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Cardiovascular Research 1999 42(1):149-161; doi:10.1016/S0008-6363(98)00300-9
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Mitochondrial metabolism of pyruvate is required for its enhancement of cardiac function and energetics1

Robert T Mallet* and Jie Sun

Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA

* Corresponding author. Tel.: +1-817-735-2260; fax: +1-817-735-5084. E-mail address: malletr@hsc.unt.edu

Received 2 January 1998; accepted 21 September 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Pyruvate augmentation of contractile function and cytosolic free energy of ATP hydrolysis in myocardium could result from pyruvate catabolism in the mitochondria or from increased ratio of the cytosolic NAD+/NADH redox couple via the lactate dehydrogenase equilibrium. Objective: To test the hypothesis that cytosolic oxidation by pyruvate is sufficient to increase cardiac function and energetics. Methods: Isolated working guinea-pig hearts received 0.2 mM octanoate±2.5 mM pyruvate as fuels. {alpha}-Cyano-3-hydroxycinnamate (COHC, 0.6 mM) was administered to selectively inhibit mitochondrial pyruvate uptake without inhibiting pyruvate’s cytosolic redox effects or octanoate oxidation. The effects of pyruvate and COHC on sarcoplasmic reticular Ca2+ handling were examined in 45Ca-loaded hearts. Results: Pyruvate increased left ventricular stroke work and power 40%, mechanical efficiency 29%, and cytosolic ATP phosphorylation potential nearly fourfold. 14CO2 formation from [1-14C]pyruvate was inhibited 65% by COHC, and octanoate oxidation, i.e. 14CO2 formation from [1-14C]octanoate, concomitantly increased threefold. COHC prevented pyruvate enhancement of left ventricular function, mechanical efficiency and cytosolic phosphorylation potential, but did not alter respective levels in pyruvate-free control hearts and augmented cytosolic oxidation by pyruvate. Pyruvate increased sarcoplasmic reticular Ca2+ turnover, i.e. Ca2+ uptake and release, as indicated by 62% decrease in caffeine-induced 45Ca release following 40 min 45Ca washout (P<0.01). In presence of COHC, pyruvate did not lower caffeine-induced 45Ca release; thus, COHC abrogated pyruvate enhancement of Ca2+ turnover (P<0.001). Conclusion: Pyruvate oxidation of cytosolic redox state is not sufficient to increase cardiac function, cytosolic energetics and sarcoplasmic reticular Ca2+ turnover when mitochondrial pyruvate transport is disabled; thus, mitochondrial metabolism of pyruvate is essential for its metabolic inotropism.

KEYWORDS Mitochondrial pyruvate transport; ATP phosphorylation potential; Citrate; Sarcoplasmic reticular calcium transport; Caffeine; Mechanical efficiency


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The working myocardium is heavily dependent on oxidation of fuels extracted from the coronary circulation to sustain metabolic energy production essential for its contractile activity. Although myocardium is capable of oxidizing a variety of substrates including fatty acids, glucose, pyruvate, lactate, ketone bodies and amino acids, shifts in substrate supply can produce appreciable changes in cardiac function and energetics [1–3]. In this respect, pyruvate has proven particularly effective as a metabolic inotropic agent. Pyruvate augments contractile function in isolated [3, 4]and in situ hearts [5]even when other fuels are available. Pyruvate-enhanced contractile performance is paralleled by increased thermodynamic state of the cytosolic free adenylate system, i.e. cytosolic ATP phosphorylation potential [3, 5, 6]; thus, in pyruvate perfused myocardium more free energy is available from ATP hydrolysis for energy-consuming processes central to contractile function. The postulated role of cytosolic phosphorylation potential in modulating cardiac function is exemplified by sarcoplasmic reticular Ca2+ transport, which is augmented in pyruvate-energized hearts but attenuated in hearts depleted of energy reserves by substrate-free perfusion [3]. In isolated rat ventricular cardiomyocytes, superfusion with 5 mM pyruvate markedly increased systolic Ca2+ transients and cell shortening relative to equimolar concentrations of lactate or acetate [7]. Thus, evidence from several different mammalian heart preparations supports the hypothesized mechanistic linkage between pyruvate enhancements of cytosolic energetics, sarcoplasmic reticular Ca2+ transport, and ventricular contractile function.

Pyruvate exerts opposite effects on NADH redox states in the cytosol vs. mitochondrial matrix. In the mitochondria, pyruvate oxidation by pyruvate dehydrogenase and the TCA cycle generates reducing equivalents in the form of NADH for oxidative phosphorylation; in this respect, pyruvate catabolism does not differ from that of other energy-yielding substrates. Unlike other fuels, pyruvate is a cytosolic oxidant; increased pyruvate concentration in this compartment increases NAD+/NADH via the lactate dehydrogenase equilibrium. Veech et al. [8]proposed a near-equilibrium between cytosolic redox state and ATP phosphorylation potential effected by the powerful enzymes lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. This mechanism could enable pyruvate to directly increase cytosolic ATP phosphorylation potential by oxidizing the redox state of the cytosolic pyridine nucleotides; such an effect would be superimposed on the energetic effects of mitochondrial pyruvate oxidation. Indeed, pyruvate has proven more effective than other fuels at increasing the cytosolic ATP phosphorylation potential [4, 6, 9].

The cytosolic redox and adenylate systems were in near-equilibrium in isolated, nonworking guinea-pig hearts metabolizing glucose [10]; however, these systems could be displaced from near-equilibrium at the higher metabolic rates necessary to sustain physiological levels of external work. Recent reports in isolated working rabbit hearts [11, 12]have challenged the cytosolic near-equilibrium hypothesis. These studies demonstrated that cytosolic ATP phosphorylation potentials generally correlated with mitochondrial NADH concentrations but not with cytosolic [NAD+]/[NADH], implying that pyruvate enhancement of cytosolic energetics might not be mediated by cytosolic oxidation. Nevertheless, pyruvate sustained higher cytosolic phosphorylation potentials than equimolar concentrations of lactate despite similar mitochondrial [NADH] during perfusion with either substrate [12].

