INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE HIBERNATING MYOCARDIUM is defined by reversible myocardial contractile dysfunction due to reduced coronary flow and decreased oxygen supply to the myocardium (26). The hallmarks of hibernation are the retention of viable myocardial tissue with residual mitochondrial functions such as pyruvate and fatty acid oxidation, electron-transport-chain flux, and oxidative phosphorylation to generate ATP (1, 29). Little is known about the function of the citric acid cycle (CAC) in myocardium during hibernation. Ischemia in isolated perfused rat hearts causes an increase in the net efflux of the CAC intermediate succinate (15, 24, 25). Although this suggests a net loss of CAC intermediates during ischemia, it is not clear whether ischemia results in depletion of the pool of CAC intermediates (11, 15, 24). The tissue content of CAC intermediates is small compared with the flux through the cycle, and loss of CAC intermediates from the cycle must be balanced by the entry of intermediates into the CAC if the pool size is to be maintained (see Fig. 1). With normal myocardial blood flow, the loss of CAC intermediates is balanced by the entry of newly synthesized intermediates into the cycle; this process is termed anaplerosis (7, 8, 11, 14, 21). The effects of acute myocardial hibernation on the rate of anaplerosis and the tissue content of CAC intermediates are not known.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Scheme of the incorporation of pyruvate into the citric acid cycle (CAC) via pyruvate carboxylation and decarboxylation. Arrows going toward the CAC are anaplerotic fluxes (feeding in); arrows going out are CAC intermediate release.

Pyruvate carboxylation is a major anaplerotic pathway in normal myocardium in vivo (21) and generates malate and oxaloacetate (OAA) via malic enzyme and pyruvate carboxylase, respectively (2, 8, 23, 35, 36) (see Fig. 1). We recently developed a method to measure in the heart of anesthetized swine the rate of pyruvate carboxylation and decarboxylation using [U-¹³C₃]lactate and [U-¹³C₃]pyruvate tracers and mass isotopomer analysis of tissue pyruvate and citrate (21). In isolated rat hearts and in vivo swine myocardium, we found that pyruvate carboxylation accounted for 2.5-8% of the citrate synthase flux (7, 8, 21, 39). We also found that the rate of pyruvate carboxylation was not significantly altered when the rate of pyruvate decarboxylation (i.e., flux through pyruvate dehydrogenase) was inhibited by >90% by infusion of octanoate. Thus constitutive pyruvate carboxylation appears to be essential for normal cardiac function as was demonstrated in isolated working rat hearts where there was a dramatic fall in ventricular power when pyruvate carboxylation was pharmacologically inhibited (28). Constitutive pyruvate carboxylation balances the loss of CAC intermediates (11, 15, 21, 39) including citrate (21, 39). Under aerobic conditions, citrate efflux from rat, swine, and human hearts ranges from 5 to 20 nmol · g¹ · min¹ (21, 38, 39). Net citrate efflux from swine myocardium amounts to ~20% of the rate of pyruvate carboxylation (21).

The extent of pyruvate carboxylation and citrate efflux in the hibernating or ischemic myocardium is not known. Opie (20) reported a 33% decrease in myocardial citrate content after 30 min of severe ischemia (an ~90% reduction in flow) in dogs; however, the actual rate of citrate efflux from the heart was not measured. Evidence for a role of CAC intermediate depletion in postischemic cardiac dysfunction was suggested by improved postischemic functional recovery when anaplerotic substrates (pyruvate, glutamate, or propionate) were administered to isolated rodent hearts (4, 6, 27, 28, 37). The functional benefits of elevated pyruvate concentration suggest that anaplerotic pyruvate carboxylation may play an important role in the correction of metabolic abnormalities during conditions of stress such as hibernation.

