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(Circulation Research. 1996;79:940-948.)
© 1996 American Heart Association, Inc.

Articles

Cardiac Efficiency Is Improved After Ischemia by Altering Both the Source and Fate of Protons

Bin Liu, Alexander S. Clanachan, Richard Schulz, Gary D. Lopaschuk

the Cardiovascular Disease Research Group, Departments of Pediatrics (B.L., R.S., G.D.L.) and Pharmacology (B.L., A.S.C., R.S., G.D.L.), The University of Alberta, Edmonton, Canada.

Correspondence and reprint requests to Dr Gary D. Lopaschuk, 423 Heritage Medical Research Bldg, The University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail gary.lopaschuk@ualberta.ca.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cardiac efficiency is decreased in hearts after severe ischemia. We determined whether reducing the production of H⁺ from glucose metabolism or inhibiting the clearance of H⁺ via Na⁺-H⁺ exchange could increase cardiac efficiency during reperfusion. This was achieved using dichloroacetate (DCA) to stimulate glucose oxidation and 5-(N,N-dimethyl)-amiloride (DMA) to inhibit Na⁺-H⁺ exchange, respectively. Isolated working rat hearts were subjected to 30 minutes of global ischemia and 60 minutes of reperfusion. Glycolysis and oxidation rates of glucose, lactate, and palmitate were measured. Recovery of cardiac work, O₂ consumption (MVO₂), and rates of acetyl-coenzyme A and ATP production during reperfusion were determined. After ischemia, cardiac work recovered to 35±5% of preischemic values in control hearts (n=23), although MVO₂, tricarboxylic acid (TCA) cycle activity, and ATP production from glycolysis and oxidative metabolism rapidly recovered to preischemic levels. This decrease in cardiac efficiency was accompanied by a substantial production of H⁺ from glucose metabolism. DCA caused a 2.2-fold increase in glucose oxidation, a 46±17% decrease in H⁺ production, a 1.6-fold increase in cardiac efficiency, and a 2.0-fold increase in cardiac work during reperfusion (n=17). Inhibition of Na⁺-H⁺ exchange with DMA did not alter TCA cycle activity and ATP production rates but did result in a 1.8-fold increase in cardiac efficiency and a 1.7-fold increase in cardiac work (n=12). These data show that cardiac efficiency and the contractile function after ischemia can be improved by either reducing the rate of H⁺ production from glucose metabolism during reperfusion or inhibiting the clearance of H⁺ via Na⁺-H⁺ exchange. Our data suggest that an increased requirement for ATP to restore ischemia-reperfusion–induced alterations in ion homeostasis contributes to the decrease in cardiac efficiency and contractile function after ischemia.

Key Words: glycolysis • glucose oxidation • lactate oxidation • Na⁺/H⁺ exchanger • fatty acid oxidation • reperfusion

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
During ischemia, oxidative metabolism is suppressed and anaerobic glycolysis becomes an important source of ATP production.¹ In severely ischemic myocardium, production of H⁺ from the hydrolysis of glycolytically derived ATP is a major contributor to the acidosis that occurs in the myocardium.² Upon reperfusion, intracellular pH quickly recovers, although this can lead to a significant increase in intracellular Na⁺ and Ca²⁺ that contributes to contractile dysfunction.^{3 4 5 6} A number of studies have now shown that intracellular acidosis during severe ischemia increases sarcolemmal Na⁺-H⁺ exchange during reperfusion (see References 7 and 8 for review). The resultant increase in intracellular Na⁺ in turn can increase Ca²⁺ accumulation within the myocyte, due to a decrease in the transsarcolemmal Na⁺ electrochemical gradient, resulting in a decreased Ca²⁺ efflux via the Na⁺-Ca²⁺ exchanger. This can lead to Ca²⁺ overload and cell death.^{7 8 9 10} Accumulation of intracellular H⁺ during ischemia is an important contributing factor to these sequelae, and continued production of H⁺ during the critical early period of reperfusion has the potential to exacerbate injury.^{11 12}

