Regulation of pyruvate dehydrogenase activity and citric acid cycle intermediates during high cardiac power generation
1 Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4970, USA
Abstract
A high rate of cardiac work increases citric acid cycle (CAC) turnover and flux through pyruvate dehydrogenase (PDH); however, the mechanisms for these effects are poorly understood. We tested the hypotheses that an increase in cardiac energy expenditure: (1) activates PDH and reduces the product/substrate ratios ([NADH]/[NAD+] and [acetyl-CoA]/[CoA-SH]); and (2) increases the content of CAC intermediates. Measurements were made in anaesthetized pigs under control conditions and during 15 min of a high cardiac workload induced by dobutamine (Dob). A third group was made hyperglycaemic (14 mM) to stimulate flux through PDH during the high work state (Dob + Glu). Glucose and fatty acid oxidation were measured with 14C-glucose and 3H-oleate. Compared with control, the high workload groups had a similar increase in myocardial oxygen consumption ( and cardiac power. Dob increased PDH activity and glucose oxidation above control, but did not reduce the [NADH]/[NAD+] and [acetyl-CoA]/[CoA-SH] ratios, and there were no differences between the Dob and Dob + Glu groups. An additional group was treated with Dob + Glu and oxfenicine (Oxf) to inhibit fatty acid oxidation: this increased [CoA-SH] and glucose oxidation compared with Dob; however, there was no further activation of PDH or decrease in the [NADH]/[NAD+] ratio. Content of the 4-carbon CAC intermediates succinate, fumarate and malate increased 3-fold with Dob, but there was no change in citrate content, and the Dob + Glu and Dob + Glu + Oxf groups were not different from Dob. In conclusion, compared with normal conditions, at high myocardial energy expenditure (1) the increase in flux through PDH is regulated by activation of the enzyme complex and continues to be partially controlled through inhibition by fatty acid oxidation, and (2) there is expansion of the CAC pool size at the level of 4-carbon intermediates that is largely independent of myocardial fatty acid oxidation.
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Introduction
Intense exercise or catecholamine stress causes a 3- to 6-fold increase in ATP turnover, citric acid cycle (CAC) flux, and myocardial oxygen consumption compared with rest (Khouri et al. 1965; Balaban et al. 1986; Mootha et al. 1997). Under unstressed aerobic conditions the oxidation of fatty acids supplies most (60–80%) of the energy for ATP synthesis. However, during increased work states there is a greater relative contribution from the oxidation of glucose and lactate (Gertz et al. 1988; Collins-Nakai et al. 1994; Goodwin et al. 1998; Chatham, 2002). Glucose and lactate oxidation is regulated by pyruvate dehydrogenase (PDH), which is inactivated by phosphorylation by PDH kinase, which is in turn inhibited by pyruvate and activated by increases in [acetyl-CoA]/[CoA-SH] and [NADH]/[NAD+] ratios (Wieland et al. 1971; Whitehouse et al. 1974; Kerbey et al. 1976; Randle & Priesman, 1996). The PDH complex also contains a PDH phosphatase that is activated by intramitochondrial [Ca2+] (McCormack et al. 1990), which is elevated by adrenergic stimulation (McCormack & Denton, 1984; McCormack et al. 1990). In skeletal muscle the activation state of PDH is low at rest (15% of max), but is rapidly activated with exercise in proportion to the increase in energy expenditure (Putman et al. 1995; Howlett et al. 1998); however, this effect is not the result of an increase in pyruvate concentration or a fall in the [acetyl-CoA]/[CoA-SH] or [NADH]/[NAD+] ratios, but is related to a fall in [ATP]/[ADP], and activation of PDH phosphatase by an increase in mitochondrial [Ca2+] (McCormack et al. 1990; Spriet & Heigenhauser, 2002).
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Little is known about the effects of increased on the regulation of myocardial PDH activity and carbohydrate oxidation. The maximal rate of pyruvate oxidation is set by the degree of phosphorylation of PDH; however, the actual flux is largely determined by the concentrations of substrates and products in the mitochondrial matrix (Hansford & Cohen, 1978). Under unstressed resting conditions PDH activity and pyruvate oxidation are regulated by fatty acid oxidation via the Randle phenomenon, with enhanced PDH activity and flux with decreased fatty acid oxidation (Randle et al. 1963; Kerbey et al. 1976; Higgins et al. 1980, 1981; Kruszynska et al. 1991; Lopaschuk et al. 1994; Schwartz et al. 1994; Clarke et al. 1996; Stanley et al. 1997). An increase in cardiac energy expenditure, however, increases pyruvate oxidation despite an increase in the [acetyl-CoA]/[CoA-SH] ratio, suggesting that this mechanism does not facilitate PDH activation or flux (Hall et al. 1996a, 1996b). It has been proposed that there is a fall in the [NADH]/[NAD+] ratio with an increase in work demand (White & Wittenberg, 1993; Ashruf et al. 1995) but this has not been examined with physiologically relevant substrates or in vivo.
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Oxidation of acetyl-CoA by the CAC is dependent upon a sufficient concentration of all of the CAC intermediates; however, little is known about the content of CAC intermediates in the myocardium at high work states. In skeletal muscle contraction increases the tissue content of CAC intermediates (Aragon & Lowenstein, 1980; Sahlin et al. 1990), which requires ‘anaplerosis’, defined as the entry of intermediates into the CAC independent of synthesis of citrate from acetyl-CoA and oxaloacetate (e.g. from pyruvate carboxylation, glutamate transamination or from propionate conversion to succinyl-CoA) (Gibala et al. 2000). In human skeletal muscle exercise causes a 2- to 4-fold increase in all of the CAC intermediates, except -ketoglutarate (-KG) which declines by approximate 40% (Gibala et al. 1997, 1998). The increase in CAC pool size with exercise is enhanced when pyruvate oxidation is reduced and fatty acid oxidation increased (Gibala et al. 2002), but is also enhanced when pyruvate oxidation is stimulated (Timmons et al. 1996; Constantin-Teodosiu et al. 1999). The myocardial content of CAC intermediates under low workloads is dependent upon the availability of acetyl-CoA, as observed by the increase in CAC intermediates when acetate (Williamson, 1965; Taegtmeyer, 1984), or octanoate (Panchal et al. 2000) is a substrate.
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In the present investigation we tested the hypotheses that during high rates of myocardial energy expenditure: (1) PDH is regulated by the degree of phosphorylation inhibition on PDH, and product inhibition of flux through the active enzyme from fatty acid oxidation; and (2) there is an increase in the myocardial content of CAC intermediates that is independent of the rate of myocardial fatty acid oxidation. Measurements were made in anaesthetized open chest pigs under control conditions, and at the end of 15 min of elevated cardiac workload induced by simultaneous dobutamine infusion, muscarinic blockade and constriction of the ascending aorta (Dob). In an attempt to stimulate glucose oxidation, a third group was made hyperglycaemic (14 mM) to stimulate flux through PDH during the high work state (Dob + Glu). An additional group was treated with oxfenicine (Oxf) (Dob + Glu + Oxf), an inhibitor of carnitine palmitoyltransferase I (CPT-I), in order to abolish inhibition on PDH from fatty acid oxidation. Tissue was analysed for PDH activity and key regulators of the enzyme complex (e.g. [acetyl-CoA]/[CoA-SH], [NADH]/[NAD+]) and CAC intermediate content, and glucose and fatty acid oxidation were measure with [U-14C]glucose and [9,10-3H]oleate.
