High rates of residual fatty acid oxidation during mild ischemia decrease cardiac work and efficiency

Journal of Molecular and Cellular Cardiology 47 (2009) 142–148

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Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y j m c c

Original article

High rates of residual fatty acid oxidation during mild ischemia decrease cardiac work and efficiency Clifford D.L. Folmes, Daniel Sowah, Alexander S. Clanachan, Gary D. Lopaschuk ⁎ Cardiovascular Research Group and Departments of Pharmacology and Pediatrics, University of Alberta, Edmonton, Alberta, Canada

a r t i c l e

i n f o

Article history: Received 13 November 2008 Received in revised form 6 March 2009 Accepted 7 March 2009 Available online 19 March 2009 Keywords: Residual metabolism Mild ischemia Glycolysis Glucose oxidation Palmitate oxidation

a b s t r a c t It is unknown what effects high levels of fatty acids have on energy metabolism and cardiac efficiency during milder forms of ischemia. To address this issue, isolated working rat hearts perfused with Krebs–Henseleit solution (5 mM glucose, 100 μU/mL insulin, and 0.4 (Normal Fat) or 1.2 mM palmitate (High Fat)) were subjected to 30 min of aerobic perfusion followed by 30 min of mild ischemia (39% reduction in coronary flow). Both groups had similar aerobic function and rates of glycolysis, however the High Fat group had elevated rates of palmitate oxidation (150%), and decreased rates of glucose oxidation (51%). Mild ischemia decreased cardiac work (56% versus 40%) and efficiency (29% versus 11%) further in High Fat hearts. Palmitate oxidation contributed a greater percent of acetyl-CoA production during mild ischemia in the High Fat group (81% versus 54%). During mild ischemia glycolysis remained at aerobic levels in the Normal Fat group, but was accelerated in the High Fat group. Triglyceride, glycogen and adenine nucleotide content did not differ at the end of mild ischemia, however glycogen turnover was double in the High Fat group (248%). Addition of the pyruvate dehydrogenase inhibitor dichloroacetate to the High Fat group resulted in a doubling of the rate of glucose oxidation and improved cardiac efficiency during mild ischemia. We demonstrate that fatty acid oxidation dominates as the main source of residual oxidative metabolism during mild ischemia, which is accompanied by suppressed cardiac function and efficiency in the presence of high fat. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Under normal aerobic conditions the heart acquires between 50% and 80% of its tricarboxylic acid (TCA) cycle acetyl-CoA from fatty acid oxidation [1]. In hearts reperfused following severe ischemia, fatty acid oxidation quickly recovers and dominates as the main pathway of mitochondrial oxidative metabolism [2–4]. This is due to the exposure of the heart to a high concentration of fatty acids and subcellular changes in the control of myocardial fatty acid oxidation [5]. However, it is unclear what effect exposure of the heart to high levels of fatty acids has on the contribution of fatty acids or glucose to residual oxidative metabolism during mild ischemia. Following severe ischemia, energy substrate preference has a significant impact on both contractile function and efficiency of O2 utilization [5–7]. During reperfusion following severe ischemia, rates of fatty acid oxidation recover rapidly and lead to a delay in the recovery of intracellular pH and cardiac efficiency due to the inhibition of glucose oxidation [8–10]. During severe ischemia overall mitochondrial oxidative metabolism is inhibited due to a decrease in oxygen supply to the heart, which leads to a competition between fatty acids

⁎ Corresponding author. 423 Heritage Medical Research Building, The University of Alberta, Edmonton, Alberta, Canada T6G 2S2. Tel.: +1780 492 2170; fax: +1780 492 9753. E-mail addresses: [email protected] (C.D.L. Folmes), [email protected] (G.D. Lopaschuk). 0022-2828/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2009.03.005

and glucose as a source of TCA cycle acetyl-CoA [8–10]. To compensate for impaired oxidative metabolism during severe ischemia, glycolysis initially increases and becomes an important source of ATP production for maintenance of ion gradients in the cardiomyocyte [6,7]. However, if the pyruvate from glycolysis is not subsequently oxidized, there is a net production of protons from the hydrolysis of glycolytically derived ATP contributing to ischemia-induced acidosis [11,12]. Intracellular acidosis leads to a sequelae of adverse events, including intracellular Na+ and Ca2+ overload, decreased cardiac pressure development, the initiation of arrhythmias, and decreased responsiveness of contractile proteins to Ca2+ [13,14]. If glycolytically derived pyruvate is aerobically metabolized (i.e. by glucose oxidation), the net production of protons from glucose metabolism is zero as an equivalent number of protons produced by glycolysis are consumed in the TCA cycle [8–12]. In addition, drug-induced optimization of glucose metabolism, which reduces proton production by stimulating glucose oxidation, has been shown to improve cardiac function and efficiency during reperfusion [5,8–10]. However, it has yet to be established what effect high levels of fatty acids have on glucose oxidation during mild ischemia. Several studies have examined the relationship between low flow ischemia and contractile function [15,16], however few have directly assessed the contribution of fatty acids and carbohydrates to residual oxidative metabolism. Neely et al. demonstrated that at a reduced coronary flow of 5 mL/min, fatty acid oxidation accounted for 70% of the oxygen consumption, while at flows of 1 mL/min fatty acid oxidation