In most clinical situations of reperfusion after ischemia, the heart muscle is exposed to high levels of fatty acids (see Reference 13 for review). If hearts are aerobically reperfused after ischemia, fatty acid oxidation quickly recovers to rates that can equal or exceed preischemic rates.^{12 14 15 16 17} High rates of fatty acid ß-oxidation dramatically inhibit glucose oxidation (see References 1 and 18 for review), which results in a marked imbalance between glycolysis and glucose oxidation.^{11 19 20} This uncoupling is a major source of H⁺ production in the heart. If glycolysis is coupled to glucose oxidation, H⁺ production from glucose metabolism is zero.^{11 21 22} However, if glycolysis is uncoupled from glucose oxidation (and pyruvate derived from glycolysis is not oxidized), there is a net production of 2 H⁺ from each glucose molecule that comes from the hydrolysis of glycolytically derived ATP. Recently, we demonstrated that in isolated working rat hearts perfused with fatty acids, mitochondrial function and overall ATP production quickly recover after a 30-minute period of severe ischemia.¹² However, overall ATP production is not efficiently translated into mechanical work, resulting in a marked decrease in cardiac efficiency in the postischemic heart. While we speculated that an increased production of H⁺ from glycolysis uncoupled from glucose oxidation may contribute to this decrease in efficiency, direct evidence to support this was lacking.

A recent study by Hata et al²³ showed that clearance of H⁺ via the Na⁺-H⁺ exchanger in aerobically perfused hearts subjected to an intracellular acid load leads to a significant decrease in cardiac efficiency. This is presumably due to the fact that the increase in intracellular Na⁺ and Ca²⁺ levels that arises after activation of the Na⁺-H⁺ and Na⁺-Ca²⁺ exchanger activity requires energy-dependent pathways to restore ion homeostasis. By inhibiting the Na⁺-H⁺ exchanger, Hata et al²³ were able to increase cardiac efficiency. Based on their observation, we hypothesized that both the production and fate of H⁺ affect cardiac efficiency in postischemic hearts. If this is the case, then either decreasing the production of H⁺ during reperfusion or inhibiting the clearance of H⁺ via the Na⁺-H⁺ exchanger should increase cardiac efficiency during reperfusion.

DCA is a PDH activator²⁴ that stimulates glucose oxidation during reperfusion of ischemic hearts.^{11 25} DCA also decreases the imbalance between glycolysis and glucose oxidation, resulting in a significant decrease in H⁺ production from glucose metabolism during reperfusion.^{11 12} In this study we determined whether decreasing this source of H⁺ production during reperfusion would be accompanied by an increase in cardiac efficiency in hearts reperfused after ischemia. We also determined whether inhibition of Na⁺-H⁺ exchange with DMA was also capable of increasing cardiac efficiency during reperfusion. Isolated working rat hearts perfused with high levels of fatty acids were subjected to a 30-minute period of global no-flow ischemia, followed by 60 minutes of aerobic reperfusion. The effects of DCA and DMA on the recovery of cardiac work, O₂ consumption, glycolysis, and overall oxidative metabolism of glucose, lactate, and fatty acid were measured. Our results demonstrate that by inhibiting the source and altering the fate of H⁺, a significant improvement in the recovery of mechanical function and cardiac efficiency is seen in the postischemic heart.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heart Perfusions
Isolated rat hearts were cannulated for working heart perfusions as described previously.¹¹ In brief, male Sprague-Dawley rats (Charles River Laboratories, Montreal, Quebec, 300 to 350 g) were anesthetized with sodium pentobarbital, and the hearts were quickly excised and immersed in ice-cold Krebs-Henseleit solution. The aorta was cannulated and retrograde perfusion at 37°C was initiated at a hydrostatic pressure of 60 mm Hg. Hearts were trimmed of excess tissue. The pulmonary artery and the opening to the left atrium were cannulated. After 10 minutes of Langendorff perfusion, hearts were switched to the working mode by clamping the aortic inflow line from the Langendorff reservoir and opening the left atrial inflow line. The perfusate was delivered from an oxygenator into the left atrium at a preload pressure of 11.5 mm Hg. The perfusate was ejected from spontaneously beating hearts into a compliance chamber and then into an aortic outflow line. The afterload was set at a hydrostatic pressure of 80 mm Hg. All working hearts were perfused with Krebs-Henseleit solution containing 2.5 mmol/L free Ca²⁺, 11 mmol/L glucose, 0.5 mmol/L lactate, 1.2 mmol/L palmitate, 100 µU·mL^-1 insulin, and 3% BSA (fraction V, Boehringer Mannheim). Palmitate was bound to the albumin as described previously.¹⁷