This investigation was undertaken to define the role of mitochondrial catabolism in mediating pyruvate’s inotropic and energetic effects in working guinea-pig myocardium. Mitochondrial pyruvate uptake was blocked with {alpha}-cyano-3-hydroxycinnamate, a selective inhibitor of the mitochondrial monocarboxylate transporter which does not impede sarcolemmal pyruvate uptake nor mitochondrial oxidation of fatty acids [13]. Inhibition of mitochondrial pyruvate oxidation blunted its favorable effects on cytosolic phosphorylation potential, sarcoplasmic reticular Ca2+ transport, and contractile function, although trans-sarcolemmal entry of exogenous pyruvate into the cytosolic compartment was unimpeded and octanoate oxidation increased sufficient to maintain the overall rate of oxidative metabolism.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Isolated working hearts
Animal experimentation was approved by the institutional Animal Care and Use Committee of the University of North Texas Health Science Center, and was performed in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, USA, 1996). Hearts, isolated from male Hartley guinea-pigs (n=67; body mass 479±9 g) and beating at intrinsic sinus rhythm, were antegradely perfused as working hearts at 10–12 cm H2O left atrial filling pressure and 90 cm H2O aortic afterload. Methodological details of working heart perfusions have been described previously [14–16]. Coronary and aortic flows were measured by timed collections, and cardiac output equalled the sum of coronary and aortic flows. Krebs–Henseleit bicarbonate perfusion media were prepared as recently described [14]. All media were fortified with 0.2 mM octanoate as oxidizable energy substrate and supplemented with 2.5 mM pyruvate and/or 0.6 mM {alpha}-cyano-3-hydroxycinnamate (COHC; Sigma, St. Louis, MO, USA) as indicated. Media were not recirculated.

2.2 Experimental protocols
2.2.1 Protocol I: pyruvate and octanoate oxidation
Rates of myocardial pyruvate and octanoate oxidation were assessed from 14CO2 formation from 14C-pyruvate and 14C-octanoate, respectively. Perfusion media contained both 0.2 mM octanoate and 2.5 mM pyruvate; radiolabelled substrate (2 mCi mol–1) was either [1-14C]octanoate (ICN, Costa Mesa, CA, USA) or [1-14C]pyruvate (Amersham, Arlington Heights, IL, USA). To measure 14CO2 formation, coronary venous effluent (4–8 ml) from the cannulated pulmonary artery was collected in 25-ml Warburg flasks, which were immediately sealed with rubber stoppers fitted with polypropylene center wells (Kontes, Vineland, NJ, USA) containing 0.4 ml of the CO2 trapping agent benzethonium hydroxide (Sigma, St. Louis, MO, USA). Effluent was then acidified by injection of 1 mEquiv. HCl to quantitatively convert HCO3– to CO2. 14CO2 trapping was facilitated by gentle oscillation of the flasks for 24 h. Essentially all of the 14C initially present in coronary effluent as dissolved 14CO2 or H14CO3– is trapped by this procedure [15]. Disintegrations per minute (dpm) of 14C were computed from counts per minute by correcting for quench. Myocardial 14CO2 release from each radiolabelled substrate stabilized within 15 min (cf. Fig. 1), indicating attainment of isotopic steady states. At 20–50 min, COHC was continuously infused to a left atrial concentration of 0.6 mM. COHC concentrations in perfusion media and coronary venous effluent were confirmed by measurements of UV absorbance at 337 nm wavelength; the extinction coefficient of COHC was 0.146 cm–1 mM–1.


Figure 1
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  		Fig. 1 Reciprocal changes in 14CO2 formation from 14C-pyruvate and 14C-octanoate during {alpha}-cyano-3-hydroxycinnamate (COHC) infusion. Isolated working guinea-pig hearts were perfused with 2.5 mM pyruvate and 0.2 mM octanoate; radioactive tracer was either [1-14C]pyruvate or [1-14C]octanoate. 14CO2 formation from [1-14C]pyruvate (broken line, open triangles) and [1-14C]octanoate (dotted line, open circles) stabilized within 15 min, indicating isotopic steady state. 0.6 mM COHC was continuously infused from 20–50 min. Stroke work (solid line, filled circles) was monitored as a measure of cardiac function. Means±S.E.M., n=6. * P<0.05 vs. 20 min.

 
2.2.2 Protocol II: cardiac function and energetics
The effects of 2.5 mM pyruvate on left ventricular contractile function and cytosolic energy metabolites were examined in the presence vs. absence of COHC. Perfusion media contained 0.2 mM octanoate alone or with 0.6 mM COHC. Baseline function and blood gas measurements were obtained at 15 min. At 20–50 min, sodium pyruvate (150 mM, pH 7.4) was continuously infused to a left atrial concentration of 2.5 mM. To control for nonspecific effects of volume infusion, 150 mM NaCl (pH 7.4) was infused at similar rates in separate experiments.

Left atrial inflow and coronary venous effluent were sampled anaerobically. Partial pressures of O2 and CO2 in these samples were measured in a Corning model 168 blood gas laboratory. Myocardial O2 consumption was computed according to the Fick principle. Intracellular H+ concentration was determined from coronary venous PCO2 as previously described [6]. Left ventricular function was assessed from stroke work (mJ g–1) and power, i.e. stroke work times heart rate (mJ min–1 g–1). Mechanical efficiency was computed as power divided by the energy equivalent of consumed O2 [17, 18], which was assumed to equal 448 J mmol O2–1 [19]. Sucrose (300 mM) was infused to a left atrial concentration of 2 mM at 45–50 min; myocardial extracellular fluid space was taken as the volume of sucrose distribution as recently described [15]. The sucrose distribution space was 0.56±0.02 ml g–1 in these hearts and was unaltered by pyruvate and/or COHC. At 50 min, hearts were stop-frozen with Wollenberger clamps precooled to constant temperature in liquid N2. Myocardial metabolites were extracted from stop-frozen hearts as described below.

2.2.3 Protocol III: sarcoplasmic reticular Ca2+ handling
The effects of pyruvate metabolism and blockade of its mitochondrial oxidation on sarcoplasmic reticular Ca2+ turnover were assessed by subjecting hearts to a 45Ca loading/washout protocol as recently described [3]and summarized as follows. After a 15-min prelabeling equilibration period, hearts were perfused with 0.2 mM octanoate-fortified labeling medium containing 45Ca (ICN) at a specific activity of 3.33 Ci mol–1. To standardize 45Ca labeling, hearts were perfused with labeling medium for 6000 heart beats, i.e. 6000 action potential-initiated 45Ca pulses. To increase the specific activity of perfusate 45Ca and facilitate radiolabeling of intracellular Ca2+ pools, medium Ca2+ concentration was lowered from 1.0 to 0.5 mM during the labeling period. Spontaneous heart rate fell from 213±4 to 165±3 min–1 and aortic pressure also fell from 90 to 65±2 cm H2O due to decreased extracellular Ca2+, but hearts remained within their autoregulatory reserve [16]throughout the labeling period.