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accounted for 90% of the oxygen consumption during moderate ischemia in an isolated working rat heart model [6]. Liedtke et al. confirmed that CO2 production from fatty acid oxidation was maintained during mild ischemia in an in vivo swine model [17]. However these studies did not have simultaneous measures of glucose metabolism, therefore little is known about the effect of a high concentration of fatty acid on the rate of flux through glycolysis and glucose oxidation and the efficiency of oxygen utilization during mild ischemia. In order to determine directly the major pathway of residual oxidative metabolism during mild ischemia, we modified a previous model of mild ischemia in the isolated working rat heart, which allows for the direct measurement of rates of oxidative metabolism, oxygen consumption and contractile function in the presence of normal or high concentrations of fatty acids. We demonstrate that high levels of fatty acids impair cardiac function and cardiac efficiency during mild ischemia due to a greater reliance on fatty acid oxidation as a source of residual oxidative metabolism. 2. Materials and methods The University of Alberta adheres to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences and complies with the Canadian Council of Animal Care guidelines. 2.1. Isolated working rat hearts Rat hearts were cannulated for isolated working heart perfusions as described previously [10]. In brief, male Sprague-Dawley rats (0.25– 0.3 kg) were anesthetized with pentobarbital sodium (60 mg/kg i.p.), the hearts were quickly excised and immersed in ice-cold Krebs– Henseleit solution (118 mM NaCl, 25 mM NaHCO3 pH 7.4, 5.9 mM KCl, 1.2 mM MgSO4 7H2O, 2.5 mM CaCl2·2H2O and 5 mM glucose). The aorta was cannulated and perfusion with Krebs–Henseleit solution (37 °C) was initiated at a hydrostatic pressure of 60 mm Hg. Hearts

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were trimmed of excess tissue, and both the pulmonary artery and the opening to the left atrium were cannulated. After 15 min 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. Oxygenated Krebs–Henseleit solution containing 5 mM glucose, and 100 μU/mL insulin and either 0.4 mM (Normal Fat) or 1.2 mM (High Fat) palmitate bound to 3% BSA (palmitate was pre-bound to the albumin as described previously [18]), was delivered to the left atrium at a preload pressure of 11.5 mm Hg. Perfusate was ejected from spontaneously beating hearts into a compliance chamber (containing 1 mL of air) and into the aortic outflow line against a hydrostatic afterload pressure of 80 mm Hg. The perfusate was recirculated, and pH was adjusted to 7.4 by surface gassing the perfusate in a glass oxygenator with a gas mixture containing 95% O2 and 5% CO2. Heart rate and aortic pressure (mm Hg) were measured with a Gould P21 pressure transducer (Harvard Apparatus) connected to the aortic outflow line. Cardiac output and aortic flow (mL/min) were measured with Transonic T206 flow probes in the preload and afterload lines, respectively. Coronary flow (mL/min) was calculated as the difference between cardiac output and aortic flow. Myocardial oxygen consumption (MVO2, μmol O2/g dry wt/min) was derived from coronary flow and the O2 content (μmol/mL) in the perfusate, which was measured with a micro-oxygen probe (YSI Life Sciences) in the left atrial inflow line and a line originating from the cannulated pulmonary artery. Cardiac work (mL/mm Hg/min 10− 2) was calculated as the product of aortic systolic pressure and cardiac output. Cardiac efficiency was defined as a ratio of cardiac work to MVO2. Data were collected using an MP100 system from AcqKnowledge (BIOPAC Systems, Inc USA). 2.2. Mild ischemia model A model of mild ischemia in the isolated working rat heart was developed using a modification of a technique first described by Neely

Fig. 1. High concentration of fatty acid impairs cardiac work and efficiency during mild ischemia. Cardiac efficiency (A), cardiac work (B), coronary flow (C), and myocardial oxygen consumption (D) during aerobic perfusion and mild ischemia. represents Normal Fat aerobic, represents High Fat aerobic, represents Normal Fat mild ischemia and represents High Fat mild ischemia. Values represent mean ± SEM of 6 High Fat perfused hearts and 7 Normal Fat perfused hearts for the 60 min aerobic controls. Values represent mean ± SEM of 28 High Fat perfused hearts and 12 Normal Fat perfused hearts for the mild ischemia group. Differences were determined using a 2-way repeated measures ANOVA with a Bonferroni post hoc test. ⁎P b 0.05, significantly different from Normal Fat group at corresponding perfusion time.