Experimental Protocols
Working hearts were initially perfused for a 30-minute period under aerobic conditions. Global no-flow ischemia was then introduced by clamping both the left atrial inflow and aortic outflow lines. After 30 minutes of no-flow ischemia, the left atrial and aortic flows were restored and the hearts were reperfused for 60 minutes under aerobic conditions. When used, DCA (BDH Chemicals Ltd) was added immediately before the reperfusion of ischemic hearts at a 1 mmol/L final concentration. DMA (Sigma Chemical Company), when used, was added 10 minutes before the onset of no-flow ischemia to a final concentration of 2 µmol/L. DMA was first dissolved in perfusion buffer in a stock concentration of 2 mmol/L and wrapped with aluminum foil. Due to the light sensitivity of DMA, these experiments were done in a darkened room to reduce the direct exposure of DMA to light.

Spontaneously beating hearts were used throughout the studies. Heart rate and aortic pressure were measured with a Gould P21 pressure transducer in the aortic outflow line. Cardiac output and aortic flow were measured with Transonic ultrasound flow probes in the preload and afterload lines, respectively. Coronary flow was calculated as the difference between cardiac output and aortic flow. The O₂ content in the perfusate was measured using YSI micro oxygen electrodes in the preload line and in a line originating from the cannulated pulmonary artery. Myocardial O₂ consumption (MVO₂) was calculated according to the Fick principle, using coronary flow rates and the arteriovenous difference in perfusate O₂ concentration. Cardiac work was calculated as the product of systolic pressure and cardiac output. Cardiac efficiency was defined as a ratio of cardiac work to MVO₂, and as the ratio of cardiac work to TCA cycle activity (the total rate of acetyl-CoA production for TCA cycle).

At the end of reperfusion, hearts were quickly frozen with Wollenberger clamps cooled to the temperature of liquid N₂. The atrial tissue was dried in an oven for 12 hours at 100°C and weighed. The frozen ventricular tissue was weighed and powdered in a mortar and pestle cooled to the temperature of liquid N₂. A portion of the powdered tissue was used to determine the dry weight–to–wet weight ratio. The dried atrial weight, frozen ventricular weight, and ventricular dry weight–to–wet weight ratio were then used to determine the total dry weight of the heart.

Measurement of Glycolysis, Glucose Oxidation, Lactate Oxidation, and Palmitate Oxidation
Glycolysis and glucose oxidation were simultaneously measured by perfusing hearts with perfusate containing [5-³H/U-¹⁴C]glucose as described previously.¹¹ The total myocardial ³H₂O production and ¹⁴CO₂ production were determined at 10-minute intervals during both the initial aerobic perfusion period and the 60-minute period of reperfusion. To measure the rates of glycolysis, ³H₂O in perfusate samples was separated from [³H]glucose and [¹⁴C]glucose using Dowex columns, which contain Dowex 1-X4 anion exchange resin (200 to 400 mesh) extensively washed with distilled H₂O after pretreatment with 0.2 mol/L potassium tetraborate. Glucose oxidation was determined by quantitative measurement of ¹⁴CO₂ production including ¹⁴CO₂ released as a gas in the oxygenation chamber and ¹⁴CO₂ dissolved as HCO₃^- in perfusate. The gaseous ¹⁴CO₂ was trapped in hyamine hydroxide solution through an exhaust line in the perfusion system. The dissolved ¹⁴CO₂ as HCO₃^- was released and trapped on a filter with hyamine hydroxide in the central well of 25-mL stoppered flasks after perfusate samples were acidified by the addition of 9N H₂SO₄.

Lactate oxidation was determined by adding [U-¹⁴C]lactate to the perfusate, and ¹⁴CO₂ was measured in a method similar to that of glucose oxidation.²⁰ Palmitate oxidation was determined by adding [9,10-³H]palmitate to BSA buffer to label the 1.2 mmol/L palmitate in the perfusate. The sampling of total ¹⁴CO₂ and ³H₂O production was also measured at 10-minute intervals during the initial aerobic perfusion and during the 60 minutes of reperfusion. The ³H₂O was separated from [³H]palmitate by using a chloroform extraction technique.¹⁹ This yielded greater than a 99% separation efficiency of ³H₂O from [³H]palmitate.

