INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

RESPIRATORY INEFFICIENCY in stunned myocardium is characterized by normal levels of oxygen consumption despite reduced contractile function (14). Consistent with normal oxygen use and apparent inefficiency in oxygen consumption (5, 27), tricarboxylic acid (TCA) cycle flux (V_TCA) has also been found to be normal in postischemic hearts (16, 17, 33). Despite normal rates, isolated hearts oxidizing ¹³C-enriched substrates indicate that carbon turnover within the NMR-detectable glutamate pool is slower in postischemic hearts relative to normal hearts because of reduced metabolite transport between the mitochondria and cytosol across the -ketoglutarate (-KG)-malate transporter (16, 33). Although clearly representing a change in the balance between oxidation in the TCA cycle and metabolite exchange between mitochondria and cytosol, the mechanism for this reduced exchange has yet to be elucidated as either a fundamental defect in transporter protein function or a consequence of competition between the oxidative rate within the TCA cycle and mitochondrial influx/efflux of carbon units. This study examines these possibilities as the reason for reduced metabolite exchange via the -KG-malate exchanger transporter and in the process examines a mechanism for maintenance of oxidative flux in response to altered TCA cycle enzyme activity in stunned myocardium.

The balance between V_TCA and the exchange of mitochondrial and cytosolic metabolites is regulated by the coordinated activity of the mitochondrial matrix enzyme, -KG dehydrogenase, and the -KG-malate transporter of the mitochondrial membrane. The oxidative reaction catalyzed by -KG dehydrogenase represents a rate-limiting step within the TCA cycle by balancing flux through two spans of the TCA cycle (23). The dehydrogenase also competes with the reversible -KG-malate transporter for exchange of carbon units between subcellular compartments (12, 13, 22). The reversible -KG-malate transporter functions independently or, working in tandem with the unidirectional glutamate-aspartate exchanger, forms the malate-aspartate shuttle (4, 25, 31). Our laboratory has previously (37) demonstrated that the rate of glutamate labeling in normal hearts, via ¹³C enrichment of TCA cycle intermediates, is responsive to an increase in flux through the -KG-malate transporter during recruitment of net forward malate-aspartate shuttle activity. Whether reduced flux through the -KG-malate transporter of stunned myocardium is also responsive to such stimulation has yet to be determined and would aid in elucidating the mechanism for this reduced transport and the altered balance between oxidative flux and metabolite influx/efflux across the mitochondrial membrane.

Therefore, we examined the mechanism for reduced metabolite exchange between the mitochondria and cytosol in postischemic hearts. The findings confirm a shift in the balance between oxidative rate and mitochondrial/cytosolic interactions, suggesting a homeostatic mechanism for preserving V_TCA during pathophysiological changes in mitochondrial dehydrogenase activity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated heart model. Hearts were excised from Dutch Belted rabbits (500 g) that were given an intraperitoneal injection of heparin (20 U) and anesthetized with ketamine (500 U) and Telozol (200 U). Before either ischemia or perfusion with labeled substrate, isolated hearts were retrograde-perfused at 100-cm hydrostatic pressure, using a modified Krebs-Henseleit buffer solution containing (in mM) 116 NaCl, 4 KCl, 1.5 CaCl₂, 1.2 MgSO₄, 1.2 NaHPO₄, 25 NaHCO₃, and 5 glucose, and oxygenated with 95% O₂-5% CO₂. The temperature of the buffer entering the heart was maintained at 37°C. A latex balloon containing water was inserted into the left ventricle, and end-diastolic pressure was set at 5-10 mmHg. The balloon was connected to a pressure transducer to monitor left ventricular developed pressure (LVDP) and heart rate (HR). At the end of the experiment, myocardial oxygen consumption (MO₂) was calculated from the difference in O₂ content of the perfusion medium in the supply line and coronary effluent (21). Coronary effluent was collected without exposure to air by inserting a catheter into the pulmonary artery and withdrawing perfusate into a syringe. Coronary flow was established by collecting the fluid dripping from the pulmonary artery and heart in a graduated cylinder for 1 min.