Following labeling, 45Ca was washed out by a 50-min perfusion with nonradioactive media containing either 0.2 mM octanoate alone, octanoate plus 2.5 mM pyruvate, or the combination of octanoate, pyruvate and 0.6 mM COHC. Ca2+ concentration of these media was restored to the physiological level of 1.0 mM, and heart rates and aortic pressures returned to pre-labeling baseline levels within 2 min. Coronary effluent was sampled in 1-min periods from 35–40 min; residual 45Ca release during this period was stable and did not differ among the three conditions, and was taken as baseline 45Ca release rate. At 40–50 min washout, 150 mM caffeine (pH 7.4) was continuously infused to 10 mM left atrial concentration to mobilize sarcoplasmic reticular Ca2+ stores [20]and coronary effluent was collected throughout the caffeine infusion. Caffeine-induced 45Ca release was determined by subtracting baseline 45Ca release from total 45Ca release during caffeine infusion. At 50 min washout, hearts were removed from the perfusion system and blotted, and left ventricles were desiccated to constant mass at 90°C. Residual 45Ca content of dried left ventricle was measured to determine the amount of radioactive calcium remaining in the heart after caffeine infusion. Desiccated left ventricles were pulverized in a porcelain mortar and radioactivity in 50-mg aliquots was determined by scintillation counting. Counting efficiency of 45Ca was 99–100% in venous effluents and 92–94% in powdered ventricle.

2.3 14C-Sucrose washout kinetics and residual myocardial 14C-sucrose content
Additional protocol III experiments (n=13) were conducted to address the possibility that between-group differences in caffeine induced 45Ca release may have arisen from 45Ca trapped in the extracellular space during the 45Ca washout period. In these experiments 45Ca was not included in the labeling medium; instead, the cardiac extracellular space was selectively labeled [21, 22]by continuous infusion of [U-14C]sucrose (672 Ci mol–1; ICN) to a left atrial concentration of 52–55·103 dpm ml–1 during the final 5 min perfusion with labeling medium. 14C-Sucrose infusion was discontinued at the onset of washout with 0.2 mM octanoate, octanoate plus 2.5 mM pyruvate, or the combination of octanoate, pyruvate and 0.6 mM COHC. At 20 min washout, left ventricles were blotted, weighed, desiccated, and pulverized as described above. 14C in coronary effluent and powered tissue was measured by liquid scintillation counting; counting efficiencies in these fractions were 91 and 43%, respectively.

The kinetics of 14C washout in each heart were determined by "curve peeling" analyses of the natural logarithms of coronary venous 14C release data (dpm min–1 g dry–1) as described by Becker et al. [22]. Three kinetic components could be resolved from the 14C washout curves. Component III was the only significant source of 14C release between 5 and 10 min washout; the contribution of this component to total 14C release at earlier time points was determined by extrapolating its linear regression at 5–10 min to the beginning of 14C washout. Subtraction of component III from total 14C release yielded a modified washout curve from which components I and II could be resolved by extrapolating component II (determined by linear regression of 14C release data between 2 and 4.5 min washout) to the beginning of washout and subtracting it from the modified curve. Half times of elimination (i.e., t1/2) and apparent distribution volumes (i.e., Vsat) of each kinetic component were computed as previously described [22].

2.4 Metabolite extractions
Myocardial metabolites in protocol II hearts were extracted by standard procedures of our laboratory [15, 23]and enzymatically assayed [24]in a Perkin-Elmer Lambda 2 UV–vis spectrophotometer. At the conclusion of each assay known amounts of metabolite standards were added to assure that the expected absorbance changes were obtained. Inorganic phosphate (Pi), pyruvate and lactate were also measured in perfusion media and coronary venous effluent [15]and averaged to determine the respective extracellular concentrations. Intracellular Pi, pyruvate, and lactate concentrations were computed by subtracting extracellular amounts of each metabolite, i.e. extracellular concentration times extracellular space, from the respective total myocardial content. Cytosolic ATP phosphorylation potential, i.e. [ATP]/([ADP][Pi]), was determined from measured creatine kinase reactants including intracellular [H+] [25, 26]The creatine kinase equilibrium constant equaled 8.14·10–10 M–1 at the cytosolic free Mg concentration of 0.5 mM we recently reported in isolated perfused guinea-pig hearts [27].

2.5 Statistics
Data are reported as means±S.E.M. Results from protocol I were analyzed by one-way repeated measures (within-subject design) analysis of variance (ANOVA). Between-group comparisons of results from protocols II and III were accomplished by two-way ANOVA in combination with Student–Newman–Keuls multiple comparison tests. P values <0.05 were taken to indicate statistically significant differences. Statistical analyses were performed with SIGMASTAT advanced statistical software (version 2; Jandel Scientific, San Rafael, CA, USA).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of {alpha}-cyano-3-hydroxycinnamate on pyruvate and octanoate oxidation
The effects of COHC, a selective inhibitor of the mitochondrial monocarboxylate transporter, were examined in hearts metabolizing 0.2 mM octanoate and 2.5 mM pyruvate as fuels. Rates of octanoate and pyruvate oxidation were assessed from formation of 14CO2 from [1-14C]octanoate and [1-14C]pyruvate, respectively, while cardiac function was monitored as stroke work. 14CO2 release in coronary venous effluent stabilized within 15 min of perfusion with 14C-octanoate or 14C-pyruvate (Fig. 1). Rates of pyruvate and octanoate oxidation computed from 14CO2 release at 20 min were 1.2±0.1 and 0.045±0.008 µmol min–1 g–1, respectively. Subsequent infusion of 0.6 mM COHC elicited a rapid decline in 14CO2 formation from [1-14C]pyruvate to 35% of baseline and a reciprocal threefold increase in 14CO2 formation from [1-14C]octanoate. At 30 min COHC infusion, rates of pyruvate and octanoate oxidation computed from 14CO2 release equaled 0.45±0.05 and 0.14±0.02 µmol min–1 g–1, respectively. Stroke work fell with pyruvate oxidation and stabilized at 70% of pre-COHC baseline. Thus, when pyruvate oxidation was inhibited 65% by COHC, the myocardium increased its oxidation of octanoate fatty acid to compensate, and cardiac function stabilized following a moderate decline.

It should be noted that rates of pyruvate oxidation computed from 14CO2 formation in these hearts are only estimates. Substantial amounts of citrate were generated in pyruvate perfused hearts (see below), indicating significant pyruvate carboxylation. Under such anaplerotic conditions, 14CO2 could be generated from [1-14C]pyruvate within the TCA cycle independent of pyruvate dehydrogenase and cannot be taken as a quantitative index of pyruvate dehydrogenase flux or of pyruvate oxidation [28]. However, these results clearly demonstrate appreciable inhibition of mitochondrial pyruvate metabolism by COHC which blunted pyruvate enhancement of cardiac function despite a compensatory increase in octanoate oxidation.