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Methods
Studies were performed on adolescent domestic pigs of either gender (35–45 kg) under the guidelines stated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. no. 85-23) and the Institutional Animal Care and Use Committee at Case Western Reserve University.
Surgical preparation
After an overnight fast, pigs were sedated with an intramuscular injection of 6 mg kg–1 Telazol (zolazepam-titletamine) and anaesthetized with isoflurane (5% by mask). A tracheotomy was performed and anaesthesia was maintained with isoflurane (0.75–1.5%) and ketamine (4 mg kg–1 h–1). Animals were mechanically ventilated with 100% oxygen adjusted to maintain blood gases in the normal range (PCO2 35–45 mmHg, and pH 7.35–7.45). A femoral artery and vein were catheterized for arterial blood sampling and infusions, respectively. The heart was exposed by midline sternotomy as previously described (Hall et al. 1996a,b), and the animal was heparinized (200 U kg–1I.V.). The cardiac anterior interventricular vein was cannulated for measurement of the venous effluent from the myocardium. A 7-Fr manometer-tipped catheter (Milar) was introduced through the left carotid artery into the left ventricle (LV) and pressure was continuously recorded. A vascular occluder (24 mm, Harvard Apparatus) was placed around the ascending aorta and inflated with saline during the increased power phase of the protocol. Two pairs of sonomicrometry crystals were embedded in the anterior left ventricular wall at midmyocardial depth to determine regional segment length and assess changes in LV external work from the LV pressure–regional segment length loop area, as previously described (Chavez et al. 2003; Chandler et al. 2002, 2003). A catheter was placed in the left atrial appendage for injection of microspheres for determination of myocardial blood flow (Hall et al. 1996a,b).
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Experimental protocol
Four experimental groups were studied: (1) a control group (n= 7) with sham instrumentation; (2) a group that simultaneously received the receptor agonist dobutamine (25 μg kg–1 min–1), atropine (2 mg I.V.), and aortic constriction (Dob; n= 6); (3) a hyperglycaemic group (glucose injection of 200 mg kg–1I.V. followed by an infusion at 15 mg kg–1 min–1) that was treated with dobutamine, atropine and aortic constriction (Dob + Glu; n= 9); and (4) a group that received Oxf (30 mg kg–1I.V. as a bolus followed by an infusion at 30 mg kg–1 h–1) to inhibit fatty acid oxidation in addition to Dob + Glu treatment (Dob + Glu + Oxf) (Fig. 1). At the beginning of the protocol [U-14C]glucose (0.4 μCi min–1) and [9,10-3H]oleate (0.2 μCi min–1) were infused for the measurement of myocardial glucose and fatty acid oxidation, and the glucose and Oxf treatments were initiated in the Dob + Glu and Dob + Glu + Oxf groups. After 50 min cardiac power was stimulated for 15 min in the Dob, Dob + Glu, and Dob + Glu + Oxf groups by injection of the atropine bolus, infusion of dobutamine, and inflation of the aortic cuff. Cuff inflation was adjusted to give a peak LV systolic pressure of approximately 170 mmHg. Simultaneous arterial and coronary venous blood samples were taken at 60 and 65 min and analysed for oxygen content, glucose and plasma free fatty acids. Additionally, samples were analysed for [14C]glucose, 14CO2, [3H]oleate and 3H2O, which were needed to calculate levels of substrate oxidation as described below. Cardiovascular measurements were taken immediately before each blood sample and recorded on a commercial on-line data acquisition system (Crystal Biotech model CBI8000 with Biopaq software). Five million stable labelled microspheres (BioPhysics Assay Laboratory, Inc.) were injected into the left atrial appendage and simultaneously withdrawn from the femoral artery by a withdrawal pump (4 ml min–1) at 40 and 58 min in order to determine blood flow before and during the treatment period (Reinhardt et al. 2001). At 65 min the protocol needle biopsies (20–30 mg) were taken from the anterior LV free wall. The isoflurane concentration was then increased to 5%, and the animal was immediately killed by severing the inferior vena cava. A large punch biopsy (4–5 g) was immediately taken from the anterior LV free wall (Hall et al. 1996b; Chandler et al. 2002). All tissue samples were immediately freeze-clamped, placed in liquid nitrogen and stored at –80°C until analysed.
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See text for abbreviations.
Analytical methods
Blood analysis. Arterial and venous pH, PCO2 and PO2 were measured on a blood gas analyser (NOVA Profile Stat 3), and haemoglobin concentration and saturation were measured on a haemoximeter (Avoximeter). Whole blood samples were deproteinized in ice-cold 1 M perchloric acid (1: 2 v/v) and analysed for glucose and lactate spectrophotometrically (Hall et al. 1996b; Chandler et al. 2002). Blood [14C]glucose specific radioactivity was measured using ion-exchange as previously described (Chandler et al. 2002). Plasma fatty acids were measured spectrophotometrically (Hall et al. 1996b) and insulin was measured with a spectrophotometric immuno-antibody method (ALPCO Diagnostics). 3H-labelled fatty acids were extracted from plasma using heptane–isopropanol (3: 7) and specific activity determined as previously described (Chandler et al. 2002). 3H2O concentration was measured by distilling 0.5 ml of plasma in modified Hickman stills (Chandler et al. 2002). Blood 14CO2 concentration was measured by releasing 14CO2 with the addition of concentrated lactic acid and trapping it in hyamine hydroxide (Chandler et al. 2002).
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Tissue analysis. Tissue [ATP] and [ADP] were measured using an ATP bioluminescent assay kit (Sigma-Aldrich). Tissue lactate and glycogen were assayed by enzymatic spectrophotometic assay (Panchal et al. 2000; Chandler et al. 2002). Levels of tissue pyruvate were measured on a gas chromotography mass spectrometer (GCMS). Homogenized extracts (1 μl) were injected into an Agilent MS engine (Agilent 5973 MS and Agilent 6890 GC system) equipped with a DB-17MS capillary column (30 m, 0.25 mm internal diameter, 0.25 μm film thickness). The following gas chromotography program was used in 40: 1 split mode: helium carrier gas at (1.0 ml min–1), column head pressure 100 kPa, column start temperature 100°C increasing at 20°C min–1 to 240°C and then held for 3 min. Pyruvate concentrations were determined by the standard curve corrected ratios of m/z 274–277 in electron ionization mode (Des Rosiers et al. 1994). Whole tissue [NAD+] and [NADH] were measured by end-point UV enzymatic assays performed on the spectrophotometer and spectroflurometer, as previously described (Bergmeyer, 1989). Active and total PDH activity (Sterk et al. 2003), and [acetyl-coA] and [CoA-SH] (Cederblad et al. 1990) were determined by a radioenzymatic assay. All tissue concentrations were expressed per gram wet weight of tissue.
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Citric acid cycle intermediates were analysed on an Agilent 5973 mass spectrometer, equipped with an Agilent 6890 gas chromatograph, using a HP-5MS 5% phenyl methyl siloxane fused silica capillary column (60 m, 250 μm i.d., 0.25 μm film thickness) according to the method of Des Rosiers et al. (1994). The internal standards were [2,2,4,4-2H]citrate, [2,2,3,3-2H]succinate, [1,2,3,4-13C]fumarate, [2,3,32H3]malate and [1,2,3,4,5-13C]-KG. The -KG standard was generated by the transamination of [1,2,3,4,5-13C]glutamate as previously described (Laplante et al. 1995).