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et al. [19]. The modified technique allowed for the direct measurement of contractile function, MVO2 and oxidative metabolism under steady state conditions. The model makes use of a ball valve in the aortic outflow tract and a diastolic backflow controller. Preliminary characterization of the experimental system determined that a 35– 40% reduction in coronary flow would result in a decrease in cardiac work to a new steady state that was maintained for a further 30 min of perfusion. 2.3. Perfusion protocol Experimental groups included: 1) a 60 min aerobic perfusion with Normal Fat or High Fat and 2) a 30 min aerobic perfusion followed by 30 min of mild ischemia with Normal Fat or High Fat, 3) a 60 min aerobic perfusion with High Fat in the presence of 3 mM dichloroacetate (DCA, BDH Chemicals Ltd.), or 4) a 30 min aerobic perfusion followed by 30 min of mild ischemia with High Fat and 3 mM DCA. At the end of mild ischemia, hearts were quickly frozen with Wollenberger clamps cooled by liquid N2, and the dry to wet ratio of the heart tissue was determined as previously described [10]. 2.4. Glycolysis, glucose and palmitate oxidation Glycolysis, glucose oxidation or palmitate oxidation was measured by perfusing hearts with [5-3H] or [U-14C]glucose or [9,10-3H] palmitate, respectively [18]. The total myocardial 3H2O production and 14CO2 production were determined at 10 min intervals during both the aerobic perfusion period and during the 30 min period of mild ischemia. To measure the rates of glycolysis and palmitate oxidation, 3H2O in perfusate samples was separated from [3H]glucose or [3H]palmitate using the dowex method as previously described [18]. Oxidation rates for glucose were determined by quantitative measurement of 14CO2 production, as described previously [18]. 2.5. Glycogen, triglyceride and nucleotide content Glycogen content and accumulation were measured as previously described [20]. Accumulation of [14C]glucose in glycogen (μmol/g dry wt) was determined by measuring [14C] accumulation in these extracts. The tissue content of triglycerides was measured in chloroform-methanol extractions using an enzymatic assay (Wako Pure Chemical Industries), as previously described [21]. The tissue content of nucleotides was measured in neutralized perchloric acid extracts of frozen tissue by HPLC, as previously described [22].

Fig. 2. High concentration of fatty acid increases glycolysis and fatty acid oxidation at the expense of glucose oxidation during mild ischemia. Glycolysis (A), glucose oxidation (B), and palmitate oxidation (C) during aerobic perfusion and mild ischemia. Aerobic values represent 10–30 min average ± SEM and mild ischemia values represent 40–60 min average ± SEM of 18 High Fat and 12 Normal Fat hearts for glycolysis, 20 High Fat and 6 Normal Fat hearts for glucose oxidation and 8 High Fat and 6 Normal Fat rat hearts for palmitate oxidation. Differences were determined using a 2-way ANOVA with a Bonferroni post hoc test. ⁎P b 0.05, significantly different from Normal Fat group.

2.6. Statistical analysis All data are presented as the mean ± S.E.M. The data were analyzed with the statistical programs Prism 4 and GB-stat. Two-way repeated measures ANOVA with a Bonferroni post hoc test was used to evaluate the statistical significance of differences among groups for functional parameters. Two-way ANOVA with a Bonferroni post hoc test was used to evaluate the statistical significance of differences among groups for the metabolic data. Values of P b 0.05 were considered significant. 3. Results 3.1. Baseline aerobic values in hearts perfused with Normal Fat and High Fat Under time-matched aerobic conditions, High Fat did not affect cardiac work, oxygen consumption, cardiac efficiency or rates of glycolysis compared to Normal Fat hearts (Figs. 1 and 2A). However, glucose oxidation rates in the High Fat group were half of the rates in the Normal Fat group (500 ± 60 versus 1010 ± 90, P b 0.05, Fig. 2B) and palmitate oxidation rates were elevated (670 ± 30 versus 450 ± 50,

P b 0.05, Fig. 2C). In the High Fat group, the contributions of palmitate oxidation to overall TCA cycle acetyl-CoA production (64 ± 7% and 85 ± 4%, P b 0.05) and to ATP production (57 ± 2% and 77 ± 3%, P b 0.05) were significantly higher compared to the Normal Fat group (Figs. 3B and D). 3.2. The effects of mild ischemia on hearts perfused with Normal Fat and High Fat Mild ischemia produced a 39 ± 2% reduction in coronary flow (P b 0.05, Fig. 1A) accompanied by a 37 ± 2% reduction in MVO2 (P b 0.05, Fig. 1C) in both groups. Heart rate, peak systolic pressure and developed pressure did not differ significantly during either aerobic perfusion or mild ischemia. Throughout mild ischemia, the High Fat group had lower cardiac work than the Normal Fat group (36 ± 3 versus 47 ± 4 mL/mm Hg/min 10− 2, P b 0.05, Fig. 1B) with no significant difference in MVO2 or coronary flows (Fig. 1C). As a result, there is a lower efficiency of oxygen utilization in the High Fat group (0.85 ± 0.07 versus 1.06 ± 0.09 cardiac work/MVO2, P b 0.05, Fig. 1D). Rates of glycolysis were accelerated (4280 ± 440 versus 3120 ±