Calculation of H⁺ Production From Glucose Utilization
If glucose passes through glycolysis to lactate, a net production of 2 H⁺ ions per molecule of glucose occurs.^{4 22} In contrast, if glycolysis is coupled to glucose oxidation, the net production of H⁺ is zero (this is because beyond the oxidation of pyruvate a net consumption of 2 H⁺ occurs). Therefore, the overall rate of H⁺ ion production derived from glucose utilization was determined by subtracting the rate of glucose oxidation from the rate of glycolysis and multiplying by 2.

Calculation of Acetyl-CoA and ATP Production Rates
The rate of acetyl-CoA production for TCA cycle was calculated assuming 1 acetyl-CoA was produced from lactate oxidation, 2 acetyl-CoA from glucose oxidation, and 8 acetyl-CoA from palmitate oxidation. ATP production from glycolysis, lactate oxidation, glucose oxidation, and palmitate oxidation was calculated and presented as a percentage of total ATP production, assuming 2 ATP are produced per glucose passing through glycolysis, 18 ATP per lactate oxidized, 36 ATP per glucose oxidized, and 129 ATP per palmitate oxidized.

Statistical Analysis
All data are mean±SEM. The data were analyzed with the statistical program SPSS/PC⁺ (SPSS Inc). Student's t test was used to determine the difference between preischemic and postischemic values. One-way ANOVA was used to compare the preischemic or postischemic values among the control, DCA-, and DMA-treated hearts. Two-way ANOVA was used to compare the values at the same reperfusion time among the control, DCA-, and DMA-treated hearts. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of DCA and DMA on the Recovery of Mechanical Function During Reperfusion of Hearts After 30 Minutes of Global No-Flow Ischemia
Fig 1A shows the effects of the addition of DCA and DMA on the recovery of cardiac work in hearts subjected to 30 minutes of global ischemia. After ischemia, the recovery of cardiac work was depressed in control hearts, returning to only 35±5% of preischemic values at 60 minutes of reperfusion. During reperfusion, heart rate recovered to preischemic values (Table 1), but peak systolic pressure, developed pressure, cardiac output, aortic flow, and coronary flow were all significantly depressed during reperfusion. During reperfusion, MVO₂ in control hearts recovered to a greater extent than cardiac work (Fig 1B), resulting in a significant decrease in cardiac efficiency throughout the entire 60-minute reperfusion period (Fig 1C).

View larger version (23K): [in this window] [in a new window]   Figure 1. Effects of DCA and DMA on the recovery of cardiac work (A), O2 consumption (B), and cardiac efficiency (C) of hearts reperfused after 30 minutes of global no-flow ischemia. Values are mean±SEM of 23 control hearts (), 17 hearts in which DCA (1 mmol/L) was added immediately before reperfusion (), or 12 hearts in which DMA (2 µmol/L) was added 10 minutes before the onset of ischemia (). *Significantly different from control hearts at the corresponding reperfusion time.

View this table: [in this window] [in a new window]   Table 1. Effects of DCA and DMA on the Recovery of Mechanical Function of Postischemic Working Rat Hearts

When 1 mmol/L DCA was added at reperfusion, cardiac work recovered quickly to 66±6% of preischemic values by 10 minutes of reperfusion and continued to recover to 81±6% of preischemic values by 30 minutes of reperfusion (Fig 1A and Table 2). At the end of 60 minutes of reperfusion, the recovery of cardiac work in DCA-treated hearts was 68±9% of preischemic values, compared with only 35±5% in control hearts (P<.05). Peak systolic pressure, developed pressure, cardiac output, aortic flow, and coronary flow also recovered to significantly greater values than in control hearts (Table 1). MVO₂ in DCA-treated hearts quickly recovered to preischemic values (Fig 1B). In the DCA-treated hearts, cardiac efficiency also recovered to preischemic values and was significantly greater at any time point of reperfusion compared with control hearts (P<.05) (Fig 1C).