The goals of the present study were to: 1) examine the effects of hibernation on the content of CAC intermediates in the heart; 2) measure pyruvate carboxylation, pyruvate decarboxylation, and citrate efflux in the myocardium during hibernation; and 3) determine the effects of pharmacological concentrations of pyruvate on the hibernating myocardium. We used [U-¹³C₃] lactate and [U-¹³C₃]pyruvate and isotopomer analysis of tissue pyruvate and citrate to measure pyruvate carboxylation and decarboxylation in the well-characterized swine model of acute myocardial hibernation (1, 18, 29).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals. Chemicals, enzymes, and coenzymes were purchased from Boehringer Mannheim (Indianapolis, IN) and Sigma-Aldrich (Milwaukee, WI). [²H₆]succinic acid, [U-¹³C₃] lactate, and [U-¹³C₃]pyruvate were obtained from Isotec (Miamisburg, OH). The derivatization agent N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide was supplied by Regis Chemical (Morton Grove, IL). Intralipid solution was obtained from Baxter Healthcare (Deerfield, IL).

Experimental model. A previously described in vivo technique was used to deliver ¹³C-labeled substrates directly into the left anterior descending (LAD) coronary artery of swine (31, 32). Overnight-fasted domestic swine (weight 27-38 kg) of either sex were sedated with Telazol (6 mg/kg im), anesthetized with pentobarbital sodium (25 mg/kg + 5 mg · kg¹ · h¹ iv), intubated via a tracheotomy, and ventilated to maintain arterial blood gas values in the normal range (PO₂ > 100 mmHg, PCO₂ of 35-45 mmHg, and pH of 7.35-7.45). A 7-Fr high-fidelity pressure transducer catheter (Millar; Houston, TX) was positioned in the left ventricle via the carotid artery. The animal was then heparinized (300 U/kg bolus + 150 U · kg¹ · h¹ iv) and infused with a 20% triglyceride emulsion (Intralipid 20%, 0.3 ml · kg¹ · h¹ iv) to increase plasma free fatty acids (FFA) to 0.6 mM (32). Coronary blood flow in the anterior wall was controlled by an extracorporeal circuit as previously described (31, 32). The anterior interventricular vein was cannulated to collect venous blood samples from the perfusion territory of the LAD. The coronary perfusion pump flow was adjusted to give an interventricular venous Hb saturation of 35-40% (31, 32).

Experimental protocols. Pigs were subjected to acute myocardial hibernation induced by decreasing the blood flow to the LAD bed by 40% (see Fig. 2). Pyruvate carboxylation and decarboxylation were measured with an intracoronary infusion of [U-¹³C₃]lactate and/or [U-¹³C₃]pyruvate for 60 min, with subsequent analysis of myocardial tissue for ¹³C isotopomers of pyruvate and CAC intermediates by gas chromatography-mass spectrometry (GC-MS) as previously described (21). Stock solutions of 99% [U-¹³C₃]lactate and/or 99% [U-¹³C₃]pyruvate were directly infused into the LAD perfusion circuit at a rate of 6.5 µl/ml of LAD blood flow (see Fig. 2). Pyruvate carboxylation was measured in hibernating myocardium under either near-normal arterial lactate and pyruvate concentrations (HIB group, n = 8), or with elevated arterial pyruvate concentration (HIB + PYR group, n = 8). In HIB animals, the concentrations of [U-¹³C₃]lactate and [U-¹³C₃]pyruvate in the infusate were 154 and 15.4 mM, respectively, so that the lactate and pyruvate concentrations in LAD blood were raised by 1.0 and 0.1 mM, respectively. In the HIB + PYR group, the concentration of [U-¹³C₃]pyruvate in the infusate was 154 mM, so that the pyruvate concentration in LAD blood was raised by 1.0 mM. Left ventricular pressure, end-diastolic pressure, peak first derivative of left ventricular (LV) pressure with time (dP/dt), heart rate, and arterial and venous blood samples were taken at all sample times (see Fig. 2). Plasma samples were stored at 80°C until further analysis. At the end of each protocol, large punch biopsies (3 g) of the LAD and circumflex (CFX) beds were quickly taken, freeze-clamped, and stored at 80°C until analysis. We have previously shown that the CFX biopsy receives normal myocardial blood flow and thus serves as control tissue (18, 31). The heart was excised, and black ink was infused down the right and left main coronary arteries to identify the LAD perfusion bed, which was dissected and weighed (37.1 ± 2.7 g).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Experimental protocol was divided into equilibration and experimental periods. At time 20, blood flow from the left anterior descending (LAD) coronary artery was reduced by 40%. Infusion of 13C isotopic substrates began at time 0 and continued to the end of the protocol. A large punch biopsy was taken at time 60 for measurement of pyruvate decarboxylation and carboxylation. Arrows designate sampling of arterial and venous blood and cardiovascular recordings. HIB, hibernating myocardium under near-normal arterial lactate and pyruvate concentrations; HIB + PYR, hibernating myocardium with elevated arterial pyruvate concentration.