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Fig. 3. Increased reliance on palmitate as a source of acetyl-CoA and ATP production in the presence of a high concentration of fatty acid. Total acetyl-CoA production (A), normalized acetyl-CoA production (B), total ATP production (C) and normalized ATP production (D) during aerobic perfusion and mild ischemia. TCA cycle activity was calculated from the rates of palmitate oxidation and glucose oxidation based on the oxidation of 1 mol of palmitate producing 8 mol of acetyl-CoA and the oxidation of 1 mol of glucose producing 2 mol of acetyl-CoA. ATP production was calculated from rates of glycolysis, glucose and palmitate oxidation based on 1 mol glucose producing 2 mol of ATP in glycolysis and 30 mol of ATP in glucose oxidation and 1 mol of palmitate producing 105 mol of ATP. Aerobic values represent 10–30 min average ± SEM and mild ischemia values represent 40–60 min average ± SEM of 20 High Fat and 12 Normal Fat hearts for glycolysis, 20 High Fat and 6 Normal Fat hearts for glucose oxidation and 8 High Fat and 8 Normal Fat hearts for palmitate oxidation. Differences were determined using a 2-way ANOVA with a Bonferroni post hoc test.

430 nmol/g dry wt/min, P b 0.05, Fig. 2A) and glucose oxidation rates were reduced (530 ± 60 versus 1230 ± 170 nmol/g dry wt/min, P b 0.05, Fig. 2B) in the High Fat group compared to the Normal Fat group. During mild ischemia, rates of palmitate oxidation did not differ between Normal Fat and High Fat groups (Fig. 2C). If hearts were reperfused following the mild ischemic episode, all hearts showed a complete recovery of cardiac function (data not shown). In order to induce the previously demonstrated detrimental effect of high levels of fat on the recovery of function during reperfusion, the hearts would need to be subjected to a more severe ischemic insult that would completely abolish cardiac function during ischemia. Total acetyl-CoA production was similar during mild ischemia in the Normal and High Fat groups, however there was a significant reduction in total acetyl-CoA production in the High Fat group (23 ± 5%, Fig. 3A) compared to the High Fat aerobic controls. During mild ischemia, the contribution of palmitate and glucose oxidation to TCA cycle activity was similar in the Normal Fat group (Fig. 3B). In hearts perfused with High Fat, palmitate oxidation predominated as a source of acetyl-CoA for the TCA cycle (81 ± 9% versus 54± 9, P b 0.05, Fig. 3B). Similar results were observed for ATP production during mild ischemia with palmitate oxidation predominating as a source of ATP in the High Fat group (71 ± 8% versus 47 ± 8%, P b 0.05, Fig. 3D). 3.3. Glycogen, triglyceride and adenine nucleotide contents in hearts perfused with Normal Fat and High Fat There were no differences in total content or accumulation of glycogen between groups during aerobic perfusion (Table 1). However, during mild ischemia incorporation of radiolabeled glucose into glycogen was elevated in the High Fat group (87 ± 21 versus 35 ± 6, μmol/g dry wt, P b 0.05, Table 1) even though the total glycogen

content did not change, suggesting that glycogen turnover may be accelerated in the High Fat hearts during mild ischemia. This increase in glycogen turnover during ischemia parallels the increase in glycolysis observed in the presence of high fat. This could be secondary to the increased metabolic stress in the High Fat hearts due to a decreased cardiac efficiency. There is no difference in triglyceride content between groups at the end of the aerobic period or mild ischemia (Table 1). AMP/ATP, ADP/ATP and Cr/PCr ratios did not differ between the Normal Fat and High Fat group either during aerobic perfusion or mild ischemia (Table 2). In addition, mild ischemia did not change ratios compared to time-matched aerobic controls. Although during aerobic perfusion absolute values of ADP, ATP, Cr and PCr were elevated in the High Fat hearts, they were suppressed during mild ischemia (Table 2). The reason for the higher ATP and Cr

Table 1 Glycogen and triglyceride contents and incorporation of radiolabelled glucose into glycogen in isolated working rat hearts subjected to aerobic perfusion or aerobic perfusion followed by mild ischemia. Aerobic perfusion

Mild ischemia

Normal Fat High Fat Normal Fat (n = 6) (n = 6) (n = 8) Glycogen content (μmol/g dry wt) Glycogen label accumulation (μmol/g dry wt) Triglyceride content (μmol/g dry wt)

High Fat (n = 7)

116 ± 18 33 ± 6

124 ± 8 43 ± 7

117 ± 7 35 ± 5

128 ± 21 87 ± 21⁎

25 ± 2

33 ± 8

25 ± 2

34 ± 4

Values are means ± SEM. Aerobic values represent the values at the end of a timematched 60 min aerobic perfusion. Mild ischemia values represent the values at the end of the 30 min mild ischemia period. Differences were determined using a 2-way ANOVA with a Bonferroni post hoc test. ⁎ P b 0.05, significantly different from Normal Fat group.

