibly because of the difficulty of detecting small percent changes. Upon increasing H₂O₂ by 25-fold, a small lactate decrease was detected after 30 minutes but only in the presence of Fe(NTA)₂^3-. Hence, lactate oxidation may have occurred to some degree, but it is important to note that during the experimental protocols, Fe(NTA)₂^3- was administered by infusion into the aortic line just proximal to the heart. Clearly, exogenous depletion of H₂O₂ via lactate oxidation would be negligible in the context of the perfused-heart experiments.

Given the much faster H₂O₂ scavenging activity of pyruvate compared with lactate, it is possible that the lactate protection could be related to an intracellular augmentation of pyruvate, occurring via the rapid and reversible lactate dehydrogenase reaction.⁶² In fact, our assays of intracellular pyruvate clearly revealed that in hearts perfused with 5 mmol/L exogenous lactate, intracellular pyruvate increased by 2.7-fold compared with the level in hearts perfused with glucose only. This is consistent with a report indicating that in the presence of 10 mmol/L lactate, intracellular pyruvate increased by 4.7-fold in the perfused rat heart.⁶³ Furthermore, pyruvate production from glycolytic sources would be likely to decrease as GAPDH inhibition progresses during H₂O₂/Fe³⁺ exposure. Although further studies are required to conclusively determine the mechanism for the protection with lactate, the experiments indicate that increased intracellular H₂O₂ scavenging via intracellular pyruvate augmentation needs to be considered. This could also play a role in the protection observed with exogenous pyruvate, because intracellular pyruvate is known to markedly increase in the presence of exogenous pyruvate.⁴⁹ This would be consistent with the decrease in postischemic myocardial free radical production reported in the presence of exogenous pyruvate.²¹

In summary, the present study demonstrates that large perturbations in cationic and energy homeostasis can occur in myocardium exposed to H₂O₂/Fe³⁺, including substantial alterations in Na⁺_i, K⁺_i, and Ca²⁺_i. However, caution should be observed in causally relating Na⁺_i and K⁺_i changes to the initial deterioration in Ca²⁺ homeostasis and function. Also, energy depletion needs to be carefully considered as playing a potentially important role in Na⁺_i and/or K⁺_i changes during oxidant stress. Finally, the data document a powerful capacity for pyruvate and lactate to attenuate H₂O₂/Fe³⁺-induced oxidant injury.

	Selected Abbreviations and Acronyms

 

5F-BAPTA = 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid GAPDH = glyceraldehyde 3-phosphate dehydrogenase LVDP = left ventricular developed pressure LVEDP = left ventricular end-diastolic pressure NMR = nuclear magnetic resonance NTA = nitrilotriacetic acid PCr = phosphocreatine Pi = inorganic phosphate SP = sugar phosphate Tm(DOTP)5- = thulium 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetramethylenephosphonate

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant R29-HL-45684 (Dr Pike). Dr Pike is an Established Investigator of the American Heart Association.

Received January 27, 1995; accepted June 14, 1995.

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