3.2 Cardiac work, power, oxygen consumption and efficiency
The effects of 2.5 mM pyruvate on cardiac function and O2 consumption were determined in the absence vs. presence of COHC. During the initial baseline period, hearts were perfused with 0.2 mM octanoate±0.6 mM COHC (Fig. 2, left side of each panel). At 20–50 min, pyruvate was continuously infused to a left atrial concentration of 2.5 mM; in separate experiments, NaCl was infused at the same rate to control for left atrial volume infusion (Fig. 2, right side of each panel). Left ventricular contractile function was assessed from stroke work (Fig. 2A) and external work per minute, i.e. power (Fig. 2B), and myocardial O2 consumption (MVO2) was measured simultaneously (Fig. 2C). Gross mechanical efficiency was taken as the ratio of power divided by the energy equivalent of consumed oxygen (Fig. 2D). None of these variables differed among the four groups during the baseline period. Thus, COHC did not alter contractile function, O2 consumption, and mechanical efficiency in the absence of exogenous pyruvate, and the levels of these variables in control and COHC hearts destined to receive pyruvate infusion did not differ from respective levels in hearts destined for NaCl infusion. Furthermore, NaCl infusion did not alter any of the variables, indicating that the volume infusion per se did not affect cardiac function, O2 demand, or efficiency.


Figure 2
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  		Fig. 2 Pyruvate enhancement of cardiac function and mechanical efficiency is abrogated by {alpha}-cyano-3-hydroxycinnamate. Hearts were perfused with 0.2 mM octanoate-fortified media; 0.6 mM {alpha}-cyano-3-hydroxycinnamate (COHC) was added to inhibit mitochondrial pyruvate uptake. In each panel, baseline data (means±S.E.M., n=6) were obtained at 20 min, and infusion data were obtained following 30 min infusion of 2.5 mM pyruvate or NaCl volume control. CON: control medium, NaCl infusion; COHC: COHC medium, NaCl infusion; PYR: control medium, pyruvate infusion; PYR+COHC: COHC medium, pyruvate infusion. Power (B) equals stroke work (A) times heart rate (min–1). Gross mechanical efficiency (D) is indexed as power divided by the energy equivalent of myocardial oxygen consumption (MVO2; C). * P<0.05 vs. baseline; {dagger} P<0.05 vs. CON; {ddagger} P<0.05, PYR+COHC vs. PYR.

 
Infusion of 2.5 mM pyruvate increased stroke work and power 40% in the absence of COHC. Despite the enhanced function, pyruvate did not significantly increase MVO2; consequently, cardiac gross mechanical efficiency increased 29% during pyruvate infusion. Although COHC did not alter contractile function in pyruvate-free hearts, it prevented the enhancements of function and mechanical efficiency by pyruvate infusion: stroke work, power, and mechanical efficiency of COHC-treated hearts during pyruvate infusion did not differ from the respective levels in untreated and COHC-treated hearts receiving NaCl infusions (Fig. 2). Thus, inhibition of mitochondrial pyruvate metabolism blunted pyruvate-evoked enhancements of cardiac function and mechanical efficiency, but did not lessen O2 demand.

3.3 Energy metabolites and cytosolic phosphorylation potential
Myocardial energy metabolites and cytosolic phosphorylation potentials were measured in control and COHC-perfused hearts receiving pyruvate or NaCl infusions. ATP content did not differ among the four groups (Table 1). Pyruvate increased phosphocreatine content 30%, lowered creatine content 32%, and decreased intracellular inorganic phosphate concentration ([Pi]i) by 44%, relative to control hearts infused with NaCl. These pyruvate-induced changes in energy metabolites were lessened or prevented by COHC. Thermodynamic measures of cytosolic energetics were enhanced by pyruvate, and COHC blunted these enhancements as well. Thus, pyruvate increased phosphocreatine potential 241%, and cytosolic ATP phosphorylation potential, assessed from the creatine kinase equilibrium, 3.5-fold. COHC did not alter energy metabolites or phosphorylation potential in the absence of exogenous pyruvate, but essentially prevented energetic enhancements by pyruvate. Thus, pyruvate did not increase phosphocreatine content, phosphocreatine potential, and cytosolic ATP phosphorylation potential in COHC-treated myocardium, relative to NaCl.


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  		Table 1 Effects of pyruvate and {alpha}-cyano-3-hydroxycinnamate (COHC) on myocardial energy metabolites

 
3.4 Cytosolic redox metabolites
Intracellular pyruvate/lactate ratio can be taken as an index of cytosolic NAD+/NADH as prescribed by the lactate dehydrogenase equilibrium [29]. Accordingly, intracellular lactate and pyruvate concentrations were measured to assess the effects of COHC and pyruvate on the cytosolic redox state (Table 1). In pyruvate-free hearts, COHC significantly increased lactate concentration but did not appreciably alter the pyruvate/lactate ratio. Exogenous 2.5 mM pyruvate increased intracellular pyruvate concentration tenfold and pyruvate/lactate approximately ninefold, indicating pronounced oxidation of the cytosolic redox state. In pyruvate-perfused hearts, COHC effected a threefold increase in intracellular pyruvate concentration to 2.2 mM and a fourfold increase in pyruvate/lactate ratio. Thus, COHC increased the cytosolic NAD+/NADH ratio in pyruvate-perfused myocardium. Despite this cytosolic oxidation, COHC abrogated pyruvate enhancement of cytosolic phosphorylation potential; thus, inhibition of mitochondrial pyruvate uptake and metabolism effectively dissociated pyruvate’s energetic and redox effects in the cytosol.

3.5 Transaminase-linked amino acids and citrate
If COHC selectively inhibited mitochondrial but not sarcolemmal pyruvate uptake, then myocardial contents of cytosolic pyruvate metabolites should have been unchanged or even increased by COHC, while products of mitochondrial pyruvate metabolism should have been lowered. Alanine, a product of cytosolic pyruvate transamination, and citrate, generated by anaplerotic flux of mitochondrial pyruvate into the TCA cycle intermediate pools, were measured in each of the four groups (control, COHC, pyruvate, and pyruvate+COHC) of protocol II. In the absence of exogenous pyruvate, COHC increased alanine content 4.6-fold (Fig. 3); thus, blockade of mitochondrial pyruvate uptake appeared to divert endogenous pyruvate to the cytosolic transamination pathway. Citrate content was unaltered by COHC in these hearts; thus, mitochondrial metabolism of endogenous pyruvate was not essential to establish the baseline level of citrate in hearts metabolizing octanoate as exogenous energy substrate.