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Table 2 AMP, ADP, ATP, Cr and PCr content of isolated working rat hearts subjected to aerobic perfusion or aerobic perfusion followed by mild ischemia. Aerobic perfusion

AMP (μmol/g dry wt) ADP (μmol/g dry wt) ATP (μmol/g dry wt) Cr (μmol/g dry wt) PCr (μmol/g dry wt) AMP/ATP ADP/ATP Cr/PCr

Table 4 Effect of 3 mM dichloroacetate (DCA) on metabolism of isolated working rat hearts subjected to aerobic perfusion followed by mild ischemia.

Mild ischemia

Aerobic perfusion

Normal Fat (n = 8)

High Fat (n = 6)

Normal Fat (n = 6)

High Fat (n = 16)

1.6 ± 0.2 6.5 ± 0.2 14.1 ± 1.1 51.6 ± 1.8 21.6 ± 3.5 0.1 ± 0.02 0.5 ± 0.04 2.8 ± 0.4

2.1 ± 0.3 9.4 ± 0.5a 19.9 ± 0.7a 65.4 ± 5.8a 35.8 ± 7.6a 0.1 ± 0.01 0.5 ± 0.04 2.4 ± 0.4

1.7 ± 0.2 6.1 ± 0.3 14.4 ± 0.9 57.0 ± 1.0 25.3 ± 2.9 0.1 ± 0.02 0.4 ± 0.02 2.4 ± 0.3

2.2 ± 0.1 7.1 ± 0.6b 16.8 ± 0.8b 44.5 ± 9.0b 31.1 ± 2.3 0.1 ± 0.01 0.4 ± 0.03 2.2 ± 0.2

Values are means ± SEM. Aerobic values represent the values at the end of a timematched 60 min aerobic perfusion. Mild ischemia values represent the values at the end of the 30 min mild ischemia period. Differences were determined using a 2-way ANOVA with a Bonferroni post hoc test. a P b 0.05, significantly different from Normal Fat value. b P b 0.05, significantly different from aerobic value.

levels during the aerobic period in High Fat hearts was not clear, but may be related to an increase in energy supply from hearts perfused with High Fat during the aerobic period (Fig. 3C). This could conceivably decrease the rate of loss of adenosine and Cr from the High Fat hearts during the initial aerobic period compared to the Low Fat hearts. It should also be noted that measured contents of ADP and AMP do not represent the free concentrations of ADP and AMP that are present in the cardiomyocytes, but rather the levels seen in the total tissue extract. 3.4. The effect of 3 mM DCA during aerobic perfusion and mild ischemia The addition of DCA, a pyruvate dehydrogenase activator, to the High Fat group resulted in mechanical function intermediate to that observed in the Normal and High Fat groups (Table 3), and was associated with a 2-fold increase in glucose oxidation rates (1160 ± 160 versus 530 ± 60 nmol/g dry wt/min, P b 0.05 (Table 4)). The addition of DCA to the High Fat hearts resulted in an increased contribution of glucose oxidation to TCA cycle activity during both the aerobic period (30 ± 6 versus 15 ± 3 %, P b 0.05) and during the mild ischemia period (36 ± 5 versus 19 ± 2 %, P b 0.05 (Table 4)). 4. Discussion A number of studies have examined in detail the importance of energy substrate preference to cardiac function during reperfusion of hearts subjected to severe ischemia [2–4,6–10,23]. In this study we developed an experimental model in which energy metabolism, oxygen consumption, contractile function and cardiac efficiency could be assessed during the actual ischemic period. Using this approach we

Glycolysis (nmol/g dry wt/min) Palmitate oxidation (nmol/g dry wt/min) Glucose oxidation (nmol/g dry wt/min) % Acetyl-CoA from palmitate oxidation % Acetyl-CoA from glucose oxidation

Mild ischemia

High Fat

DCA

High Fat

DCA

3150 ± 320 (n = 18) 670 ± 30 (n = 8) 500 ± 60 (n = 20) 85 ± 7

3440 ± 420 (n = 15) 680 ± 70 (n = 5) 1160 ± 120b (n = 10) 70 ± 8

4400 ± 420a (n = 18) 530 ± 70a (n = 8) 530 ± 60 (n = 20) 81 ± 8

4400 ± 480a (n = 15) 550 ± 60 (n = 5) 1160 ± 160b (n = 10) 64 ± 8

15 ± 3

30 ± 6a

19 ± 2

36 ± 5a

Values are means ± SEM. Aerobic values represent the average value for 10 to 30 min values of aerobically perfused hearts. Mild ischemia values represent the average for 40 to 60 min values for mild ischemia. Differences were determined using a 2-way ANOVA with a Bonferroni post hoc test. a Denotes P b 0.05, significantly different from corresponding aerobic value. b Denotes P b 0.05, significantly different from corresponding High Fat value.