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Calculations
and the rates of glucose and fatty acid oxidation were calculated as previously described (Recchia et al. 2002). The rate of glucose oxidation was calculated as the product of myocardial blood flow and the venous – arterial 14CO2 concentration divided by the arterial glucose specific activity (Wisneski et al. 1985b; Wisneski et al. 1985a). The LV pressure-segment length loop area was calculated off-line as previously described (Chavez et al. 2003; Chandler et al. 2002). The LV pressure -segment length loop area times heart rate was used as an index of anterior wall external power, and was expressed as a percent of the pretreatment values (Fig. 2). The LV mechanical efficiency was calculated as the LV pressure -segment length loop area times heart rate divided by , and was expressed as a per cent of the value during the baseline period, as previously described (Chavez et al. 2003; Chandler et al. 2003).
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*P < 0.05, significantly different from control group. There were no other statistical differences among Dob, Dob + Glu and Dob + Glu + Oxf groups.
Statistical analysis
The control and Dob groups were compared using a non-parametric t test, and differences among the Dob, Dob + Glu and Dob + Glu + Oxf were determined using a Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks with a Dunn's pair-wise multiple comparison. Results are presented as means ± standard error (S.E.M.). A P value < 0.05 was considered significant.
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Results
Haemodynamics
The Dob, Dob + Glu and Dob + Glu + Oxf groups had a significantly greater heart rate, peak LV pressure, myocardial blood flow and anterior wall power compared with control (Table 1, Fig. 2), and there were no statistically significant differences between the Dob, Dob + Glu and Dob + Glu + Oxf groups. The LV mechanical efficiency at the end of the protocol was 86 ± 6% of the value during the baseline period in the control group. The change in efficiency in response to increase cardiac power was highly variable (90 ± 27, 65 ± 11 and 50 ± 8% of the baseline period for the Dob, Dob + Glu and Dob + Glu + Oxf groups, respectively) and there were no statistical differences among groups.
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Arterial concentrations
The arterial concentrations of glucose and insulin were significantly elevated in the Dob + Glu and Dob + Glu + Oxf compared with Dob and control groups (Table 2). Arterial plasma free fatty acid concentration was significantly elevated in the Dob compared with the control group, and they were significantly lower in the Dob + Glu and Dob + Glu + Oxf groups compared with the Dob group. Arterial lactate concentration was similar among all treatment groups (Table 2).
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PDH activity
There were no differences in the total PDH activity between treatment groups. The per cent of PDH in the active form increased from 30 ± 4% in the control group to 62 ± 6% in the Dob group (P < 0.001; Fig. 3). There were no differences in PDH activity among the Dob, Dob + Glu and Dob + Glu + Oxf groups.
*P < 0.001 compared with the control group. There were no other statistical differences among Dob, Dob + Glu and Dob + Glu + Oxf groups.
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Tissue metabolites
The myocardial [pyruvate], [NADH], [NAD+], [ATP] and [ADP] were not different among the treatment groups (Table 3), nor were there differences in the [ATP]/[ADP] and [NADH]/[NAD+] ratios (Fig. 4). The tissue [lactate] and the [lactate]/[pyruvate] ratio were increased in the Dob group compared with control (P < 0.001) and there were no differences among the Dob, Dob + Glu and Dob + Glu + Oxf groups (Table 3). Myocardial free CoA content was decreased by 50% in the Dob group compared with control (P < 0.001), without any significant difference in acetyl-CoA (Table 3), resulting in a 2.3-fold increase in the [acetyl-CoA]/[CoA-SH] ratio (P < 0.0005) (Fig. 4). There were no differences among the Dob, Dob + Glu and Dob + Glu + Oxf groups in acetyl-CoA content or the [acetyl-CoA]/[CoA-SH] ratio (Table 3, Fig. 4); however, the free CoA content was 2.1-fold higher in the Dob + Glu + Oxf group compared with the Dob group (P < 0.05) (Table 3). Glycogen concentration was 42% lower in the Dob group compared with control (P < 0.05), and there were no differences among the Dob, Dob + Glu and Dob + Glu + Oxf groups (Table 3).
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*P < 0.0005 compared with the control group. There were no other statistical differences among Dob, Dob + Glu and Dob + Glu + Oxf groups.
Substrate oxidation
At 42 and 47 min of tracer infusions steady state values were achieved for arterial [14C]glucose and [3H]oleate specific activities, and arterial and venous 14CO2, and 3H2O concentrations (data not shown). Plasma free fatty acid oxidation by the myocardium was greater in the Dob group than the control group (Fig. 5). Hyperglycaemia in the Dob + Glu group did not significantly reduced fatty acid oxidation compared with Dob; however, pharmacological inhibition of fatty acid oxidation with Oxf in the Dob + Glu + Oxf group resulted in near-complete suppression of fatty acid oxidation (Fig. 5). Glucose oxidation was significantly increased in the Dob group compared with the control group (Fig. 5). Hyperglycaemia and hyperinsulinaemia in the Dob + Glu group had no effect on glucose oxidation; however, inhibition of fatty acid oxidation with oxfencine resulted in a 1.9-fold increase in the rate of glucose oxidation compared with the Dob group.
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*P < 0.05 compared with the control group; P < 0.05 compared with the Dob group during increased cardiac power.
Citric acid cycle intermediates
Compared with the control group, the Dob group had approximately a 3-fold increase in the myocardial content of succinate, fumarate and malate, but no increase in citrate or -KG content (Table 4, Fig. 6). There were no differences between the Dob, Dob + Glu and Dob + Glu + Oxf treatment groups in the tissue content for -KG, succinate, fumarate or malate. Citrate content was lower in the Dob + Glu + Oxf compared with the Dob + Glu group (Table 4, Fig. 6).
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The myocardial content of citrate (CIT), -ketoglutarate (-KG), succinate (SUC), fumarate (FUM) and malate (MAL) in the Dob, Dob + Glu, and Dob + Glu + Oxf groups expressed as a fraction of the mean value in the control group. *Significantly different from the Dob + Glu group.
Discussion
This study demonstrates that a large increase in cardiac power and energy expenditure increases glucose oxidation and activates PDH independent of changes in the tissue content of the classic activators of PDH (increase in pyruvate content, decrease in [ATP]/[ADP], decrease in [NADH]/NAD+], and decrease in [acetyl-CoA]/[CoA-SH]). Furthermore, pharmacological suppression of fatty acid oxidation under hyperglycaemic conditions by inhibition of CPT-I increased [CoA-SH], resulted in greater glucose oxidation (Fig. 5, Table 3); however, this increase in flux through PDH occurred without further activation of the enzyme (Fig. 3). Thus at high rates of cardiac energy expenditure fatty acid oxidation still exerts inhibition on myocardial glucose oxidation independent of allosteric inhibition of PDH activity.
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Our results are similar to those obtained during the transition from rest to exercise in skeletal muscle: an increase in energy expenditure causes an activation of PDH (Fig. 3), but does not result in changes in the regulators of PDH kinase in a manner that would dephosphorylate and activate PDH (Putman et al. 1995; Howlett et al. 1998; Spriet & Heigenhauser, 2002). We did not observe a fall in the [ATP]/[ADP], [NADH]/[NAD+] or [acetyl-CoA]/[CoA-SH] ratios, or an increase in myocardial pyruvate content in the Dob group compared with control (Table 3, Fig. 4), thus the activity of PDH kinase was probably unchanged. In fact, we observed a fall in free CoA content and an increase in the [acetyl-CoA]/[CoA-SH] ratio, which should activate PDH kinase and inhibit PDH; however, we observed an increase in PDH activity. This suggests that the activation of PDH in the Dob group was due to stimulation of PDH phosphatase activity, presumably due to a greater cytosolic Ca2+ transient and a rise in mitochondrial [Ca2+], as has been previously observed in the isolated rat heart in response to adrenergic stimulation (McCormack & Denton, 1984; McCormack et al. 1990; Spriet & Heigenhauser, 2002).