Figure 3
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  		Fig. 3 Citrate and amino acid contents in myocardium treated with pyruvate and/or 0.6 mM {alpha}-cyano-3-hydroxycinnamate (COHC). Metabolites were measured in protocol II hearts; experimental groups and abbreviations as in Fig. 2. * P<0.05 vs. CON; {dagger} P<0.05, PYR+COHC vs. PYR.

 
Pyruvate increased myocardial alanine content 18-fold in the absence of COHC. The increase in alanine content (10.1 µmol g dry mass–1) was essentially matched by a decrease of 11.6 µmol g dry mass–1 in the combined contents of glutamate plus aspartate, the sources of amino groups for alanine formation via glutamate–pyruvate and glutamate–oxaloacetate transaminases. In pyruvate-perfused myocardium, COHC did not lower and even tended to increase alanine content. COHC also augmented the pyruvate-induced declines in glutamate and aspartate contents (P<0.05; Fig. 3). In the absence of COHC, pyruvate increased myocardial citrate content 50-fold, from 0.12±0.04 to 6.2±0.5 µmol g dry mass–1. COHC did not alter citrate content of control hearts, but lowered that of pyruvate-treated hearts 80%, to 1.24±0.24 µmol g dry mass–1. Thus, COHC selectively inhibited formation of products of mitochondrial pyruvate metabolism without inhibiting cytosolic metabolism of pyruvate.

3.6 Sarcoplasmic reticular Ca2+ turnover
The effects of pyruvate vs. COHC on sarcoplasmic reticular Ca2+ turnover were examined in hearts prelabeled with 45Ca according to protocol III. Hearts were perfused with 0.2 mM octanoate fortified medium containing 3.33 Ci mol–1 45Ca for 6000 heart beats to effect radiolabeling of intracellular Ca2+. 45Ca was then washed out by perfusion with 0.2 mM octanoate alone (control) or with additional 2.5 mM pyruvate in the absence or presence of 0.6 mM COHC. At 35 min washout, pyruvate elevated stroke work 40%, and COHC prevented pyruvate enhancement of stroke work (Fig. 4A), as would be expected from the results of protocols I and II. Importantly, levels of stroke work in each of the three protocol III groups were similar to those of the corresponding groups from protocol II (see Fig. 2A), indicating that cardiac performance was unimpaired and remained responsive to pyruvate and COHC despite antecedent 30–40 min perfusion with lowered Ca2+ concentration in protocol III.


Figure 4
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  		Fig. 4 Effects of pyruvate and {alpha}-cyano-3-hydroxycinnamate on sarcoplasmic reticular Ca2+ turnover. Following 45Ca labeling for 6000 heart beats, 45Ca was washed out by 40 min tracer-free perfusion, then 10 mM caffeine was infused to mobilize residual intracellular 45Ca according to protocol III. Stroke work (A) was measured at 35 min 45Ca washout. Pre-caffeine baseline 45Ca release (nCi min–1 g dry–1) at 37–40 min 45Ca washout did not differ among the three groups (P>0.2): control (CON) 7.5±0.7; pyruvate (PYR) 9.0±1.2; PYR+{alpha}-cyano-3-hydroxycinnamate (COHC) 7.6±1.2. Caffeine-mobilized 45Ca (B) was determined by subtracting baseline from total 45Ca release during caffeine infusion. Residual tissue 45Ca content (C) was measured immediately following caffeine infusion. Means±S.E.M., n=6. * P<0.05 vs. CON; {dagger} P<0.05 vs. PYR.

 
Caffeine was infused from 40–50 min washout to mobilize sarcoplasmic reticular Ca2+. Baseline 45Ca release during the 3 min prior to caffeine infusion was essentially stable and did not differ among the three groups (nCi min–1 g dry mass–1): control 7.5±0.7; pyruvate 9.0±1.2; pyruvate+COHC 7.6±1.2. In control hearts, caffeine released 0.280±0.038 µCi 45Ca g dry mass–1 (Fig. 4B). In the absence of COHC, pyruvate lowered caffeine-induced 45Ca release 62%, to 0.105±0.029 µCi g dry mass–1, indicating increased turnover and washout of sarcoplasmic reticular 45Ca during the preceding 40 min of tracer-free perfusion. COHC abrogated this pyruvate-dependent reduction of caffeine induced 45Ca release: COHC increased caffeine induced 45Ca release threefold to 0.315±0.012 µCi g dry mass–1, a level not significantly different from control.

It could be argued that pyruvate lessened caffeine induced 45Ca release by inhibiting caffeine-dependent mobilization of sarcoplasmic reticular Ca2+, rather than by increasing sarcoplasmic reticular Ca2+ turnover and 45Ca release prior to caffeine. If this were the case, the amount of 45Ca remaining in the myocardium following caffeine infusion would be increased by approximately 0.15–0.2 µCi g dry mass–1 in pyruvate-perfused vs. control hearts. To address this possibility, residual 45Ca was measured in myocardium following 10 min caffeine infusion (Fig. 4C). Pyruvate did not increase residual myocardial 45Ca content; indeed, post-caffeine 45Ca content was nearly identical among the three groups. Thus, pyruvate did not lessen caffeine-induced 45Ca release by sequestering 45Ca in the sarcoplasmic reticulum. These results further substantiate the concept that pyruvate increased sarcoplasmic reticular Ca2+ turnover, and that inhibition of mitochondrial pyruvate metabolism attenuated pyruvate enhancement of Ca2+ turnover.

3.7 14C-Sucrose washout kinetics
Two additional alternative explanations for the observed effects of pyruvate and COHC on caffeine induced 45Ca release could be proposed, if it is assumed that regions of inadequate coronary flow developed during washout that trapped 45Ca in cardiac extracellular spaces, and that reflow of these regions during caffeine infusion liberated the trapped 45Ca. In the first hypothetical scenario, pyruvate and/or COHC altered the kinetics of 45Ca washout from the extracellular space, such that different amounts of 45Ca were trapped in the poorly perfused regions. In the second scenario, the 45Ca washout kinetics were unaltered by pyruvate and/or COHC, but caffeine induced reflow of smaller portions of the pyruvate hearts than the other two groups. To test these alternatives, a modified protocol III was employed in which 45Ca was deleted from the labeling medium; instead, [U-14C]sucrose was continuously infused during the final 5 min of labeling perfusion. 14C-Sucrose infusion was discontinued at the onset of washout perfusion with 0.2 mM octanoate alone or with additional 2.5 mM pyruvate in the absence or presence of 0.6 mM COHC.