View this table: [in this window] [in a new window]   Table 2. Effects of DCA and DMA on the Recovery of Cardiac Work, O2 Consumption, and Cardiac Efficiency in Postischemic Working Rat Hearts

The effects of DMA (2 µmol/L) on the recovery of mechanical function are also shown in Fig 1 and Tables 1 and 2. Previous studies have shown that Na⁺-H⁺ exchange inhibitors must be added before ischemia to exert their beneficial effects during reperfusion (see References 7 and 8 for review). We therefore added DMA to the perfusate 10 minutes before the onset of ischemia. Preliminary experiments were performed to determine a concentration of DMA that had no direct effect on cardiac function under aerobic conditions. In cumulative concentration-response experiments in aerobically perfused hearts, DMA had no significant effects on peak systolic pressure, cardiac output, or MVO₂ at concentrations between 1 and 20 µmol/L. However, heart rate decreased significantly at DMA concentrations >5 µmol/L (heart rate was 249±9, 237±16, 231±18, 202±23, 191±17, and 173±25 bpm at 0, 1, 2, 5, 10, and 20 µmol/L DMA, respectively, n=6). Because of this, we used a concentration of 2 µmol/L DMA in all further experiments. As shown in Fig 1A, DMA also significantly improved the rate and extent of recovery of cardiac work. The recoveries of peak systolic pressure, developed pressure, cardiac output, and aortic flow were also all significantly increased during reperfusion in the DMA-treated hearts compared with control hearts (Table 1). DMA had no effect on MVO₂ (Fig 1B), but due to the enhanced recovery of function, cardiac efficiency was increased during reperfusion in the DMA-treated hearts (Fig 1C and Table 2).

Effects of DCA and DMA on Glycolysis, Glucose Oxidation, Lactate Oxidation, and Palmitate Oxidation During Reperfusion of Hearts After Ischemia
Fig 2 shows the amount of substrates metabolized versus time via glycolysis (panel A), glucose oxidation (panel B), lactate oxidation (panel C), and palmitate oxidation (panel D) during the 60-minute period of reperfusion. In all experimental groups a constant rate of glycolysis and glucose oxidation was observed during reperfusion. Lactate oxidation was linear until 40 minutes, after which the rate increased and reached a new steady state. DCA and DMA did not have any significant effects on glycolysis (Fig 2A) or palmitate oxidation (Fig 2D) in the reperfusion period. DMA was also without effects on either glucose oxidation or lactate oxidation. In contrast, addition of DCA at reperfusion resulted in a significant increase in the amount of substrates metabolized via glucose oxidation (Fig 2B) and lactate oxidation (Fig 2C), which would be expected due to its known actions as a PDH complex activator.

View larger version (25K): [in this window] [in a new window]   Figure 2. The effects of DCA and DMA on the time course of glycolysis (A), glucose oxidation (B), lactate oxidation (C), and palmitate oxidation (D) in hearts reperfused after 30 minutes of no-flow ischemia. Values are mean±SEM of 15 control hearts (), 7 hearts in which DCA (1 mmol/L) was added immediately before reperfusion (), or 9 hearts in which DMA (2 µmol/L) was added 10 minutes before the onset of ischemia (). *Significantly different from control hearts at the corresponding reperfusion time.

The effects of DCA and DMA on steady state rates of glycolysis, glucose oxidation, lactate oxidation, and palmitate oxidation are shown in Table 3. Steady state rates were calculated as the averages of values at each time period between 10 and 60 minutes of reperfusion shown in Fig 2. In control hearts, glycolysis recovered to preischemic rates. DCA and DMA had no significant effects on the rates of glycolysis during reperfusion compared with control hearts.

View this table: [in this window] [in a new window]   Table 3. Effects of DCA and DMA on the Steady State Rates of Glycolysis, Glucose Oxidation, Lactate Oxidation, Palmitate Oxidation, and H+ Production From Glucose Utilization in Postischemic Working Rat Hearts

As expected, during the preischemic period, the steady state rates of glucose oxidation in control hearts were substantially lower than the rate of glycolysis (Table 3). This parallels previous observations in isolated rat hearts perfused with high levels of fatty acid^{11 12 19 26} and is similar to the rate of glucose oxidation observed recently in the in vivo pig heart.²⁷ During reperfusion of control hearts, glucose oxidation recovered to preischemic rates. The addition of DCA at reperfusion resulted in a marked increase in glucose oxidation during reperfusion compared with control hearts. In contrast, DMA had no effect on glucose oxidation rates in the postischemic period.

During reperfusion, lactate oxidation rates in control hearts were significantly reduced compared with rates in the preischemic period (Table 3). The addition of DCA resulted in a significant increase in lactate oxidation during reperfusion compared with control hearts. DMA had no effect on lactate oxidation during reperfusion compared with control hearts.