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A limitation of this study is the inability to measure changes in the key metabolites in the local mitochondrial compartment where PDH resides. It is possible that concentrations of the regulators of PDH that we measured in whole tissue homogenates do not reflect the difference among treatment groups at the cellular site of PDH. All studies of this type are unable to provide data on the concentration of the regulators of PDH in the mitochondria (Newsholme & Randle, 1964; Kerbey et al. 1976; McCormack & Denton, 1984; Kruszynska et al. 1991; Hall et al. 1996b; Stanley et al. 1996; Howlett et al. 1998). Since PDH activity and flux is regulated by the local mitochondrial concentrations of metabolites, analysis of tissue homogenates may obscure local changes in the concentrations of PDH substrates and products, or in the [ATP]/[ADP] ratio. Clearly future work needs to address the important issue of tissue compartmentalization.
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We did not observe a significant increase in myocardial lactate uptake in the groups subjected to an increase in cardiac power. This differs from our previous studies where we observed a significant 2- to 3-fold increase in net lactate uptake with an infusion of dobutamine (Hall et al. 1995, 1996a,b), and during humans during exercise (Gertz et al. 1988; Stanley, 1991). The major difference between the present investigation and our earlier work was the absence of aortic constriction and atropine in our earlier studies, suggesting that the greater after-load with aortic banding might preferentially trigger an increase in glucose oxidation over lactate oxidation. There are no apparent biochemical mechanisms to explain such an effect. We also observed a 2- to 3-fold increase in the tissue [lactate]/[pyruvate] ratio with increased cardiac power, as previous observed with increase ventricular power in the perfused rat heart (Neely et al. 1976) and in the coronary venous effluent of humans subjected to intense physical exercise (Keul et al. 1967). This suggests that cytosolic [NADH]/[NAD+] ratio increased with increased power, while mitochondrial [NADH]/[NAD+] (which is largely reflected in the total tissue values (Fig. 4) (Sies, 1982; White & Wittenberg, 1993; Ashruf et al. 1995)) did not change.
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Changes in CAC intermediate content
The results of this study show that an increase in cardiac energy expenditure causes an expansion of the CAC pool size at the level of 4-carbon intermediates (succinate, fumarate and malate), but does not effect the content of the 5- and 6-carbon intermediates -KG and citrate (Fig. 6, Table 4). This differs from the response in human skeletal muscle to exercise, where there is a 2- to 4-fold increase in all of the CAC intermediates except -KG (Gibala et al. 1997, 1998). The reason for the differences in the response to a step increase in metabolic demand between cardiac and skeletal muscle are unclear, but could be due to the difference in mitochondrial density and structure between the two muscle types, and the smaller relative increase in metabolic rate in our studies than in the transition from rest to intense exercise in human leg muscle.
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Stimulation of glucose oxidation and near-complete inhibition of fatty acid oxidation had minimal effects on the increase in 4-carbon CAC intermediates induced by increased cardiac power, thus this response does not require myocardial fatty acid oxidation. Interestingly, the turnover time of 4-carbon intermediates was relatively unchanged by increased CAC flux. One can estimate the turnover time of the individual citric acid cycle intermediates from the tissue content divided by the rate of CAC flux (as estimated from the (Panchal et al. 2000; Vincent et al. 2004)). Under normal conditions the turnover times were 12.8, 3.5 and 8.5 s for succinate, fumarate and malate, respectively, and did not differ from the Dob group (9.4, 3.5 and 7.7 s, respectively). The values were unaffected by either hyperglycaemia or CPT-I inhibition. On the other hand, the turnover times of citrate and -KG were reduced from control conditions (24.7 and 1.6 s, respectively) to increased cardiac power (8.3 and 0.32 s, respectively). Glucose infusion alone had no affect; however, inhibition of CPT-I reduced citrate and -KG content and thus further reduced the turnover time for citrate and -KG (6.3 and 0.23 s, respectively). We have previously observed in pigs that infusing the medium chain fatty acid octanoate doubles the citrate concentration (Panchal et al. 2000), suggesting that by-passing CPT-I control of fatty acid oxidation has the opposite effect of CPT-I inhibition. Taken together, the initial span of the CAC (citrate to -KG) is more sensitive to alterations in CAC flux and substrate supply that the terminal span, as previously suggested (Cooney et al. 1981).
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An expansion of the CAC intermediate pool requires either greater entry of intermediates via anaplerosis and/or a decrease in loss of CAC intermediates. The main sites of anaplerosis in the heart are pyruvate carboxylation, glutamate transamination or conversion of propionate to succinyl-CoA (Gibala et al. 2000). The myocardium readily generates succinyl-CoA from propionate; however, under normal physiological conditions arterial propionate is very low and this process is unlikely to occur at a measurable rate (Martini et al. 2003; Reszko et al. 2003). Pyruvate carboxylation accounts for 3–6% of the CAC flux under normal and low flow conditions in pig heart (Panchal et al. 2001, 2000), but the effect of increased CAC flux on pyruvate carboxylation is unknown. In addition, little is known about the rate of anaplerosis from glutamate in the heart. Studies in human skeletal muscle suggest that increasing plasma concentration of glutamate amplifies the increase in the CAC pool size induced by exercise (Bruce et al. 2001). The lack of increase in tissue -KG also suggests this is not a major route for CAC pool expansion. Another possibility is that the increase in pool size is due to less efflux of CAC intermediates from the cycle, namely as citrate. Citrate efflux from the heart is relatively low (equal to approximately 1% of CAC flux) (Panchal et al. 2000, 2001), thus even if this rate were reduced to near zero it would not have much effect on pool size over such a short duration. Clearly additional work is needed to identify the mechanism(s) responsible for the increase in CAC intermediates.
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In the present study we made our measurements in the morning after an overnight fast. It is important to note that we may have obtained different results if the studies were performed in the fed condition, with high insulin and low free fatty acid. In the fed state there would probably have been greater uptake and oxidation of glucose and lactate and less fatty acid oxidation under both baseline and increased work conditions, but the effects of treatment with glucose and oxfencine would probably have been similar.
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In summary, the activity of PDH in the myocardium, like skeletal muscle, is increased when there is an increase in workload and energy expenditure. Furthermore, this activation occurs independent of changes in the concentrations of substrates and products that are known to inhibit PDH kinase (pyruvate, acetyl-CoA, free CoA, NADH, NAD+) and activate PDH. Pharmacological inhibition of myocardial fatty oxidation increased [CoA-SH] and the rate of glucose oxidation at high rates of myocardial energy expenditure without further activation of PDH. Thus fatty acid oxidation continues to play a critical role in the regulation of glucose oxidation at high rates of myocardial energy expenditure independent of PDH activation state. An increase in cardiac energy expenditure caused an expansion of the CAC pool size at the level of 4-carbon intermediates, but does not effect the content of the 5- and 6-carbon intermediates -KG and citrate. Stimulation of glucose oxidation and near-complete inhibition of fatty acid oxidation had minimal effects on the myocardial content of CAC intermediates, thus this response does not require increased myocardial fatty acid oxidation.