Experimental protocol. All hearts were initially perfused with modified Krebs-Henseleit buffer containing 5 mM unlabeled glucose. Coronary effluent was discarded, and the hearts were given 10 min to stabilize rate-pressure product (RPP = HR × LVDP). ³¹P NMR spectra were acquired to establish myocardial viability based on high-energy phosphate content (phosphocreatine, ATP). Isolated hearts were subdivided into four experimental groups perfused with 2.5 mM acetate, with or without 2.5 mM lactate: acetate control group (n = 7), acetate + lactate control group (n = 6), acetate reperfusion group (after 10 min ischemia, n = 5), and acetate + lactate reperfusion group (after 10 min ischemia, n = 7). Exogenous lactate was added to augment cytosolic redox state (NADH/NAD⁺) (24, 26, 37). In reperfused hearts, global ischemia was induced by stopping the perfusion supply pump and clamping the aortic line. Immediately after glucose perfusion or the ischemic period, the buffer was switched to the unenriched substrates listed for each group. All hearts were perfused for 10 min, and background ¹³C NMR spectra were acquired. The buffer substrate was then switched from unlabeled acetate to a recirculated reservoir of 2.5 mM 2-¹³C-enriched acetate (Isotec, Miamisburg, OH). Subsequent sequential ¹³C NMR spectra (1.3- or 2.6-min blocks) were acquired for 30-40 min. MO₂ measurements were taken after the heart was removed from the magnet. The heart was then freeze-clamped and prepared for biochemical assays and high-resolution ¹³C NMR analysis.

NMR measurements. NMR parameters required for acquisition of ³¹P and ¹³C NMR spectra are as previously reported (16, 22, 38). Briefly, perfused hearts were positioned in a 20-mm broadband probe in the 9.4-T/89-mm vertical-bore superconducting NMR magnet. Magnetic field homogeneity was optimized by shimming to a proton linewidth of 15-30 Hz. A ³¹P spectrum of heart was acquired to confirm normal energetic status based on phosphocreatine, -, -, and -ATP content. Carbon spectra were then acquired at 100 MHz with bilevel broadband decoupling and subtraction of endogenous signal from naturally abundant ¹³C (16, 22, 38). In addition, in vitro ¹³C NMR high-resolution spectra were acquired from perchloric acid extracts of myocardium in a 5-mm probe to determine the fraction of 2-¹³C-labeled acetyl-CoA.

Tissue chemistry. Perchloric acid extracts were obtained from ventricular muscle as previously described. Glutamate, -KG, citrate, and aspartate concentrations were determined from ultraviolet spectrophotometric and fluorometric techniques (3, 34).

Kinetic model. For purposes of data analysis, a simple kinetic model of nine differential equations, describing known biochemistry and the isotopic enrichment of key metabolic pools, was applied as previously described in great detail (16, 22, 36, 37). The model has been previously used under appropriate experimental conditions to examine both V_TCA and metabolite transport in normal and postischemic hearts (16, 22, 37). V_TCA and the interconversion rate between cytosolic glutamate and mitochondrial -KG (F₁) were determined by nonlinear least-squares fitting of the model to ¹³C NMR data of the second and fourth carbons of glutamate (C-2 and C-4) enrichment.

Statistical analysis. Data set comparisons were performed with Student's unpaired two-tailed t-test. Differences in mean values were considered statistically significant at a probability level of <5% (P < 0.05).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Contractile function and MO₂. All hearts were perfused with glucose before the start of each protocol under either control conditions or ischemia-reperfusion. In this manner the ischemic insult was standardized for substrate availability, and we were able to focus on a comparison of metabolic flux during oxidation of acetate in normal or postischemic hearts. All isolated heart preparations were essentially the same before an experimental fate was delineated. Hearts were randomly chosen for each of the experimental groups, and no difference in mechanical work was evident between eventual groups of hearts before the start of any of the protocols. During the initial setup period of perfusion with glucose, before the initiation of any experimental protocol, the mean RPP of hearts entering the control protocol and the mean RPP for hearts before the ischemic protocol were 17,347 ± 2,597 and 17,031 ± 2,313 mmHg · beats · min¹, respectively.

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Rate-pressure product (RPP) over the course of perfusion. , Values for normal hearts; , values for postischemic hearts. A: RPP from hearts perfused with acetate during normal perfusion or reperfusion (means ± SD). B: RPP from hearts perfused with acetate supplemented with lactate during normal perfusion or reperfusion. Postischemic hearts displayed significantly reduced RPP throughout the reperfusion periods (P < 0.05). bpm, Beats/min.