Steady state rates of palmitate oxidation during reperfusion are also shown in Table 3. After ischemia, rates in control hearts returned to preischemic values, confirming our previous findings.^{12 15 19} DCA and DMA had no effects on palmitate oxidation rates during reperfusion.

Effects of DCA and DMA on TCA Acetyl-CoA and ATP Production Rates During Reperfusion of Hearts After Ischemia
In order to evaluate the TCA cycle activity of ischemic hearts during reperfusion, the rate of acetyl-CoA production for TCA cycle from glucose oxidation, lactate oxidation, and palmitate oxidation was calculated accordingly. As shown in Table 4, during reperfusion the total rate of TCA acetyl-CoA production in control hearts recovered to preischemic levels despite a dramatic decrease in the recovery of cardiac work. The addition of DCA at reperfusion significantly increased acetyl-CoA production from glucose and lactate oxidation pathways. However, DCA did not significantly change the total rate of acetyl-CoA production for the TCA cycle during reperfusion. Treatment with DMA had no significant effects on the TCA cycle activity.

View this table: [in this window] [in a new window]   Table 4. Effects of DCA and DMA on the Steady State Rates of TCA Acetyl-CoA Production From Substrate Metabolism in Postischemic Rat Hearts

ATP production from glycolysis, glucose oxidation, lactate oxidation, and palmitate oxidation is shown in Table 5. In the preischemic period the majority of ATP was derived from palmitate oxidation, which accounted for 79±9% of total ATP production. During reperfusion, the contribution of glycolysis, glucose oxidation, and palmitate oxidation was similar to preischemic values, except that the contribution of lactate oxidation to ATP production was significantly lower.

View this table: [in this window] [in a new window]   Table 5. Effects of DCA and DMA on the Rate of ATP Production From Substrate Metabolism in Postischemic Working Rat Hearts

The primary effect of adding DCA at reperfusion was to increase the contribution of glucose oxidation to total ATP production from 12±1% to 27±4% (Table 5). DMA had no effect on overall ATP production in the reperfusion period or on the relative contribution of each of the substrates to ATP production.

Effects of DCA and DMA on H⁺ Production From Glucose Metabolism and Cardiac Efficiency During Reperfusion
Fig 3 shows the cumulative H⁺ production from glucose metabolism during reperfusion of ischemic hearts, calculated from rates of glycolysis and glucose oxidation presented in Fig 2. As shown in Table 3, a substantial uncoupling of glycolysis from glucose oxidation occurs in hearts perfused with high levels of fatty acids. The result is a substantial production of H⁺ from glucose metabolism.^{11 21 22} Over the course of the 60 minutes of reperfusion, >300 µmol^.g dry wt^-1 of H⁺ was produced from glucose metabolism in control hearts (Fig 3). By selectively increasing glucose oxidation rates, DCA improved the coupling between glycolysis and glucose oxidation, resulting in a significant decrease in H⁺ production during reperfusion (Table 3). Although DMA slightly decreased H⁺ production early in reperfusion, no significant changes in H⁺ production occurred during reperfusion compared with control hearts (Fig 3 and Table 3).

View larger version (21K): [in this window] [in a new window]   Figure 3. The effects of DCA and DMA on time course of H+ production from glucose metabolism in hearts reperfused after 30 minutes of no-flow ischemia. Hearts were perfused with dual-labeled [5-3H/U-14C] glucose for simultaneous measurement of glycolysis and glucose oxidation. The H+ production from glucose metabolism was calculated as indicated in "Materials and Methods." Values are the mean±SEM of 15 control hearts, 7 hearts in which DCA (1 mmol/L) was added immediately before reperfusion, or 9 hearts in which DMA (2 µmol/L) was added 10 minutes before the onset of ischemia. *Significantly different from control hearts at the corresponding reperfusion time.

Effects of DCA and DMA on the Coupling of Cardiac Work to TCA Cycle Activity During Reperfusion of Ischemic Hearts
Fig 4 shows the relationship between cardiac work and rates of total TCA acetyl-CoA production during reperfusion of ischemic hearts. In control hearts, a significant decrease in cardiac work/TCA acetyl-CoA occurred during reperfusion compared with preischemic values. The addition of either DCA or DMA to the perfusate resulted in a significant improvement in cardiac work/TCA acetyl-CoA produced during reperfusion compared with control hearts.