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Abstract
A high rate of cardiac work increases citric acid cycle (CAC) turnover and flux through pyruvate dehydrogenase (PDH); however, the mechanisms for these effects are poorly understood. We tested the hypotheses that an increase in cardiac energy expenditure: (1) activates PDH and reduces the product/substrate ratios ([NADH]/[NAD+] and [acetyl-CoA]/[CoA-SH]); and (2) increases the content of CAC intermediates. Measurements were made in anaesthetized pigs under control conditions and during 15 min of a high cardiac workload induced by dobutamine (Dob). A third group was made hyperglycaemic (14 mM) to stimulate flux through PDH during the high work state (Dob + Glu). Glucose and fatty acid oxidation were measured with 14C-glucose and 3H-oleate. Compared with control, the high workload groups had a similar increase in myocardial oxygen consumption ( and cardiac power. Dob increased PDH activity and glucose oxidation above control, but did not reduce the [NADH]/[NAD+] and [acetyl-CoA]/[CoA-SH] ratios, and there were no differences between the Dob and Dob + Glu groups. An additional group was treated with Dob + Glu and oxfenicine (Oxf) to inhibit fatty acid oxidation: this increased [CoA-SH] and glucose oxidation compared with Dob; however, there was no further activation of PDH or decrease in the [NADH]/[NAD+] ratio. Content of the 4-carbon CAC intermediates succinate, fumarate and malate increased 3-fold with Dob, but there was no change in citrate content, and the Dob + Glu and Dob + Glu + Oxf groups were not different from Dob. In conclusion, compared with normal conditions, at high myocardial energy expenditure (1) the increase in flux through PDH is regulated by activation of the enzyme complex and continues to be partially controlled through inhibition by fatty acid oxidation, and (2) there is expansion of the CAC pool size at the level of 4-carbon intermediates that is largely independent of myocardial fatty acid oxidation.
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Introduction
Intense exercise or catecholamine stress causes a 3- to 6-fold increase in ATP turnover, citric acid cycle (CAC) flux, and myocardial oxygen consumption compared with rest (Khouri et al. 1965; Balaban et al. 1986; Mootha et al. 1997). Under unstressed aerobic conditions the oxidation of fatty acids supplies most (60–80%) of the energy for ATP synthesis. However, during increased work states there is a greater relative contribution from the oxidation of glucose and lactate (Gertz et al. 1988; Collins-Nakai et al. 1994; Goodwin et al. 1998; Chatham, 2002). Glucose and lactate oxidation is regulated by pyruvate dehydrogenase (PDH), which is inactivated by phosphorylation by PDH kinase, which is in turn inhibited by pyruvate and activated by increases in [acetyl-CoA]/[CoA-SH] and [NADH]/[NAD+] ratios (Wieland et al. 1971; Whitehouse et al. 1974; Kerbey et al. 1976; Randle & Priesman, 1996). The PDH complex also contains a PDH phosphatase that is activated by intramitochondrial [Ca2+] (McCormack et al. 1990), which is elevated by adrenergic stimulation (McCormack & Denton, 1984; McCormack et al. 1990). In skeletal muscle the activation state of PDH is low at rest (15% of max), but is rapidly activated with exercise in proportion to the increase in energy expenditure (Putman et al. 1995; Howlett et al. 1998); however, this effect is not the result of an increase in pyruvate concentration or a fall in the [acetyl-CoA]/[CoA-SH] or [NADH]/[NAD+] ratios, but is related to a fall in [ATP]/[ADP], and activation of PDH phosphatase by an increase in mitochondrial [Ca2+] (McCormack et al. 1990; Spriet & Heigenhauser, 2002).
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Little is known about the effects of increased on the regulation of myocardial PDH activity and carbohydrate oxidation. The maximal rate of pyruvate oxidation is set by the degree of phosphorylation of PDH; however, the actual flux is largely determined by the concentrations of substrates and products in the mitochondrial matrix (Hansford & Cohen, 1978). Under unstressed resting conditions PDH activity and pyruvate oxidation are regulated by fatty acid oxidation via the Randle phenomenon, with enhanced PDH activity and flux with decreased fatty acid oxidation (Randle et al. 1963; Kerbey et al. 1976; Higgins et al. 1980, 1981; Kruszynska et al. 1991; Lopaschuk et al. 1994; Schwartz et al. 1994; Clarke et al. 1996; Stanley et al. 1997). An increase in cardiac energy expenditure, however, increases pyruvate oxidation despite an increase in the [acetyl-CoA]/[CoA-SH] ratio, suggesting that this mechanism does not facilitate PDH activation or flux (Hall et al. 1996a, 1996b). It has been proposed that there is a fall in the [NADH]/[NAD+] ratio with an increase in work demand (White & Wittenberg, 1993; Ashruf et al. 1995) but this has not been examined with physiologically relevant substrates or in vivo.
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Oxidation of acetyl-CoA by the CAC is dependent upon a sufficient concentration of all of the CAC intermediates; however, little is known about the content of CAC intermediates in the myocardium at high work states. In skeletal muscle contraction increases the tissue content of CAC intermediates (Aragon & Lowenstein, 1980; Sahlin et al. 1990), which requires ‘anaplerosis’, defined as the entry of intermediates into the CAC independent of synthesis of citrate from acetyl-CoA and oxaloacetate (e.g. from pyruvate carboxylation, glutamate transamination or from propionate conversion to succinyl-CoA) (Gibala et al. 2000). In human skeletal muscle exercise causes a 2- to 4-fold increase in all of the CAC intermediates, except -ketoglutarate (-KG) which declines by approximate 40% (Gibala et al. 1997, 1998). The increase in CAC pool size with exercise is enhanced when pyruvate oxidation is reduced and fatty acid oxidation increased (Gibala et al. 2002), but is also enhanced when pyruvate oxidation is stimulated (Timmons et al. 1996; Constantin-Teodosiu et al. 1999). The myocardial content of CAC intermediates under low workloads is dependent upon the availability of acetyl-CoA, as observed by the increase in CAC intermediates when acetate (Williamson, 1965; Taegtmeyer, 1984), or octanoate (Panchal et al. 2000) is a substrate.
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In the present investigation we tested the hypotheses that during high rates of myocardial energy expenditure: (1) PDH is regulated by the degree of phosphorylation inhibition on PDH, and product inhibition of flux through the active enzyme from fatty acid oxidation; and (2) there is an increase in the myocardial content of CAC intermediates that is independent of the rate of myocardial fatty acid oxidation. Measurements were made in anaesthetized open chest pigs under control conditions, and at the end of 15 min of elevated cardiac workload induced by simultaneous dobutamine infusion, muscarinic blockade and constriction of the ascending aorta (Dob). In an attempt to stimulate glucose oxidation, a third group was made hyperglycaemic (14 mM) to stimulate flux through PDH during the high work state (Dob + Glu). An additional group was treated with oxfenicine (Oxf) (Dob + Glu + Oxf), an inhibitor of carnitine palmitoyltransferase I (CPT-I), in order to abolish inhibition on PDH from fatty acid oxidation. Tissue was analysed for PDH activity and key regulators of the enzyme complex (e.g. [acetyl-CoA]/[CoA-SH], [NADH]/[NAD+]) and CAC intermediate content, and glucose and fatty acid oxidation were measure with [U-14C]glucose and [9,10-3H]oleate.