Analytic methods. The concentrations of plasma FFA, blood glucose, lactate, pyruvate, and citrate, as well as tissue lactate, pyruvate, and malate were determined using spectrophotometric enzymatic assays (3, 33, 40). Tissue pyruvate concentrations were measured immediately after homogenization in neutralized perchloric acid extracts to prevent loss of pyruvate from freeze-thaw (40). Tissue concentrations of ATP and ADP were measured using the ATP Bioluminescent Assay Kit (Sigma-Aldrich).

Calculations. The myocardial blood flow was calculated as the LAD perfusion pump flow divided by the mass of the LAD perfusion bed. Mass isotopomers of metabolites containing 0-n ¹³C atoms are identified as Mi, where i = 0, 1,...,n. The relative rates of pyruvate carboxylation and decarboxylation were calculated as previously described (7) from 1) the M3 enrichment of tissue pyruvate, and 2) the M2 and M3 enrichments of the acetyl-CoA and OAA moieties of citrate, respectively (21). Mass isotopomers were adjusted for the natural abundance measured in myocardial tissue samples from pigs (n = 4) that were not infused with labeled substrates (5, 9). The measured enrichment of the M3 OAA moiety of citrate was corrected for 1) the fraction of M3 OAA molecules coming from some citrate isotopomers metabolized in the CAC and 2) the dilution of ¹³C in the CAC, as described in detail by Comte and co-workers (Eqs. 8-10 in Ref. 7). The absolute rates of pyruvate carboxylation and decarboxylation were calculated from the relative rates of pyruvate carboxylation and decarboxylation and the absolute rate of CAC flux. The latter was calculated from the myocardial oxygen consumption (MO₂) and the stoichiometric relationships between oxygen consumption and citrate formation from fat and carbohydrate as previously described (21).

Statistical analysis. Data are presented as means ± SE. The hemodynamic variables were compared between the two protocols using repeated-measures ANOVA. Statistical significance was determined using paired and unpaired t-tests as appropriate.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cardiovascular parameters. There were no significant changes over the course of the experiment or between groups at any time point in peak systolic LV pressure, peak LV dP/dt, heart rate (see Fig. 3), or LV end-diastolic pressure (data not shown). In the HIB and HIB + PYR groups, MO₂ decreased by ~38% after 80 min of flow reduction (see Fig. 3).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Left ventricular (LV) peak systolic pressure (A), peak first derivative of LV pressure with time (dP/dt, B), heart rate (C), and myocardial oxygen consumption (D).

Metabolite concentrations and enrichments. With the onset of flow reduction, there was a metabolic switch from net lactate uptake to lactate production (see Fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Net myocardial lactate production from 40 to 0 min. *P < 0.05 compared with samples at 40 to 25 min.

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View larger version (14K): [in this window] [in a new window]   Fig. 5.   Rate of citrate release (A) and pyruvate carboxylation (B). Data for the aerobic values are from previous work (21); *P < 0.05.