The time courses of coronary venous 14C-sucrose release were nearly identical among the three groups, and 14C release fell to near the detection limit by 10 min washout in each group. Analyses of washout curves from individual experiments revealed three kinetic components (Table 2). Importantly, the t1/2 and Vsat values of these components were similar in each group; thus, neither pyruvate nor COHC altered washout of radioactive tracers from the extracellular space. The anatomical origins of these kinetic components cannot be determined with certainty. However, comparison of these t1/2 and Vsat values with those of kinetic components delineated in the isolated heart study of Becker et al. [22]suggests that component I may originate from the vasculature, component II from the interstitium, and the smaller, slowly released component III from intracellular compartments, indicating sucrose may have entered the cells to a limited extent.


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  		Table 2 Kinetic components of coronary venous washout of 14C-sucrose

 
Residual myocardial 14C content at 20 min washout was nearly identical in the three groups (Fig. 5 inset). When adjusted for the differences in left atrial inflow radioactivities during the labeling periods of the 14C vs. 45Ca experiments, the approximately 3500 dpm 14C g dry–1 at 20 min washout would be equivalent to no more than 0.1 µCi 45Ca g dry–1, even in the unlikely event that no additional 14C-sucrose release would have occurred at 20–40 min washout. These data indicate that the bulk of the caffeine-released 45Ca did not originate from the extracellular compartment, and that the effects of pyruvate and COHC on caffeine induced 45Ca release could not be ascribed to differences in residual extracellular 45Ca content prior to caffeine infusion as proposed in scenario 1. The small amount of extracellular 45Ca also cannot account for the differences in 45Ca release in even the most extreme case of scenario 2, wherein caffeine produces complete reflow of control and COHC treated hearts, but no reflow of pyruvate treated hearts. Moreover, the near equivalence of stroke work in the protocol III hearts (Fig. 4A) with that of the analogous protocol II hearts (Fig. 2A), which were not subjected to low Ca2+ labeling periods, is incompatible with the presence of underperfused regions during 45Ca washout, which would likely have impaired cardiac function due to ischemia.


Figure 5
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  		Fig. 5 14C-sucrose washout and residual myocardial 14C-sucrose content. [U-14C]sucrose was infused during the final 5 min of the protocol III labeling period. 14C-sucrose infusion was then discontinued (broken vertical line) and coronary venous 14C release was measured during perfusion with 0.2 mM octanoate (CON: circles; n=5), octanoate+2.5 mM pyruvate (PYR: squares; n=4), or octanoate+pyruvate+0.6 mM {alpha}-cyano-3-hydroxycinnamate (PYR+COHC: triangles; n=4). Inset: Residual myocardial 14C content was measured at 20 min washout. Data from individual experiments and means±S.E.M. of each group are shown. No statistically significant differences between groups were found for 14C-sucrose release or myocardial 14C content.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This investigation was undertaken to determine if the cytosolic redox effects of pyruvate in working myocardium were sufficient to enhance cytosolic ATP phosphorylation potential and contractile function. Mitochondrial metabolism of pyruvate was inhibited by {alpha}-cyano-3-hydroxycinnamate (COHC), a selective blocker of the mitochondrial monocarboxylate transporter [13]. Hearts were energetically stabilized with octanoate, a fatty acid substrate metabolized by COHC-independent pathways, which prevented cytosolic energy depletion and impairment of baseline cardiac function by COHC in the absence of pyruvate. As expected and in conformance with previous investigations [3, 6], pyruvate effected parallel increases in cytosolic ATP phosphorylation potential, sarcoplasmic reticular Ca2+ transport, and cardiac function. Pyruvate also increased cytosolic NAD+/NADH ratio; according to the proposed near-equilibrium between cytosolic redox state and ATP phosphorylation potential [8, 10], this cytosolic oxidation should have increased cytosolic ATP potential independently of mitochondrial pyruvate metabolism. However, 65% inhibition of mitochondrial pyruvate metabolism abrogated pyruvate enhancement of cytosolic ATP phosphorylation potential, sarcoplasmic reticular Ca2+ handling and cardiac function, although oxidative metabolism was maintained by octanoate oxidation and cytosolic NAD+/NADH ratio was augmented. These results indicate that the enhancements of cardiac function and cytosolic phosphorylation potential by pyruvate can be dissociated from its oxidation of the cytosolic NAD+/NADH redox state when mitochondrial pyruvate metabolism is inhibited. Thus, mitochondrial metabolism of pyruvate appears essential for pyruvate’s metabolic inotropism, even when other oxidizable fuels are provided. These findings confirm and extend recent reports in isolated working rabbit heart [11, 12]that cytosolic redox state and phosphorylation potential were poorly correlated.

We [3, 6]and others [4]have found pyruvate to be more effective than other fuels including octanoate, lactate, acetate and glucose in augmenting cytosolic energetics, although these other fuels are also readily oxidized in the mitochondria and, like pyruvate, are potent mitochondrial reductants [11, 12, 30]. These other fuels differ from pyruvate in that they do not increase cytosolic NAD+/NADH, nor do they increase cytosolic energetics or contractile function to the same extent as pyruvate. It appears that the cytosolic redox effects of pyruvate might still be important, provided mitochondrial pyruvate oxidation is intact, and could produce discrete changes in cytosolic energetics when superimposed on the relatively larger effects of mitochondrial oxidative metabolism. The results of this investigation suggest a peculiarity of mitochondrial pyruvate metabolism could be responsible for pyruvate’s metabolic inotropism. However, it is unclear whether mitochondrial pyruvate oxidation is sufficient to increase cytosolic energetics if pyruvate’s cytosolic redox effects are selectively inhibited.