Metabolite content and isotopic enrichment. Steady state metabolite contents are listed in Table 1 for all experimental groups. Metabolite contents were similar to those of previously published results (16), showing the expected drop in postischemic glutamate content to 40% (P < 0.005) lower than values in normal hearts.

¹³C NMR spectroscopy, isotope kinetics, and metabolic flux. A representative sequential ¹³C NMR spectrum acquired from normal hearts oxidizing [2-¹³C]acetate is shown in Fig. 2. Similar spectra (not shown) were acquired for normal and reperfused hearts provided acetate supplemented with lactate. Figure 3 graphically displays the time course of ¹³C enrichment of glutamate (means ± SD) at C-2 and C-4 from spectra of normal and reperfused acetate hearts. The time course of ¹³C enrichment of glutamate at C-2 and C-4 from spectra of normal and reperfused acetate + lactate hearts is shown in Fig. 4. Note that the flux parameters shown in Fig. 5 are obtained from data combining such isotope enrichment curves with the corresponding metabolite pool sizes. The least-squares fitting of the kinetic model to the data is also shown in Figs. 3 and 4. The correlation coefficient between the data and the fit was 0.98. Output from the model provided V_TCA and F₁.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Dynamic 13C NMR spectra from isolated rabbit hearts during the pre-steady-state enrichment with [2-13C]acetate. Spectra are from a normal heart (A) and from a postischemic heart (B). Peak assignments: GLU-C2, 2nd carbon of glutamate; GLU-C3, 3rd carbon of glutamate; GLU-C4, 4th carbon of glutamate; and ACE-C2, 2nd carbon of acetate. PPM, parts per million. Final glutamate signal in reperfused hearts was slightly lower because of lower glutamate content in these hearts (see Table 1).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Time course of glutamate 13C enrichment from both NMR measurements and kinetic analysis for normal perfused (A) and postischemic (B) hearts oxidizing 2.5 mM [2-13C]acetate. Signal intensities from dynamic 13C NMR spectra are normalized to steady-state enrichment levels of the GLU-C4. , GLU-C4 enrichment; , GLU-C2 enrichment. Solid lines, modeled enrichment curves from least-squares fitting. Each experimental group displayed a significantly different time constant for the 2 labeling curves; graphs shown are for illustrative purposes. For determination of flux values, glutamate enrichment curves were analyzed by least-squares fitting to an established kinetic model that was employed on each set of enrichment curves from each single heart (see MATERIALS AND METHODS).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Time course of glutamate 13C enrichment from both NMR measurements and kinetic analysis for hearts oxidizing 2.5 mM [2-13C]acetate supplemented with 2.5 mM unlabeled lactate in normal perfused (A) and postischemic (B) hearts. , GLU-C4 enrichment; , GLU-C2 enrichment; solid lines, modeled enrichment curves from least-squares fitting.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   TCA cycle flux (VTCA) and metabolite interconversion rates (F1) in normal perfused (A) and postischemic (B) hearts. Solid bars, hearts oxidizing 2.5 mM [2-13C]acetate; open bars, hearts oxidizing 2.5 mM [2-13C]acetate supplemented with 2.5 mM unlabeled lactate. Values are means ± SD. In both normal and postischemic hearts, hearts oxidizing acetate + lactate display a significantly higher F1. * P < 0.05 vs. hearts oxidizing acetate alone;  F1 is significantly reduced in postischemic hearts compared with normal hearts (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study explores the regulation of mitochondrial oxidative function in intact, stunned hearts with ¹³C NMR at an investigative level previously restricted to isolated mitochondria (11-13, 23, 25, 31). Sequential ¹³C NMR spectra were obtained from intact hearts under conditions of normal and postischemic perfusion with [2-¹³C]acetate, with or without elevated cytosolic redox potential. TCA cycle rate and the rate of -KG efflux from the mitochondria for interconversion with cytosolic glutamate (-KG-malate transport rate) were determined by fitting a kinetic model to the dynamic ¹³C enrichment data of glutamate (16, 22, 37, 38). As previously reported, net V_TCA was unchanged between controls and reperfused hearts perfused with acetate, whereas -KG-malate transport rate was significantly reduced in the postischemic hearts perfused with buffer containing acetate (16).