have made a number of novel and important findings. In the presence of a high fatty acid concentration normally seen in vivo during and following clinically relevant ischemia, fatty acid oxidation provides the major source of residual oxidative metabolism during mild ischemia. During mild ischemia, approximately 80% of the acetylCoA for mitochondrial TCA cycle activity originates from fatty acid oxidation. Secondly, while glucose oxidation can be a major pathway of energy production during mild ischemia, in the presence of high levels of fatty acids, the rate of glucose oxidation is very low. This suggests that the Randle cycle continues to function during mild ischemia in the heart [24]. An unexpected finding is the inability of a high concentration of fatty acid to inhibit glycolysis during mild ischemia, which may be due to an activation of the AMP-activated protein kinase independent of changes in the AMP/ATP ratio, which we have previously reported in this model [25]. The final important observation is that exposure of the heart to high levels of fatty acids during mild ischemia results in a decrease in both cardiac efficiency and cardiac function due to an increase in the uncoupling of glycolysis from glucose oxidation. The exact mechanism responsible for the lower cardiac work during ischemia in the hearts perfused with high fat cannot be determined with certainty. We speculate that it is the consequence of the greater proton production that decreases cardiac efficiency. Indeed, decreasing proton production from glucose metabolism during reperfusion of ischemic hearts improves both cardiac efficiency and function [9]. While we cannot rule out a greater mitochondrial uncoupling in the presence of high fatty acid concentrations for the decreased efficiency, we have previously observed that improving the coupling of glycolysis to glucose oxidation can improve cardiac

Table 3 Effect of 3 mM dichloroacetate (DCA) on mechanical function, myocardial oxygen consumption and cardiac efficiency of isolated working rat hearts subjected to aerobic perfusion followed by mild ischemia. Aerobic perfusion Heart rate (beats/min) Peak systolic pressure (mm Hg) Coronary flow (mL/min) Cardiac output (mL/min) Cardiac work (mL/mm Hg/min 10− 2) Myocardial O2 consumption (μmol O2/g dry wt/min) Cardiac efficiency (cardiac work per MVO2)

Mild ischemia

Normal Fat (n = 16)

High Fat (n = 20)

High Fat + DCA (n = 15)

Normal Fat

High Fat

High Fat + DCA

223 ± 6 153 ± 3 27 ± 1 50 ± 1 77 ± 4 63 ± 4 1.25 ± 0.06

233 ± 6 159 ± 3 28 ± 1 49 ± 1 77 ± 2 66 ± 4 1.25 ± 0.06

244 ± 8 156 ± 3 28 ± 1 51 ± 1 77 ± 3 60 ± 3 1.32 ± 0.06

220 ± 9 134 ± 3a 17 ± 1a 34 ± 1a 47 ± 4a 43 ± 2a 1.06 ± 0.09a

221 ± 7 126 ± 3a 15 ± 1a 25 ± 2ab 32 ± 2ab 38 ± 2a 0.85 ± 0.07a

228 ± 8 127 ± 4a 18 ± 1a 28 ± 2a 36 ± 3ab 38 ± 3a 1.02 ± 0.10a

Values are means ± SEM. Aerobic values represent the average value for the 10–30 min time of aerobic perfusion. Mild ischemia values represent the average for 40–60 min time of mild ischemia. Differences were determined using a 2-way ANOVA with a Bonferroni post hoc test. a Denotes P b 0.05, significantly different from aerobic time control. b Denotes P b 0.05, significantly different from aerobic time control.