4.1 Mechanism of metabolic inotropism
The effects of pyruvate and COHC on sarcoplasmic reticular Ca2+ turnover were investigated according to the protocol we recently developed [3]: 45Ca loading was accomplished by perfusing hearts with 45Ca for a fixed number of beats in presence of 0.2 mM octanoate as exogenous substrate, followed by washout of 45Ca by nonradioactive perfusion with octanoate, octanoate+2.5 mM pyruvate, or octanoate+pyruvate+0.6 mM COHC. The effects of pyruvate and COHC on sarcoplasmic reticular uptake and release of Ca2+ were indexed by the extent of 45Ca turnover during the washout period, which was assessed from caffeine-induced 45Ca release at 40–50 min washout. Thus, the greater the rate of sarcoplasmic reticular Ca2+ turnover, the greater is the extent of sarcoplasmic reticular 45Ca washout prior to caffeine infusion, such that less 45Ca remains in the sarcoplasmic reticulum to be released by caffeine. Pyruvate enhanced sarcoplasmic reticular Ca2+ transport as evidenced by increased depletion of the caffeine-mobilized 45Ca pool. This increased Ca2+ turnover was attenuated by COHC. Thus, pyruvate enhancement of sarcoplasmic reticular Ca2+ turnover, like its enhancement of function and energetics, required its mitochondrial metabolism; increased concentrations of pyruvate in the cytosolic compartment appear insufficient to augment sarcoplasmic reticular Ca2+ handling when mitochondrial pyruvate metabolism is blocked. These results demonstrate that pyruvate does not augment sarcoplasmic reticular Ca2+ transport by a direct effect of pyruvate on the Ca2+ ATPase or on the Ca2+ release channel. Instead, these findings provide further support for a direct link between cytosolic energetics and sarcoplasmic reticular Ca2+ transport as a mechanism for pyruvate enhancement of cardiac inotropism [3]. However, it is recognized that these results do not eliminate alternative scenarios wherein cytosolic pyruvate directly activates the sarcoplasmic reticular Ca2+ ATPase and/or sarcolemmal Ca2+ channel, and COHC directly antagonizes these effects. Such hypothetical direct actions of pyruvate and COHC on Ca2+ transport mechanisms could produce the inotropic effects observed in this study. Although such direct effects of pyruvate and COHC on Ca2+ ATPases and channels have not been demonstrated in myocardium, further research is necessary to delineate the specific mediators of the linkage between mitochondrial pyruvate oxidation and sarcoplasmic reticular function in order to exclude these alternative mechanisms.

4.2 Pyruvate enhancement of cardiac mechanical efficiency
Under aerobic conditions, cardiac mechanical efficiency is defined as the portion of the enthalpy of consumed oxygen expressed as external work [17, 18], equal to the ratio of stroke work to the energy equivalent of consumed O2, estimated at 448 J mmol O2–1 [19]. Mechanical efficiency was approximately 11% in control hearts oxidizing octanoate as exogenous fuel, a level comparable to previously reported efficiencies of isolated, crystalloid-perfused working heart preparations [17, 31]. Pyruvate increased stroke work 40% without increasing MVO2, and, thus, increased mechanical efficiency to nearly 15%. In these efficiency calculations, glycolytic ATP production, which is independent of MVO2, is assumed to be negligible. Indeed, glycolytic flux was likely to be low in these hearts: exogenous glucose was not provided, and fatty acid oxidation inhibits glycolysis [32]. Pyruvate oxidation does not increase and likely further inhibits glycolysis by elevating citrate, an inhibitor of phosphofructokinase [33]and by lowering cytosolic concentrations of the phosphofructokinase activators ADP, AMP and Pi secondary to enhancement of cytosolic energetics.

The principal mechanism of pyruvate enhancement of mechanical efficiency may be mediated by the reduction in intracellular Pi concentration. In skinned cardiac muscle fibres, Pi has been found to decrease the amount of active force developed per ATP hydrolyzed due to a reduction in myofilament Ca2+ sensitivity [34, 35], i.e. the contractile force developed by actin–myosin crossbridges at a given Ca2+ concentration. Thus, the reduction in intracellular Pi by pyruvate could produce a Ca2+-sensitizing effect on the myofilaments which may lower the amount of ATP hydrolyzed per unit of developed force and thereby increase mechanical efficiency. Conversely, COHC appears to prevent pyruvate enhancement of cardiac efficiency by preventing the reduction in Pi.

A large fraction of metabolic energy is expended as heat during excitation–contraction coupling, tension development and muscle shortening, and is not expressed as external work [17, 18]. Thus, cardiac mechanical efficiency is influenced by hemodynamic loading conditions and heart rate, the principal determinants of heat production associated with cardiac mechanical activity [17]. In this study, preload, afterload and heart rate were essentially identical in the different groups, so it is unlikely that pyruvate increased efficiency by decreasing contractile heat. Cardiac efficiency can also be modulated to a limited extent by changes in substrate selection due to modest differences in the ratio of ATP synthesized to oxygen reduced, i.e. the P/O ratio. The P/O ratio of pyruvate is 3.0, 8% above the octanoate P/O ratio of 2.77. It is doubtful, however, that the slightly higher P/O ratio is the principal factor responsible for the increased mechanical efficiency of hearts metabolizing pyruvate. The P/O ratio for glucose is 3.17, even greater than that of pyruvate, yet mechanical efficiencies of glucose perfused hearts in our laboratory are approximately 10%, i.e. no greater than that of control hearts oxidizing octanoate, and well below that of hearts metabolizing pyruvate.

Enhancement of sarcoplasmic reticular Ca2+ transport secondary to increased cytosolic phosphorylation potential [3]could support increased mechanical efficiency by enabling the sarcoplasmic reticular Ca2+ pump to maintain higher transmembrane Ca2+ gradients resulting in augmented diastolic relaxation and systolic contractile force. Arguably, a higher rate of oxidative phosphorylation and, thus, MVO2 would be required to maintain a higher cytosolic energetic state, if mitochondrial catabolism of pyruvate were solely responsible for pyruvate’s energetic effects. On the other hand, if cytosolic oxidation by pyruvate can elevate cytosolic energetics above the level sustained by oxidative phosphorylation, as occurs when mitochondrial pyruvate metabolism is unimpeded, sarcoplasmic reticular Ca2+ uptake could be augmented without an increase in O2 demand. According to this scenario, both mitochondrial catabolism and cytosolic oxidation could contribute to the favorable effects of pyruvate on cardiac function and efficiency.

4.3 Mechanisms of citrate formation in hearts metabolizing pyruvate
Two distinct pathways could catalyze citrate formation in pyruvate-perfused myocardium. In the cytosolic compartment, pyruvate transamination with glutamate, catalyzed by glutamate–pyruvate transaminase, generates {alpha}-ketoglutarate, from which citrate is formed via the cytosolic isoforms of NADP+-dependent isocitrate dehydrogenase and aconitase [36]. Several investigations in mammalian myocardium have demonstrated significant activities of the mitochondrial enzymes pyruvate carboxylase [28, 37, 38]and/or malic enzyme, [1, 39]which carboxylate pyruvate to generate the 4-carbon TCA cycle intermediates oxaloacetate and malate, respectively. The anaplerotic flux generated by these mitochondrial enzymes increases the steady state levels of TCA cycle intermediates including citrate. In our study, cytosolic transaminases were not inhibited by COHC (alanine content was unchanged, if not increased), yet COHC lowered myocardial citrate content 80% in pyruvate perfused hearts. Thus, the mitochondrial anaplerotic pathways appear to be the predominant mechanisms for citrate formation in myocardium metabolizing pyruvate and fatty acid as exogenous fuels. Furthermore, large increases in myocardial citrate content were not associated with significant changes in MVO2, in agreement with previous reports [38, 40].