In this study, increasing cytosolic redox state with addition of lactate caused -KG-malate exchange to increase in both normal and reperfused hearts. Despite countering of the reduced rate of -KG-malate exchange in stunned hearts to demonstrate that the transporters remained responsive to cytosolic redox state in the stunned heart, contractility remained depressed during reperfusion. Consequently, we were able to demonstrate that despite reduced -KG-malate transporter activity in stunned myocardium, the transporter, as part of the malate-aspartate shuttle, remains responsive to redox state changes. Thus the results show that the reduced exchange of metabolites across the mitochondrial membrane is not caused by dysfunction of the -KG-malate exchange protein but rather indicates a change in the balance between -KG transport and -KG oxidation, as described above. Because transporter function appears to be intact, the cause for the reduced metabolite exchange rates across the mitochondrial membrane of the stunned myocardium must be a response to altered rates of oxidation at the -KG dehydrogenase reaction. This finding indicates a metabolic component of stunning at the level of mitochondrial dehydrogenase activity, affecting metabolite exchange across the mitochondrial membrane. The mechanisms of metabolic flux homeostasis that account for these findings are discussed below.

The regulatory enzymes that control the rate of V_TCA in hearts oxidizing acetate have been discussed previously by Randle et al. (23). They described two separate spans of the TCA cycle that are regulated by different rate-limiting enzymes: acetyl-CoA to -KG, which is controlled by citrate synthase; and -KG to oxaloacetate, which is controlled by -KG dehydrogenase. At steady state, metabolite concentrations attain stability as the rates of the two cycle spans become equal. One mechanism for attaining equilibrium among the TCA cycle intermediate pools and maintaining coordinated flux through the two spans is the competition between the oxidation of -KG and the transport of -KG between the mitochondria and cytosol by the reversible -KG-malate exchanger (12, 13, 22). Thus this competition for -KG between the -KG dehydrogenase and the reversible -KG-malate transporter serves as a balance point for V_TCA homeostasis.

The balance between V_TCA and the exchange of metabolite between the mitochondria and cytosol is regulated by the mitochondrial redox state (4, 35), intramitochondrial calcium and hydrogen ion levels (6, 9), and substrate availability (23). Although the -KG dehydrogenase is sensitive to all these factors, the pH-independent -KG-malate transporter is sensitive to substrate availability and, indirectly, to cytosolic redox state (NADH/NAD⁺) as part of the malate-aspartate shuttle. Whereas it is clear from our earlier work that this -KG-malate transport rate is reduced in postischemic heart (16), it is not clear whether the transporter is dysfunctional or simply responds to regulation. The results of the present study demonstrate that the transporter remains responsive in stunned myocardium and does not limit the availability of metabolite to the mitochondria for the oxidative processes of the TCA cycle and respiratory chain.

The -KG-malate transporter also functions as part of the malate-aspartate shuttle. The activity of the shuttle is key to coordinating exchange of metabolites between the mitochondria and cytosol and transferring reducing equivalents from the cytosol into the mitochondria. Net forward flux through the malate-aspartate shuttle involves the coordinated activity of the reversible -KG-malate exchanger and the unidirectional glutamate-aspartate exchanger.

The exchange of labeled -KG from the mitochondria with the cytosolic glutamate pool does not require both transporters of the malate-aspartate transporter. Instead, the reversible -KG-malate exchanger need only be available to enable the interconversion of labeled -KG with cytosolic glutamate. This was evident in our earlier work in which the observed efflux of labeled -KG from the mitochondria was delayed in the postischemic hearts provided [2-¹³C]acetate (16). In that study, supplying postischemic hearts with both acetate and labeled glucose produced negligible glycolytic activity for cytosolic NADH production, thus indicating very little involvement of the malate-aspartate shuttle. In the present study, increasing cytosolic redox state with lactate induced net forward flux through this shuttle, as demonstrated in a previously published report (37).

For the hearts in the current study that were oxidizing acetate, the TCA cycle was the primary source of reducing equivalents that entered the respiratory chain to account for the measured oxygen use. Other contributions to respiration include the reducing equivalents produced in the mitochondria from a small amount of -oxidation of endogenous fatty acids as well as reducing equivalents that were produced in the cytosol from glycolysis or by metabolism of exogenous lactate. Normal and reperfused hearts oxidizing acetate alone were nearly solely reliant on the TCA cycle as the source of reducing equivalents, and the measured V_TCA rate accounted for 100% of the measured oxygen consumption in these hearts. Thus, as expected, net forward flux across the aspartate-malate shuttle is essentially zero at this relatively low cytosolic redox state and does not significantly contribute to oxidative energy production. This finding is important because it confirms that the observed F₁ value, for -KG efflux/influx across the mitochondrial membrane under the condition of acetate as sole substrate, represents a basal level of exchange across the -KG-malate carrier and not malate-aspartate shuttle activity.