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Methods
Studies were performed on adolescent domestic pigs of either gender (35–45 kg) under the guidelines stated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. no. 85-23) and the Institutional Animal Care and Use Committee at Case Western Reserve University.
Surgical preparation
After an overnight fast, pigs were sedated with an intramuscular injection of 6 mg kg–1 Telazol (zolazepam-titletamine) and anaesthetized with isoflurane (5% by mask). A tracheotomy was performed and anaesthesia was maintained with isoflurane (0.75–1.5%) and ketamine (4 mg kg–1 h–1). Animals were mechanically ventilated with 100% oxygen adjusted to maintain blood gases in the normal range (PCO2 35–45 mmHg, and pH 7.35–7.45). A femoral artery and vein were catheterized for arterial blood sampling and infusions, respectively. The heart was exposed by midline sternotomy as previously described (Hall et al. 1996a,b), and the animal was heparinized (200 U kg–1I.V.). The cardiac anterior interventricular vein was cannulated for measurement of the venous effluent from the myocardium. A 7-Fr manometer-tipped catheter (Milar) was introduced through the left carotid artery into the left ventricle (LV) and pressure was continuously recorded. A vascular occluder (24 mm, Harvard Apparatus) was placed around the ascending aorta and inflated with saline during the increased power phase of the protocol. Two pairs of sonomicrometry crystals were embedded in the anterior left ventricular wall at midmyocardial depth to determine regional segment length and assess changes in LV external work from the LV pressure–regional segment length loop area, as previously described (Chavez et al. 2003; Chandler et al. 2002, 2003). A catheter was placed in the left atrial appendage for injection of microspheres for determination of myocardial blood flow (Hall et al. 1996a,b).
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Experimental protocol
Four experimental groups were studied: (1) a control group (n= 7) with sham instrumentation; (2) a group that simultaneously received the receptor agonist dobutamine (25 μg kg–1 min–1), atropine (2 mg I.V.), and aortic constriction (Dob; n= 6); (3) a hyperglycaemic group (glucose injection of 200 mg kg–1I.V. followed by an infusion at 15 mg kg–1 min–1) that was treated with dobutamine, atropine and aortic constriction (Dob + Glu; n= 9); and (4) a group that received Oxf (30 mg kg–1I.V. as a bolus followed by an infusion at 30 mg kg–1 h–1) to inhibit fatty acid oxidation in addition to Dob + Glu treatment (Dob + Glu + Oxf) (Fig. 1). At the beginning of the protocol [U-14C]glucose (0.4 μCi min–1) and [9,10-3H]oleate (0.2 μCi min–1) were infused for the measurement of myocardial glucose and fatty acid oxidation, and the glucose and Oxf treatments were initiated in the Dob + Glu and Dob + Glu + Oxf groups. After 50 min cardiac power was stimulated for 15 min in the Dob, Dob + Glu, and Dob + Glu + Oxf groups by injection of the atropine bolus, infusion of dobutamine, and inflation of the aortic cuff. Cuff inflation was adjusted to give a peak LV systolic pressure of approximately 170 mmHg. Simultaneous arterial and coronary venous blood samples were taken at 60 and 65 min and analysed for oxygen content, glucose and plasma free fatty acids. Additionally, samples were analysed for [14C]glucose, 14CO2, [3H]oleate and 3H2O, which were needed to calculate levels of substrate oxidation as described below. Cardiovascular measurements were taken immediately before each blood sample and recorded on a commercial on-line data acquisition system (Crystal Biotech model CBI8000 with Biopaq software). Five million stable labelled microspheres (BioPhysics Assay Laboratory, Inc.) were injected into the left atrial appendage and simultaneously withdrawn from the femoral artery by a withdrawal pump (4 ml min–1) at 40 and 58 min in order to determine blood flow before and during the treatment period (Reinhardt et al. 2001). At 65 min the protocol needle biopsies (20–30 mg) were taken from the anterior LV free wall. The isoflurane concentration was then increased to 5%, and the animal was immediately killed by severing the inferior vena cava. A large punch biopsy (4–5 g) was immediately taken from the anterior LV free wall (Hall et al. 1996b; Chandler et al. 2002). All tissue samples were immediately freeze-clamped, placed in liquid nitrogen and stored at –80°C until analysed.
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See text for abbreviations.
Analytical methods
Blood analysis. Arterial and venous pH, PCO2 and PO2 were measured on a blood gas analyser (NOVA Profile Stat 3), and haemoglobin concentration and saturation were measured on a haemoximeter (Avoximeter). Whole blood samples were deproteinized in ice-cold 1 M perchloric acid (1: 2 v/v) and analysed for glucose and lactate spectrophotometrically (Hall et al. 1996b; Chandler et al. 2002). Blood [14C]glucose specific radioactivity was measured using ion-exchange as previously described (Chandler et al. 2002). Plasma fatty acids were measured spectrophotometrically (Hall et al. 1996b) and insulin was measured with a spectrophotometric immuno-antibody method (ALPCO Diagnostics). 3H-labelled fatty acids were extracted from plasma using heptane–isopropanol (3: 7) and specific activity determined as previously described (Chandler et al. 2002). 3H2O concentration was measured by distilling 0.5 ml of plasma in modified Hickman stills (Chandler et al. 2002). Blood 14CO2 concentration was measured by releasing 14CO2 with the addition of concentrated lactic acid and trapping it in hyamine hydroxide (Chandler et al. 2002).
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Tissue analysis. Tissue [ATP] and [ADP] were measured using an ATP bioluminescent assay kit (Sigma-Aldrich). Tissue lactate and glycogen were assayed by enzymatic spectrophotometic assay (Panchal et al. 2000; Chandler et al. 2002). Levels of tissue pyruvate were measured on a gas chromotography mass spectrometer (GCMS). Homogenized extracts (1 μl) were injected into an Agilent MS engine (Agilent 5973 MS and Agilent 6890 GC system) equipped with a DB-17MS capillary column (30 m, 0.25 mm internal diameter, 0.25 μm film thickness). The following gas chromotography program was used in 40: 1 split mode: helium carrier gas at (1.0 ml min–1), column head pressure 100 kPa, column start temperature 100°C increasing at 20°C min–1 to 240°C and then held for 3 min. Pyruvate concentrations were determined by the standard curve corrected ratios of m/z 274–277 in electron ionization mode (Des Rosiers et al. 1994). Whole tissue [NAD+] and [NADH] were measured by end-point UV enzymatic assays performed on the spectrophotometer and spectroflurometer, as previously described (Bergmeyer, 1989). Active and total PDH activity (Sterk et al. 2003), and [acetyl-coA] and [CoA-SH] (Cederblad et al. 1990) were determined by a radioenzymatic assay. All tissue concentrations were expressed per gram wet weight of tissue.
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Citric acid cycle intermediates were analysed on an Agilent 5973 mass spectrometer, equipped with an Agilent 6890 gas chromatograph, using a HP-5MS 5% phenyl methyl siloxane fused silica capillary column (60 m, 250 μm i.d., 0.25 μm film thickness) according to the method of Des Rosiers et al. (1994). The internal standards were [2,2,4,4-2H]citrate, [2,2,3,3-2H]succinate, [1,2,3,4-13C]fumarate, [2,3,32H3]malate and [1,2,3,4,5-13C]-KG. The -KG standard was generated by the transamination of [1,2,3,4,5-13C]glutamate as previously described (Laplante et al. 1995).