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efficiency independent of changes in mitochondrial proton leak [26]. Further studies are needed to explain the exact mechanism responsible for the decrease in cardiac function and efficiency. Myocardial energy substrate preference is an important determinant of cardiac contractile recovery following severe ischemia [2–4,8–10,17,27,28]. Previous studies have demonstrated that high levels of fatty acid oxidation markedly inhibit glucose oxidation during reperfusion of severely ischemic hearts. Importantly, the resulting increase in proton production from uncoupled glucose metabolism delays the recovery of pHi during reperfusion of ischemic hearts [8,9,29]. This contributes to a fatty acid-induced decrease in recovery of both mechanical function and cardiac efficiency during reperfusion. These previous observations are most clinically relevant to lessening lethal reperfusion injury following global ischemia, however, hitherto technical issues have made it difficult to assess accurately energy metabolism and cardiac efficiency during the actual ischemic period. For instance, measurements of fatty acid and glucose oxidation are ideally performed under conditions of stable cardiac work, a situation difficult to maintain during severe ischemia. Furthermore, measurements of cardiac efficiency during ischemia not only require measurements of oxygen consumption, but also assessment of contractile function. In order to assess directly the contribution of fatty acids and glucose to residual oxidative metabolism, as well as to measure cardiac efficiency during mild ischemia, we developed a working heart model of mild ischemia, based on an aortic outflow ball-valve design of Neely et al. [19]. Making use of the oneway ball valve and a diastolic backflow controller, a reproducible degree of mild ischemia was introduced to the perfused hearts, which was associated with a reproducible decrease in coronary flow and myocardial oxygen consumption, while maintaining a new depressed steady state level of cardiac work. This model is most clinically relevant to a mild effort-induced angina and allows for testing the hypothesis that modulating myocardial energy metabolism may decrease the detrimental effects of concurrent ischemia, as opposed to the previous studies with global ischemia that are more relevant to reperfusion injury. Of particular interest, the antianginal drug, trimetazidine, has been demonstrated to reduce fatty acid oxidation and improve functional recovery following ischemia/reperfusion [30]. Using a direct measure of metabolism we demonstrate that fatty acid oxidation is the major residual source of oxidative metabolism during mild ischemia [6,17] and this occurs at the expense of glucose oxidation. Interestingly, in the presence of a normal concentration of fatty acid, a 39% reduction in coronary flow suppresses cardiac function and efficiency independent of significant changes in AMP/ ATP, Cr/PCr and the utilization of endogenous (triglyceride and glycogen) and exogenous energy substrates. Hearts subjected to mild ischemia in the presence of high fat have a greater reduction in cardiac work and efficiency with a reduction in ATP and PCr, as well an increased incorporation of radioactive glucose into glycogen implying an accelerated glycogen turnover. Unexpectedly, the high concentration of fatty acid has little effect on glycolysis with a slight acceleration during mild ischemia, however it had a dramatic effect on the rate of glucose oxidation, reducing rates to less than half of the value found in hearts perfused with a normal concentration of fatty acid. This significantly changes the contribution of palmitate and glucose to mitochondrial TCA cycle activity and ATP production during mild ischemia. Hearts perfused with a normal concentration of fatty acid produced equal amounts of acetyl-CoA from palmitate and glucose oxidation. In comparison, in hearts perfused with a high concentration of fatty acid 81 ± 9% of TCA cycle acetyl-CoA originates from palmitate oxidation and only 19 ± 2% from glucose oxidation. This greater reliance on palmitate in the High Fat group is associated with reduced cardiac function and efficiency during mild ischemia. As hydrolysis of glycolytically derived ATP is a major source of acidosis in the severely ischemic heart [11,12], low glucose oxidation rates during ischemia can increase the accumulation of metabolic

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byproducts, such as protons, which are associated with poor recovery of mechanical function [2,8–10,12,17]. If glycolysis is completely coupled to glucose oxidation, overall proton production is zero, since an equivalent number of protons are consumed by the oxidation of pyruvate [11,12]. However, as we show in this study, glucose oxidation is decreased during ischemia in the presence of a high concentration of fatty acid, despite an increase in the rate of glycolysis. As a result, more of the pyruvate is directed towards lactate production. Proton production calculated from uncoupled rates of glycolysis and glucose oxidation has previously been demonstrated to correlate well with intracellular pH in a global ischemia model [8]. Using this calculation there is a 40% higher rate of proton production from glucose metabolism in the High Fat group during mild ischemia. We hypothesize that the resultant production of protons and the associated Na+ and Ca2+ overload due to activation of the Na+–H+ and Na+–Ca2+ exchangers is an important contributor to the decrease in cardiac efficiency seen during mild ischemia in the presence of high levels of fatty acids [13,14,31,32]. As a result, similar to what is observed during reperfusion following severe ischemia [2,8–10,29], high rates of fatty acid oxidation decrease cardiac function and efficiency during ischemia secondary to inhibition of glucose oxidation. Of note, rates of proton production were calculated from rates of glycolysis and glucose oxidation, therefore this hypothesis must be further examined using actual measures of intracellular pH. However, in a previous study we have observed a good correlation between proton production arising from glycolysis uncoupled from glucose oxidation and directly measured intracellular pH (by NMR) in the reperfused ischemic heart [8]. This uncoupling of glycolysis from glucose oxidation also produces the expected rate of lactate efflux from the heart [33]. Alternatively, accelerated rates of glycolysis have been suggested to be cardioprotective in the setting of ischemia/reperfusion by providing an anaerobic source of ATP in close association with the cell membrane, which is utilized to maintain ionic homeostasis particularily via the sodium/potassium ATPase [34]. Studies have demonstrated that inhibition of glycolysis during ischemia or early reperfusion impairs functional recovery during reperfusion [35,36], however inhibition of glycolysis has also been demonstrated to be cardioprotective [37,38]. The study of Opie and Bricknell [35] is most relevant to the present study as the effects of glycolytic inhibition were observed during the actual ischemic period as opposed to during reperfusion following transient ischemia [36–38]. However, the present study would confirm the latter of these observations as glycolytic flux was greater in the High Fat group (Figs. 2 and 3) and this was associated with poorer cardiac function and efficiency during mild ischemia. The reason for the greater glycolytic flux during ischemia in the High Fat hearts is not clear, but may be related to the decrease in cardiac efficiency observed in the presence of high fat. The increased metabolic stress in the High Fat hearts may be increasing glycolysis. Our study also suggests that the mildly ischemic heart has the capacity to switch its residual oxidative metabolism from fatty acid oxidation to glucose oxidation. In the presence of the pyruvate dehydrogenase kinase inhibitor DCA, we found a significant switch in oxidative metabolism from fatty acids to glucose, as it produced a 2fold increase in glucose oxidation. This stimulation of glucose oxidation resulted in an intermediate cardiac efficiency lying between the observed values from the Normal and High Fat perfused hearts in this model of mild ischemia. As the reduction in cardiac efficiency is small in this model, the beneficial effects of metabolic modulation may be better observed in a more severe model of ischemia. We demonstrate that during mild ischemia the major contributor to residual oxidative metabolism is fatty acid oxidation. The presence of a high concentration of fatty acid during mild ischemia results in a decrease in glucose oxidation, despite an increase in glycolysis. This results in an increase in proton production from uncoupled glucose metabolism, which may lead to a decrease in cardiac function and