View larger version (22K): [in this window] [in a new window]   Figure 4. The effects of DCA and DMA on the recovery of cardiac work per unit TCA acetyl-CoA production in hearts reperfused after 30 minutes of no-flow ischemia. Values are mean±SEM of 23 control hearts, 17 hearts in which DCA (1 mmol/L) was added immediately before reperfusion, or 12 hearts in which DMA (2 µmol/L) was added 10 minutes before the onset of ischemia. *Significantly different from aerobic values. +Significantly different from control hearts during reperfusion period.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
During reperfusion of the ischemic heart, a rapid recovery of MVO₂ and TCA acetyl-CoA production rates occurred despite a significant decrease in the recovery of contractile function. As a result, a significant decrease in cardiac efficiency was observed in the postischemic period. This decrease in cardiac efficiency is similar to that observed in both pig and rat hearts reperfused after ischemia with high levels of fatty acids.^{12 27 28} Paralleling our recent results,¹² neither TCA cycle activity nor mitochondrial respiration was impaired after 30 minutes of severe no-flow ischemia, but rather an uncoupling between contractile function and mitochondrial ATP production was observed. Results from the present study suggest that the production of H⁺ from glucose metabolism is an important contributor to the impaired recovery of mechanical function and to the decrease in cardiac efficiency, as is the pathway by which H⁺ is cleared from the heart during reperfusion. Decreasing H⁺ production from glucose metabolism during reperfusion with DCA significantly improved cardiac efficiency, as did inhibiting Na⁺-H⁺ exchanger activity with DMA.

DCA has been shown by a number of investigators to improve the functional recovery of hearts after ischemia.^{11 25 29 30 31} In this and previous studies,^{11 25 30} DCA was only present during the actual reperfusion period. These beneficial effects of DCA can be attributed to a stimulation of PDH activity, the rate-limiting enzyme for glucose oxidation. PDH is part of a mitochondrial enzyme complex that converts pyruvate, derived from either glycolysis or lactate dehydrogenase, to acetyl-CoA and CO₂. As a result, both glucose oxidation and lactate oxidation are stimulated by DCA (Fig 2). The activity of PDH is regulated by a phosphorylation and dephosphorylation cycle, with phosphorylation of PDH resulting in an inhibition of its activity. As part of the PDH complex, PDH kinase phosphorylates and inactivates PDH, and PDH phosphatase reverses this inhibition.^{18 32} DCA increases PDH activity by inhibiting PDH kinase, thus increasing the proportion of PDH in the active form.²⁴

Although DCA is capable of stimulating both glycolysis and glucose oxidation in the aerobic heart,¹¹ in the postischemic period DCA selectively stimulates glucose oxidation (Fig 2, and Reference 11). Therefore, the beneficial effects of DCA appear to occur directly due to a stimulation of glucose oxidation and not to a stimulation of glycolysis. A number of other pharmacological agents both stimulate glucose oxidation and have a beneficial effect on reperfusion recovery of the postischemic heart. These include L-carnitine,³³ propionyl-L-carnitine,³⁴ ranolazine,³⁵ and the carnitine palmitoyltransferase I inhibitor etomoxir.^{15 36} Reperfusion of previously ischemic hearts with high concentrations of pyruvate also improves the recovery of mechanical function that is related to an increased oxidative flux due to PDH activation.³⁷

The mechanisms by which stimulation of PDH (or glucose oxidation) improves mechanical function during reperfusion are unknown. We speculate that the effects of PDH stimulation can be explained by an improved coupling between glycolysis and glucose oxidation during reperfusion. In the presence of high levels of fatty acids, glucose oxidation rates are 5-fold to 10-fold lower than glycolytic rates in the heart.^{11 12 26 35 38} Selective stimulation of glucose oxidation can lead to a reduction in H⁺ production, thereby improving the coupling of glycolysis to glucose oxidation.^{11 13 21 22}