In hearts oxidizing acetate in the presence of lactate to increase cytosolic reducing equivalents, the TCA cycle accounted for 62% of the oxygen consumed in both normal and reperfused groups. Within experimental error, the remainder of oxygen use can be attributed primarily to the recruitment of net forward aspartate-malate shuttle flux. In this case, the shuttle served as a significant source of reducing equivalents entering the mitochondria during the lactate-induced increase in cytosolic redox state.

Results from reperfusion experiments reveal that the -KG-malate transporter is responsive to stimulation in stunned myocardium. Whereas V_TCA is not statistically different from that in respective nonischemic hearts, metabolite exchange is increased nearly threefold in hearts provided acetate + lactate compared with hearts supplied with acetate alone (9.8 ± 3.7 vs. 3.4 ± 1.0 µmol · min¹ · g dry wt¹, respectively). This result suggests that there is not a fundamental defect in transporter protein function. The reduced exchange rate observed in reperfused hearts provided acetate alone is more likely to be a result from substrate competition between the transporter and the -KG dehydrogenase enzyme of the mitochondria.

This hypothesis is supported by a recent finding from our laboratory (22). In the recent study, we examined the effects of increasing cytosolic and intramitochondrial Ca²⁺ and H⁺ content on -KG dehydrogenase flux and metabolite exchange across the mitochondrial membrane in both intact hearts and isolated mitochondria. The results indicated that elevated Ca²⁺ and H⁺ content increased -KG oxidation and reduced -KG efflux from the mitochondria. This apparent competition for substrate by the -KG transporter and -KG dehydrogenase is attributed to their relative Michaelis constant (K_m) values (12, 28-30). Earlier work reported that the -KG-malate transporter of the mitochondrial membrane has a relative K_m of 1.5 mM for -KG on the matrix side of the carrier (28), whereas the K_m of -KG dehydrogenase for -KG was reported as 0.67 mM (12). In our recent study (22) with isolated mitochondria incubated at low pH, the apparent K_m of the -KG dehydrogenase decreased by 50% relative to mitochondria incubated at normal pH. This makes both oxidation and efflux very sensitive to regulation by the -KG concentration in the mitochondrial matrix.

Whereas pH is reduced during ischemia, pH recovers immediately after reperfusion (1, 15) and is not likely to stimulate -KG dehydrogenase activity. However, mitochondrial dehydrogenase activity is also sensitive to Ca²⁺ content (6, 8, 9, 18, 32). If mitochondrial Ca²⁺ overload persists after reperfusion (2, 7, 10, 19, 20), dehydrogenase activity can be stimulated. Thus a decrease in mitochondrial -KG content (16) paralleled with an increase in -KG dehydrogenase activity, caused by elevated Ca²⁺, shifts the efflux of -KG from the mitochondria to oxidation by the dehydrogenase in postischemic myocardium. This altered balance between V_TCA and metabolite exchange observed in stunned hearts suggests a homeostatic mechanism for preserving V_TCA during pathophysiological changes in mitochondrial dehydrogenase activity.

In conclusion, sequential ¹³C NMR spectra were obtained from intact hearts under conditions of normal and postischemic perfusion with [2-¹³C]acetate, with or without elevated cytosolic redox potential. Dynamic ¹³C NMR analysis revealed the balance between mitochondrial TCA cycle rates and metabolite exchange between the mitochondria and cytosol. The increase in -KG-malate transport rate observed in postischemic hearts at elevated redox state denotes that the malate-aspartate shuttle remains responsive in stunned myocardium and can be recruited by high NADH/NAD⁺ in the cytosol. This result indicates that the reduction in metabolite transport, observed in postischemic hearts, is not caused by a fundamental defect in transporter protein function but rather reflects an adjustment to a new balance in carbon flux in maintaining homeostasis between the first and second spans of the TCA cycle.