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Calculations
and the rates of glucose and fatty acid oxidation were calculated as previously described (Recchia et al. 2002). The rate of glucose oxidation was calculated as the product of myocardial blood flow and the venous – arterial 14CO2 concentration divided by the arterial glucose specific activity (Wisneski et al. 1985b; Wisneski et al. 1985a). The LV pressure-segment length loop area was calculated off-line as previously described (Chavez et al. 2003; Chandler et al. 2002). The LV pressure -segment length loop area times heart rate was used as an index of anterior wall external power, and was expressed as a percent of the pretreatment values (Fig. 2). The LV mechanical efficiency was calculated as the LV pressure -segment length loop area times heart rate divided by , and was expressed as a per cent of the value during the baseline period, as previously described (Chavez et al. 2003; Chandler et al. 2003).
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*P < 0.05, significantly different from control group. There were no other statistical differences among Dob, Dob + Glu and Dob + Glu + Oxf groups.
Statistical analysis
The control and Dob groups were compared using a non-parametric t test, and differences among the Dob, Dob + Glu and Dob + Glu + Oxf were determined using a Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks with a Dunn's pair-wise multiple comparison. Results are presented as means ± standard error (S.E.M.). A P value < 0.05 was considered significant.
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Results
Haemodynamics
The Dob, Dob + Glu and Dob + Glu + Oxf groups had a significantly greater heart rate, peak LV pressure, myocardial blood flow and anterior wall power compared with control (Table 1, Fig. 2), and there were no statistically significant differences between the Dob, Dob + Glu and Dob + Glu + Oxf groups. The LV mechanical efficiency at the end of the protocol was 86 ± 6% of the value during the baseline period in the control group. The change in efficiency in response to increase cardiac power was highly variable (90 ± 27, 65 ± 11 and 50 ± 8% of the baseline period for the Dob, Dob + Glu and Dob + Glu + Oxf groups, respectively) and there were no statistical differences among groups.
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Arterial concentrations
The arterial concentrations of glucose and insulin were significantly elevated in the Dob + Glu and Dob + Glu + Oxf compared with Dob and control groups (Table 2). Arterial plasma free fatty acid concentration was significantly elevated in the Dob compared with the control group, and they were significantly lower in the Dob + Glu and Dob + Glu + Oxf groups compared with the Dob group. Arterial lactate concentration was similar among all treatment groups (Table 2).
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PDH activity
There were no differences in the total PDH activity between treatment groups. The per cent of PDH in the active form increased from 30 ± 4% in the control group to 62 ± 6% in the Dob group (P < 0.001; Fig. 3). There were no differences in PDH activity among the Dob, Dob + Glu and Dob + Glu + Oxf groups.
*P < 0.001 compared with the control group. There were no other statistical differences among Dob, Dob + Glu and Dob + Glu + Oxf groups.
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Tissue metabolites
The myocardial [pyruvate], [NADH], [NAD+], [ATP] and [ADP] were not different among the treatment groups (Table 3), nor were there differences in the [ATP]/[ADP] and [NADH]/[NAD+] ratios (Fig. 4). The tissue [lactate] and the [lactate]/[pyruvate] ratio were increased in the Dob group compared with control (P < 0.001) and there were no differences among the Dob, Dob + Glu and Dob + Glu + Oxf groups (Table 3). Myocardial free CoA content was decreased by 50% in the Dob group compared with control (P < 0.001), without any significant difference in acetyl-CoA (Table 3), resulting in a 2.3-fold increase in the [acetyl-CoA]/[CoA-SH] ratio (P < 0.0005) (Fig. 4). There were no differences among the Dob, Dob + Glu and Dob + Glu + Oxf groups in acetyl-CoA content or the [acetyl-CoA]/[CoA-SH] ratio (Table 3, Fig. 4); however, the free CoA content was 2.1-fold higher in the Dob + Glu + Oxf group compared with the Dob group (P < 0.05) (Table 3). Glycogen concentration was 42% lower in the Dob group compared with control (P < 0.05), and there were no differences among the Dob, Dob + Glu and Dob + Glu + Oxf groups (Table 3).
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*P < 0.0005 compared with the control group. There were no other statistical differences among Dob, Dob + Glu and Dob + Glu + Oxf groups.
Substrate oxidation
At 42 and 47 min of tracer infusions steady state values were achieved for arterial [14C]glucose and [3H]oleate specific activities, and arterial and venous 14CO2, and 3H2O concentrations (data not shown). Plasma free fatty acid oxidation by the myocardium was greater in the Dob group than the control group (Fig. 5). Hyperglycaemia in the Dob + Glu group did not significantly reduced fatty acid oxidation compared with Dob; however, pharmacological inhibition of fatty acid oxidation with Oxf in the Dob + Glu + Oxf group resulted in near-complete suppression of fatty acid oxidation (Fig. 5). Glucose oxidation was significantly increased in the Dob group compared with the control group (Fig. 5). Hyperglycaemia and hyperinsulinaemia in the Dob + Glu group had no effect on glucose oxidation; however, inhibition of fatty acid oxidation with oxfencine resulted in a 1.9-fold increase in the rate of glucose oxidation compared with the Dob group.
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*P < 0.05 compared with the control group; P < 0.05 compared with the Dob group during increased cardiac power.
Citric acid cycle intermediates
Compared with the control group, the Dob group had approximately a 3-fold increase in the myocardial content of succinate, fumarate and malate, but no increase in citrate or -KG content (Table 4, Fig. 6). There were no differences between the Dob, Dob + Glu and Dob + Glu + Oxf treatment groups in the tissue content for -KG, succinate, fumarate or malate. Citrate content was lower in the Dob + Glu + Oxf compared with the Dob + Glu group (Table 4, Fig. 6).
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The myocardial content of citrate (CIT), -ketoglutarate (-KG), succinate (SUC), fumarate (FUM) and malate (MAL) in the Dob, Dob + Glu, and Dob + Glu + Oxf groups expressed as a fraction of the mean value in the control group. *Significantly different from the Dob + Glu group.
Discussion
This study demonstrates that a large increase in cardiac power and energy expenditure increases glucose oxidation and activates PDH independent of changes in the tissue content of the classic activators of PDH (increase in pyruvate content, decrease in [ATP]/[ADP], decrease in [NADH]/NAD+], and decrease in [acetyl-CoA]/[CoA-SH]). Furthermore, pharmacological suppression of fatty acid oxidation under hyperglycaemic conditions by inhibition of CPT-I increased [CoA-SH], resulted in greater glucose oxidation (Fig. 5, Table 3); however, this increase in flux through PDH occurred without further activation of the enzyme (Fig. 3). Thus at high rates of cardiac energy expenditure fatty acid oxidation still exerts inhibition on myocardial glucose oxidation independent of allosteric inhibition of PDH activity.
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Our results are similar to those obtained during the transition from rest to exercise in skeletal muscle: an increase in energy expenditure causes an activation of PDH (Fig. 3), but does not result in changes in the regulators of PDH kinase in a manner that would dephosphorylate and activate PDH (Putman et al. 1995; Howlett et al. 1998; Spriet & Heigenhauser, 2002). We did not observe a fall in the [ATP]/[ADP], [NADH]/[NAD+] or [acetyl-CoA]/[CoA-SH] ratios, or an increase in myocardial pyruvate content in the Dob group compared with control (Table 3, Fig. 4), thus the activity of PDH kinase was probably unchanged. In fact, we observed a fall in free CoA content and an increase in the [acetyl-CoA]/[CoA-SH] ratio, which should activate PDH kinase and inhibit PDH; however, we observed an increase in PDH activity. This suggests that the activation of PDH in the Dob group was due to stimulation of PDH phosphatase activity, presumably due to a greater cytosolic Ca2+ transient and a rise in mitochondrial [Ca2+], as has been previously observed in the isolated rat heart in response to adrenergic stimulation (McCormack & Denton, 1984; McCormack et al. 1990; Spriet & Heigenhauser, 2002).