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Tissue metabolites. Tissue lactate and pyruvate content and the lactate-to-pyruvate ratio were similar between the two groups (see Table 1) and were not different from previously published values from normal-flow animals subjected to the same isotope-infusion protocol (21). Thus despite lactate production at the onset of flow reduction, we observed normal lactate and lactate-to-pyruvate ratios after 80 min of reduced flow. Furthermore, the concentrations of ATP and ADP and the ATP-to-ADP ratio were unchanged between the hibernating LAD and CFX beds or between the two groups for a given bed (see Table 2), which confirms the observation of Schulz et al. (29) that there are normal ATP levels after ~80 min of moderate flow reduction in swine myocardium.

Pyruvate carboxylation and decarboxylation. The relative rates of pyruvate decarboxylation and carboxylation were calculated as previously described (7, 21). There were no changes in pyruvate decarboxylation between the groups (see Table 4) nor were these values different from values obtained in aerobic hearts (21). The relative rates of pyruvate carboxylation during hibernation were unaffected by elevated arterial pyruvate concentrations; however, values from hibernating myocardium were 50% lower than those previously found in aerobic and aerobic + pyruvate animals (see Table 4). Because there was not a significant change in pyruvate decarboxylation, the ratio of carboxylation to decarboxylation was significantly decreased by hibernation in both groups (see Table 4).

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Malic enzyme and pyruvate carboxylase activity. Owing to the lower rate of pyruvate carboxylation in hibernating myocardium, we examined the possibility that there was a decrease in the activity of malic enzyme and/or pyruvate carboxylase. The activity of malic enzyme was unchanged between the LAD perfusion bed (840 ± 50 nmol · g¹ · min¹) and the CFX bed (830 ± 50 nmol · g¹ · min¹) in the HIB group. There was also no difference between the LAD and CFX beds in the activity of pyruvate carboxylase (32 ± 7 and 31 ± 9 nmol · g¹ · min¹, respectively) in the HIB group. Similar values were obtained in the HIB + PYR group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The key finding of this study is that a 40% reduction in MO₂ results in a decrease in both pyruvate carboxylation and citrate efflux with the maintenance of tissue levels of CAC intermediates. The absolute rate of pyruvate carboxylation decreased by 65% when there was only a 38% drop in MO₂ (see Fig. 3). If the decrease in malate and/or OAA supply to the CAC via pyruvate carboxylation were not matched by a decrease in the rate of the net efflux of CAC intermediates from the cycle, then the concentration of CAC intermediates in the myocardium would decrease. Hibernation had no effect on the tissue contents of citrate, succinate, fumarate, or malate, which suggests that the decrease in citrate release reflects a decrease in other efflux pathways for CAC intermediates, thus preserving the integrity of the CAC and the production of reducing equivalents, electron-transport-chain flux, and ATP formation, albeit at decreased rates.

An acute reduction in coronary blood flow of ~40% results in an initial acceleration of glycolysis, a decrease in ATP, and a subsequent resetting of the metabolism to better match the reduced oxygen supply (1, 10, 22, 29). After 60-90 min of coronary blood flow reduction, the myocardium is in a relative state of hibernation with restored ATP content and reduced lactate production (1, 10, 29). The results of the present investigation extend the understanding of the acute phase of myocardial hibernation and demonstrate that the heart adjusts its metabolism to maintain the tissue content of CAC intermediates by a decrease in pyruvate carboxylation and citrate release. Thus the mitochondria conserve the constituents of the CAC. This is reflected in the greater degree of carbon recycling in the CAC as assessed by mass isotopomer analysis. It is important to note that prolonged hibernation may result in impaired CAC function, as recently suggested by Schulz and colleagues (30), who observed a progressive decrease in MO₂ during the course of 24 h of hibernation (an ~40% reduction in LAD flow) in a similar swine model.