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efficiency in the ischemic myocardium. Strategies that switch residual oxidative metabolism during ischemia from palmitate oxidation to glucose oxidation, such as by lowering circulating free fatty acids, inhibiting fatty acid oxidation, or directly stimulating glucose oxidation, may be a therapeutic approach to improve cardiac work and efficiency during mild ischemia. Acknowledgments CDLF holds a Natural Sciences and Engineering Research Council of Canada scholarship, an Alberta Heritage Foundation for Medical Research studentship and a Canadian Institutes for Health Research (CIHR) doctoral award. This research was funded by a CIHR grant to GDL and ASC. GDL is a Medical Scientist of the AHFMR. We thank Mary Collinson for excellent technical assistance. References [1] Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev 2005;85:1093–129. [2] Lopaschuk GD, Spafford MA, Davies NJ, Wall SR. Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia. Circ Res 1990;66:546–53. [3] Liedtke AJ, Demaison L, Eggleston AM, Cohen LM, Nellis SH. Changes in substrate metabolism and effects of excess fatty acids in reperfused myocardium. Circ Res 1988;62:535–42. [4] Lerch R, Tamm C, Papageorgiou I, Benzi RH. Myocardial fatty acid oxidation during ischemia and reperfusion. Mol Cell Biochem 1992;116:103–9. [5] Folmes CD, Clanachan AS, Lopaschuk GD. Fatty acid oxidation inhibitors in the management of chronic complications of atherosclerosis. 2005; 7: 63–70. [6] Neely JR, Liedtke AJ, Whitmer JT, Rovetto MJ. Relationship between coronary flow and adenosine triphosphate production from glycolysis and oxidative metabolism. Recent Adv Stud Cardiac Struct Metab 1975;8:301–21. [7] Cross HR, Opie LH, Radda GK, Clarke K. Is a high glycogen content beneficial or detrimental to the ischemic rat heart? A controversy resolved. Circ Res 1996;78: 482–91. [8] Liu Q, Docherty JC, Rendell JC, Clanachan AS, Lopaschuk GD. High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation. J Am Coll Cardiol 2002;39:718–25. [9] Liu B, Clanachan AS, Schulz R, Lopaschuk GD. Cardiac efficiency is improved after ischemia by altering both the source and fate of protons. Circ Res 1996;79:940–8. [10] Lopaschuk GD, Wambolt RB, Barr RL. An imbalance between glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of ischemic hearts. J Pharmacol Exp Ther 1993; 264:135–44. [11] Hochachka PW, Mommsen TP. Protons and anaerobiosis. Science 1983;219:1391–7. [12] Dennis SC, Gevers W, Opie LH. Protons in ischemia: where do they come from; where do they go to? J Mol Cell Cardiol 1991;23:1077–86. [13] Carmeliet E. Cardiac ionic currents and acute ischemia: from channels to arrhythmias. Physiol Rev 1999;79:917–1017. [14] Tani M, Neely JR. Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+–Na+ and Na+–Ca2+ exchange. Circ Res 1989;65:1045–56. [15] Brooks WM, Haseler LJ, Clarke K, Willis RJ. Relation between the phosphocreatine to ATP ratio determined by 31P nuclear magnetic resonance spectroscopy and left ventricular function in underperfused guinea-pig heart. J Mol Cell Cardiol 1986;18: 149–55.

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