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A limitation of this study is the inability to measure changes in the key metabolites in the local mitochondrial compartment where PDH resides. It is possible that concentrations of the regulators of PDH that we measured in whole tissue homogenates do not reflect the difference among treatment groups at the cellular site of PDH. All studies of this type are unable to provide data on the concentration of the regulators of PDH in the mitochondria (Newsholme & Randle, 1964; Kerbey et al. 1976; McCormack & Denton, 1984; Kruszynska et al. 1991; Hall et al. 1996b; Stanley et al. 1996; Howlett et al. 1998). Since PDH activity and flux is regulated by the local mitochondrial concentrations of metabolites, analysis of tissue homogenates may obscure local changes in the concentrations of PDH substrates and products, or in the [ATP]/[ADP] ratio. Clearly future work needs to address the important issue of tissue compartmentalization.
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We did not observe a significant increase in myocardial lactate uptake in the groups subjected to an increase in cardiac power. This differs from our previous studies where we observed a significant 2- to 3-fold increase in net lactate uptake with an infusion of dobutamine (Hall et al. 1995, 1996a,b), and during humans during exercise (Gertz et al. 1988; Stanley, 1991). The major difference between the present investigation and our earlier work was the absence of aortic constriction and atropine in our earlier studies, suggesting that the greater after-load with aortic banding might preferentially trigger an increase in glucose oxidation over lactate oxidation. There are no apparent biochemical mechanisms to explain such an effect. We also observed a 2- to 3-fold increase in the tissue [lactate]/[pyruvate] ratio with increased cardiac power, as previous observed with increase ventricular power in the perfused rat heart (Neely et al. 1976) and in the coronary venous effluent of humans subjected to intense physical exercise (Keul et al. 1967). This suggests that cytosolic [NADH]/[NAD+] ratio increased with increased power, while mitochondrial [NADH]/[NAD+] (which is largely reflected in the total tissue values (Fig. 4) (Sies, 1982; White & Wittenberg, 1993; Ashruf et al. 1995)) did not change.
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Changes in CAC intermediate content
The results of this study show that an increase in cardiac energy expenditure causes an expansion of the CAC pool size at the level of 4-carbon intermediates (succinate, fumarate and malate), but does not effect the content of the 5- and 6-carbon intermediates -KG and citrate (Fig. 6, Table 4). This differs from the response in human skeletal muscle to exercise, where there is a 2- to 4-fold increase in all of the CAC intermediates except -KG (Gibala et al. 1997, 1998). The reason for the differences in the response to a step increase in metabolic demand between cardiac and skeletal muscle are unclear, but could be due to the difference in mitochondrial density and structure between the two muscle types, and the smaller relative increase in metabolic rate in our studies than in the transition from rest to intense exercise in human leg muscle.
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Stimulation of glucose oxidation and near-complete inhibition of fatty acid oxidation had minimal effects on the increase in 4-carbon CAC intermediates induced by increased cardiac power, thus this response does not require myocardial fatty acid oxidation. Interestingly, the turnover time of 4-carbon intermediates was relatively unchanged by increased CAC flux. One can estimate the turnover time of the individual citric acid cycle intermediates from the tissue content divided by the rate of CAC flux (as estimated from the (Panchal et al. 2000; Vincent et al. 2004)). Under normal conditions the turnover times were 12.8, 3.5 and 8.5 s for succinate, fumarate and malate, respectively, and did not differ from the Dob group (9.4, 3.5 and 7.7 s, respectively). The values were unaffected by either hyperglycaemia or CPT-I inhibition. On the other hand, the turnover times of citrate and -KG were reduced from control conditions (24.7 and 1.6 s, respectively) to increased cardiac power (8.3 and 0.32 s, respectively). Glucose infusion alone had no affect; however, inhibition of CPT-I reduced citrate and -KG content and thus further reduced the turnover time for citrate and -KG (6.3 and 0.23 s, respectively). We have previously observed in pigs that infusing the medium chain fatty acid octanoate doubles the citrate concentration (Panchal et al. 2000), suggesting that by-passing CPT-I control of fatty acid oxidation has the opposite effect of CPT-I inhibition. Taken together, the initial span of the CAC (citrate to -KG) is more sensitive to alterations in CAC flux and substrate supply that the terminal span, as previously suggested (Cooney et al. 1981).
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An expansion of the CAC intermediate pool requires either greater entry of intermediates via anaplerosis and/or a decrease in loss of CAC intermediates. The main sites of anaplerosis in the heart are pyruvate carboxylation, glutamate transamination or conversion of propionate to succinyl-CoA (Gibala et al. 2000). The myocardium readily generates succinyl-CoA from propionate; however, under normal physiological conditions arterial propionate is very low and this process is unlikely to occur at a measurable rate (Martini et al. 2003; Reszko et al. 2003). Pyruvate carboxylation accounts for 3–6% of the CAC flux under normal and low flow conditions in pig heart (Panchal et al. 2001, 2000), but the effect of increased CAC flux on pyruvate carboxylation is unknown. In addition, little is known about the rate of anaplerosis from glutamate in the heart. Studies in human skeletal muscle suggest that increasing plasma concentration of glutamate amplifies the increase in the CAC pool size induced by exercise (Bruce et al. 2001). The lack of increase in tissue -KG also suggests this is not a major route for CAC pool expansion. Another possibility is that the increase in pool size is due to less efflux of CAC intermediates from the cycle, namely as citrate. Citrate efflux from the heart is relatively low (equal to approximately 1% of CAC flux) (Panchal et al. 2000, 2001), thus even if this rate were reduced to near zero it would not have much effect on pool size over such a short duration. Clearly additional work is needed to identify the mechanism(s) responsible for the increase in CAC intermediates.
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In the present study we made our measurements in the morning after an overnight fast. It is important to note that we may have obtained different results if the studies were performed in the fed condition, with high insulin and low free fatty acid. In the fed state there would probably have been greater uptake and oxidation of glucose and lactate and less fatty acid oxidation under both baseline and increased work conditions, but the effects of treatment with glucose and oxfencine would probably have been similar.
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In summary, the activity of PDH in the myocardium, like skeletal muscle, is increased when there is an increase in workload and energy expenditure. Furthermore, this activation occurs independent of changes in the concentrations of substrates and products that are known to inhibit PDH kinase (pyruvate, acetyl-CoA, free CoA, NADH, NAD+) and activate PDH. Pharmacological inhibition of myocardial fatty oxidation increased [CoA-SH] and the rate of glucose oxidation at high rates of myocardial energy expenditure without further activation of PDH. Thus fatty acid oxidation continues to play a critical role in the regulation of glucose oxidation at high rates of myocardial energy expenditure independent of PDH activation state. An increase in cardiac energy expenditure caused an expansion of the CAC pool size at the level of 4-carbon intermediates, but does not effect the content of the 5- and 6-carbon intermediates -KG and citrate. Stimulation of glucose oxidation and near-complete inhibition of fatty acid oxidation had minimal effects on the myocardial content of CAC intermediates, thus this response does not require increased myocardial fatty acid oxidation.
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