Each molecule of glucose that passes through glycolysis that is not subsequently oxidized results in the production of 2 H⁺. Each H⁺ originates from the hydrolysis of glycolytically derived ATP.^{21 22} Although this glycolytically derived ATP is still produced, when glycolysis is coupled to glucose oxidation, the overall oxidation of pyruvate to CO₂ (from both PDH and TCA cycle activity) results in the consumption of 2 H⁺. As a result, glycolysis coupled to glucose oxidation is a H⁺ neutral process. We speculate that an increase in H⁺ load during the critical period of reperfusion may contribute to the well-documented Ca²⁺ overload in the postischemic heart that occurs due to an increase in Na⁺-H⁺ exchange activity coupled with Na⁺-Ca²⁺ exchange.^{7 8} By stimulating glucose (and lactate) oxidation with DCA during reperfusion, the H⁺ production from glucose utilization is markedly reduced (Fig 3 and Table 3). Associated with the marked decrease in H⁺ production during reperfusion was a significant improvement in cardiac efficiency (both cardiac work versus O₂ consumption and cardiac work versus TCA acetyl-CoA production). An improvement in cardiac efficiency by DCA has also been recently observed in postischemic rabbit hearts.³⁰ Since the driving force for the Na⁺-H⁺ exchange is decreased because of a reduction of H⁺ production by DCA, Na⁺-H⁺ exchange activity and thus Na⁺-Ca²⁺ exchange activity are expected to be reduced during reperfusion. It should also be noted that excess H⁺ production could also exert other direct effects on the myocardium to decrease cardiac efficiency, including a direct depressant effect on contractile protein function.³⁹

An alternative mechanism by which H⁺ production can be decreased is by inhibiting glycolysis. In a recent study we demonstrated that adenosine can improve the coupling of glycolysis to glucose oxidation primarily by inhibiting glycolytic rates.^{26 40} The resultant decrease in H⁺ production is also accompanied by a significant improvement in the recovery of heart function after ischemia.⁴⁰

Hata et al²³ have recently demonstrated that inhibition of Na⁺-H⁺ exchange activity with DMA can decrease the oxygen cost of contractility in hearts recovering from acidosis. They suggest that this is the result of a smaller proportion of ATP being used to re-establish Na⁺ and Ca²⁺ homeostasis. We therefore also determined whether altering the fate of H⁺, as opposed to the source of H⁺ production, could improve cardiac efficiency. DMA has previously been shown to be effective in increasing reperfusion recovery of ischemic hearts.^{10 41 42} Its protective effect on contractile function during reperfusion has been correlated to its potent inhibitory action on the Na⁺-H⁺ exchanger. Under our experimental conditions, 2 µmol/L DMA had no significant effects on energy substrate metabolism (Fig 2 and Table 3) or overall TCA acetyl-CoA and ATP production (Tables 4 and 5) in postischemic hearts. Similarly, H⁺ production from glucose utilization was also not affected (Fig 3 and Table 3). However, DMA significantly improved the recovery of cardiac function (Fig 1) and improved cardiac efficiency during reperfusion (Figs 1 and 4). This beneficial effect of DMA can be explained by its selective inhibitory effects on Na⁺-H⁺ exchange, since the concentration used was far below the concentration known to inhibit other ionic transport pathways.⁴³

Our results with DMA are consistent with the hypothesis that by reducing H⁺ production and thereafter Na⁺ and Ca²⁺ overload during reperfusion, cardiac efficiency and recovery of contractile function in postischemic hearts can be improved. Direct measurements of the rate of recovery of intracellular pH, as well as levels of intracellular Na⁺ and Ca²⁺ during reperfusion, would provide unequivocal proof that this is the case. Regardless, this study demonstrates that altering either the rate of H⁺ production during the actual reperfusion period or the clearance of H⁺ via the Na⁺-H⁺ exchange can increase cardiac efficiency.

In summary, cardiac efficiency and the recovery of contractile function in postischemic rat hearts can be improved by a reduction of H⁺ production from glycolysis uncoupled from glucose oxidation or by a selective inhibition of Na⁺-H⁺ exchange with DMA. This suggests that pharmacological strategies that alter either the source or fate of H⁺ have potential application as novel strategies for the treatment of ischemic heart disease.

	Selected Abbreviations and Acronyms

 

CoA = coenzyme A DCA = dichloroacetate DMA = 5-(N,N-dimethyl)-amiloride PDH = pyruvate dehydrogenase TCA = tricarboxylic acid

	Acknowledgments

 
This study was supported by a grant from CIBA-GEIGY Canada, Ltd. Dr Lopaschuk is a Medical Research Council of Canada Scientist and an Alberta Heritage Foundation for Medical Research Senior Scholar. Dr Schulz is a Scholar of the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada.

Received May 23, 1996; accepted July 31, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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