4.4 Specificity of cinnamate inhibition of pyruvate transport
In this study, assessment of the role of mitochondrial pyruvate metabolism in augmented function and energetics required the use of an agent that selectively inhibited mitochondrial uptake of pyruvate without affecting sarcolemmal pyruvate transport or metabolism of alternative fuels [13]. In preliminary experiments (data not shown), we determined that 0.6 mM COHC blunted pyruvate enhancement of stroke work but did not decrease baseline function. However, when higher concentrations (1 and 2 mM) of COHC were infused, pre-pyruvate baseline function was appreciably depressed, suggesting that, at these higher concentrations, COHC could have exerted nonspecific effects on oxidative metabolism. Accordingly, 0.6 mM COHC was employed in this investigation. 0.6 mM COHC inhibited mitochondrial pyruvate metabolism by 65% as judged from the decrease in 14CO2 production from [1-14C]pyruvate. Importantly, octanoate oxidation was not lowered, and indeed increased severalfold when COHC was applied. Moreover, COHC did not lower function, energetics or MVO2 of pyruvate-free control hearts. Thus, it could be concluded that COHC did not inhibit sarcolemmal or mitochondrial uptake of the fatty acid. Moreover, it was evident that COHC did not inhibit sarcolemmal pyruvate uptake: although COHC sharply decreased citrate content in pyruvate-perfused hearts, it did not lower and even tended to increase content of alanine, a metabolite generated from pyruvate in the cytosolic compartment. Thus, COHC at the concentration used in this investigation (0.6 mM) selectively inhibited mitochondrial uptake and metabolism of pyruvate.

4.5 Limitations
4.5.1 Assessment of cytosolic energetics and redox state
Due to extensive binding and compartmentation of ADP, Pi, NAD+ and NADH in myocardium, cytosolic ATP phosphorylation potential and redox state cannot be directly measured and must be computed from measured creatine kinase and lactate dehydrogenase reactants. The creatine kinase equilibrium constant is modulated by cytosolic free Mg2+ concentration [25, 26]. We recently estimated cytosolic free Mg2+ (Mgf) in isolated perfused guinea-pig hearts at 0.6 mM by indicator metabolite techniques and 0.4 mM by 31P NMR [27]and, accordingly, Mgf was assumed to equal 0.5 mM for calculations of ATP phosphorylation potential in all four perfusion conditions of this study. Citrate is a chelator of divalent cations including Mg2+, and the observed increase in citrate content in pyruvate hearts may have lowered Mgf sufficiently to lower KCK. Laughlin and Heineman recently reported that perfusion of isolated working rabbit hearts with 10 mM pyruvate decreased Mgf from 0.5 to 0.34 mM [11]. If such a decrease in Mgf occurred in our study due to 2.5 mM pyruvate, the resulting increase in KCK from 8.14·10–10 to 9.27·10–10 M–1 would produce a 12% reduction in ATP potential, from 34.7±2.9 to 30.5±2.5 mM–1. This revised ATP potential is nevertheless severalfold higher than that of the other perfusion conditions; moreover, phosphocreatine potential, a Mg2+-independent measure of cytosolic energetics, is also increased by pyruvate in the absence of COHC. Thus, a moderate decrease in cytosolic free Mg due to citrate would not alter the fundamental findings that pyruvate increases cytosolic energetics and that this increase is blunted when mitochondrial pyruvate oxidation is inhibited. With regard to assessment of cytosolic redox state, the report of disequilibrium between cytosolic redox and phosphorylation states by Laughlin and Heineman [11]suggested that the lactate dehydrogenase might be displaced from equilibrium in working myocardium metabolizing exogenous pyruvate or lactate. In such cases, the intracellular [pyruvate]/[lactate] ratio cannot be used to quantify cytosolic [NAD+]/[NADH] [41]. Nevertheless, the large increases in intracellular [pyruvate]/[lactate] in the pyruvate-perfused hearts of this study could still be taken to indicate appreciable oxidation of cytosolic [NAD+]/[NADH], even if lactate dehydrogenase were displaced from equilibrium.

4.5.2 Assessment of pyruvate oxidation
14CO2 formation from [1-14C]pyruvate is not a quantitative measure of pyruvate oxidation via pyruvate dehydrogenase when anaplerotic flux of pyruvate into other TCA cycle intermediate pools is significant [28]. Carboxylation of [1-14C]pyruvate and randomization of label in fumarate will generate oxaloacetate labeled in the 1 and 4 positions. Condensation of this oxaloacetate with acetyl CoA will produce citrate labeled in carbons 1 and 6, which will yield 14CO2 in the subsequent turn of the TCA cycle. Thus, 14CO2 production in this setting cannot be assumed to originate entirely from oxidative decarboxylation of pyruvate by pyruvate dehydrogenase. On the other hand, both of these mechanisms of 14CO2 production from [1-14C]pyruvate are confined to the mitochondria and would be equally affected by inhibition of mitochondrial pyruvate uptake. Thus, the COHC-induced reduction in 14CO2 production from [1-14C]pyruvate can be taken to indicate inhibition of mitochondrial pyruvate transport, even though pyruvate oxidation cannot be quantified in these experiments.

Time for primary review 25 days.


   Acknowledgements
 
This study was supported by a grant from the National Heart, Lung and Blood Institute (HL 50441).


   Notes
 
1 Abbreviations: COHC, {alpha}-cyano-3-hydroxycinnamate; Cr, creatine; KCK, equilibrium constant of creatine kinase; Mgf, cytosolic free magnesium; MVO2, myocardial oxygen consumption; NMR, nuclear magnetic resonance spectroscopy; PCr, phosphocreatine; Pi, inorganic phosphate; TCA, tricarboxylic acid. Enzymes: Aconitase (EC 4.2.1.3 [EC] ), creatine kinase (EC 2.7.3.2 [EC] ), glutamate–oxaloacetate transaminase (EC 2.6.1.1 [EC] ), glutamate–pyruvate transaminase (EC 2.6.1.2 [EC] ), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12 [EC] ), isocitrate dehydrogenase (EC 1.1.1.41 [EC] ), lactate dehydrogenase (EC 1.1.1.27 [EC] ), NAD(P+)-dependent malic enzyme (EC 1.1.1.39/40), phosphofructokinase (EC 2.7.1.11 [EC] ), phosphoglycerate kinase (EC 2.7.2.3 [EC] ), pyruvate carboxylase (EC 6.4.1.1 [EC] ), pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.1 [EC] 2, EC 3.1.3.4 [EC] 3). Back


   References
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 1 Introduction
 2 Methods
 3 Results
 4 Discussion
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