It has been suggested that with reduced coronary blood flow the rate of anaplerosis does not match the rate of CAC intermediate efflux, and there is a significant depletion of CAC intermediates (11). This is clearly not the case in acutely hibernating swine myocardium (see Table 3). As noted above, the decrease in anaplerotic pyruvate carboxylation matched with a decrease in net citrate efflux suggests that the CAC intermediate release is balanced by anaplerosis during hibernation. We measured net citrate efflux values from the heart of 2.5 and 1.4 nmol · g¹ · min¹, which account for 10% and 6% of the absolute pyruvate carboxylation flux for HIB and HIB + PYR animals, respectively. This suggests that a decrease in the efflux of other metabolites such as succinate (15) plays a role in maintaining the total pool size of CAC intermediates. It is important to note that net citrate release may underestimate the true rate of citrate loss from the CAC. Cytosolic ATP-citrate lyase could cleave citrate into OAA and acetyl-CoA (12); therefore citrate loss from the CAC may be greater than the net citrate release and account for a larger fraction of the pyruvate carboxylation unless the carbon of OAA returns to the mitochondria.

Another possible site of release from the CAC is the decarboxylation of malate by malic enzyme (13). However, the energetics of the reaction toward decarboxylation are unfavorable (35), and ex vivo experiments suggest that malic enzyme is mainly a carboxylating enzyme (8, 36, 39). In addition, we observed no M1 labeling of pyruvate with either normal flow (21) or with hibernation, which indicates that malate was not being decarboxylated by malic enzyme to produce pyruvate.

Myocardial hibernation did not affect the relative contribution of pyruvate decarboxylation (via pyruvate dehydrogenase) to the CAC flux (see Table 4). This confirms the work of Liedtke (16), which used ¹⁴C-labeled substrate to demonstrate that a 60% reduction in LAD flow in swine decreases CAC flux but does not alter the relative contributions of fatty acids and carbohydrates to oxidative metabolism. This same observation was made using ¹³C-labeled glucose and NMR analysis in dogs subjected to a 30% reduction in coronary flow for 3-4 h (19). Thus with acutely hibernating myocardium, there is no change in the contribution of carbohydrate to oxidative metabolism.

The elevated pyruvate concentrations in the HIB + PYR group did not elicit an increase in tissue pyruvate, pyruvate decarboxylation, or pyruvate carboxylation (see Tables 1 and 4; Fig. 5) or an increase in tissue M3 enrichment of pyruvate (data not shown). The lack of effect of elevated arterial pyruvate may be attributed to a low uptake of pyruvate into the myocardium. Studies infusing pyruvate at higher concentrations (5 mM) have reported an increased contribution of pyruvate to citrate after ischemia and improved contractile function (6, 37). In this study, with an arterial pyruvate concentration of 1.21 mM, we did not see any effect of pyruvate on pyruvate carboxylation or decarboxylation compared with an arterial pyruvate concentration of 0.28 mM. Further experimentation will be necessary to determine whether higher arterial concentrations of pyruvate will increase the contribution of pyruvate to citrate during hibernation.

The present results suggest that there is a fine interregulation among the rates of CAC flux, pyruvate carboxylation, and release of CAC intermediates and the CAC pool size; however, the biochemical mechanisms are unclear. We did not observe a decrease in the in vitro activities of malic enzyme and/or pyruvate carboxylase, which suggests that there was not stable covalent modification of these enzymes, though it is possible that the effect of in vivo modification was lost when measured in vitro at near maximal activity. Hibernation does not appear to cause gross changes in some of the regulators of malic enzyme or pyruvate carboxylase such as pyruvate, NADP⁺/NADPH, ATP, ADP, malate, and acetyl-CoA (see Tables 1-3) (21, 32). Thus the biochemical mechanisms for the decrease in pyruvate carboxylation remain to be elucidated.

In conclusion, the acute hibernation response is accompanied by a decrease in both pyruvate carboxylation and citrate efflux and the maintenance of tissue levels of CAC intermediates. Thus the integrity of the CAC is maintained during the early phase of myocardial hibernation.