Cardiovascular Researclt, 1976, 10, 427-436.
Effects of dichloroacetate on myocardial substrate extraction, epicardial ST-segment elevation, and ventricular blood flow following coronary occlusion in dogs’ 0 . D . M J Q S 2 , N. E. MILLER3, R . A . RIEMERSMA, and M . F . O L I V E R From the Departments of Cardiology and Medicine and the Lipid Research Laboratory, Department of Clinical Chemistry, Royal Injirmary, Edinburgh
Under normal circumstances the energy requirements of the heart are derived principally from the oxidation of free fatty acids (FFA) (Lassers et al, 1972). During myocardial ischaemia the oxidation of FFA is impaired to a greater extent than that of glucose, and the heart becomes more dependent upon both aerobic and anaerobic glucose metabolism for its energy re-
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Supported by grants from the Medical Research Council and the Wcllcome Trust. Reprint requests to M.F.O.,Department of Cardiology, Royal Infirmary. Edinburgh. lPresent address: Institute for Medical Biology, University ofTromso, Tromse 9000. Norway. Present address: Division of Metabolic Disease. Department of Medicine, University of California at San Diego, La Jolla. California 92037, USA.
quirements (Opie et al, 1973). Under such circumstances an increased delivery of FFA to the heart has been associated experimentally with the development of ventricular arrhythmias (Kurien et al, 1971) and with further deterioration in cellular function (Kjekshus and Mjm, 1972; de Leiris et al, 1974). For these reasons it was proposed that the survival of the ischaemic myocardium might be improved by measures which promote the utilization of glucose and decrease that of FFA (Kurien and Oliver, 1970; Opie, 1970). Recent animal studies have supported this hypothesis, and intravenous (iv) infusions of glucose and insulin were reported by Maroko ef a1 (1972) to
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Glucose metabolism in the healthy heart is stimulated by dichloroacetate (DCA). The possibility has been examined in dogs that DCA, by increasing glucose utilization, might limit the severity of acute myocardial ischaemic injury. Intravenous administration of DCA reduced the degree of epicardial ST-segment elevation induced by subsequent coronary occlusion, both under basal conditions and during isoprenaline infusion. A similar result was obtained when DCA was given during an established coronary occlusion. This effect could not be explained by changes in mean aortic blood pressure, heart rate, or regional myocardial blood flow as measured by radioactive microspheres. Measurements in arterial and coronary sinus blood demonstrated an increase in the extraction of glucose and a decrease in that of FFA by the heart. Glucose extraction also tended to be increased in the ischaemic zone, as shown by the differences in the concentrations of these substrates between arterial blood and blood obtained from the local vein draining that zone. Lactate release by the ischaemic zone was markedly reduced. AUTHORS’ SYNOPSIS
420 Mj0s, Miller, Rieniersma, and Oliver
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reduce myocardial ischaemic injury following occlusions of the artery were performed with a coronary occlusion in dogs; and similar results releasable metal clip. The left femoral vein was were obtained by Kjekshus and Mjpls (1973) cannulated as a route for infusion, and the left when arterial FFA concentrations were lowered femoral artery for measurement of mean aortic blood pressure (AP). KP was monitored with a Staby [+pyridyl-carbinol and by Mjm et a1 (1976) tham P23Db transducer. A catheter for withdrawal using clofibrate to reduce myocardial F F A of arterial blood was inserted into the right femoral extraction. artery. Coronary sinus blood was sampled through a I n 1962, Lorini and Ciman reported that di- catheter introduced via the left jugular vein under isopropyl-ammonium dichloroacetate raised the fluoroscopic guidance. In 10 experiments the local respiratory quotient in alloxan-diabetic rats, vein draining the ischaemic area was cannulated for suggesting a n increased utilization of glucose the assessment of metabolic changes in the ischaemic relative to that of FFA. Stacpoole and Felts myocardium. (1970; 1971) demonstrated that the active component was dichloroacetate (DCA), and that Experimental designs this stimulated glucose oxidation and inhibited EJects of' DCA on the response to sirhseqrient F F A oxidation in rat diaphragm muscle. coronary occlusion McAllister et a1 ( 1973) subsequently showed Experiments were carried out both under basal conthat DCA had similar effects on the metabolism ditions and during a continuous iv infusion of isoof the isolated perfused rat heart and the prenaline. In six animals the experimental design intact dog heart. Associated changes in conformed to procedure 1 of Fig. 1 . In one animal a lactate and pyruvate metabolism and in intra- similar sequence of occlusions was performed without the use of isoprenaline; in another animal isocellular intermediates suggested that DCA prenaline was given during all four occlusions. In was stimulating both glycolysis and glucose three animals the experimental design was reversed oxidation by increasing the activities of phospho- in accordance with procedure 2 of Fig. 1. In these fructokinase and pyruvate dehydrogenase. An experiments DCA was given before the first and activation of pyruvate dehydrogenase in rat myo- second occlusions, and a period of 95 rnin was then cardium by DCA was observed by Whitehouse allowed for the metabolic effects of DCA to diminish before repeating the occlusions. The effects of DCA and Randle (1973). In the present study we have investigated were assessed by paired analysis of the data for all further the metabolic and haemodynamic effects 1 I experiments. In five separate animals the effect of DCA (120 of DCA in dogs, and have examined the possimgikg) on the metabolic pattern across the ischaemic bility that the stimulation of myocardial glucose zone was studied during basal lipolysis. A constant utilization by DCA might limit the severity of infusion of albumin bound :'H-oleate (Radiochemiacute myocardial ischaemic injury during experi- cal Centre, Amersham), prepared as described by mental coronary occlusion Opie et al (1973) was commenced at least 90 min before and maintained throughout the experiment. Between 5 and I5 rnin after the occlusion of the branch of the LAD coronary artery, arterial, local Methods venous, and coronary sinus blood samples were Animal preparation taken for the measurement of FFA, :'H-FFA, gluExperiments were performed on 29 healthy mongrel cose, and lactate. After release and recovery DCA dogs of both sexes (12-19 kg body weight). Each was administered and the occlusion repeated 10 min dog was fasted overnight and anaesthetized with later. Care was taken that blood samples were obsodium pentobarbitone (25 mgikg, iv), followed by tained at the same relative times during the two maintenance doses of 50 mg. Ventilation was main- occlusions. In another five animals the identical tained throughout a cuffed endotracheal tube with procedure was repeated except that now the occlua positive pressure respirator (Harvard Apparatus sions were preceded by a continuous isoprenaline Co Inc, hdass, USA). Thoracotomy was performed infusion (0.1-0. I5 yg.kg-l/min). In four additional animals the effects of DCA on through the left fifth intercostal space and the heart suspended in a pericardial cradle. A branch of the regional myocardial blood flow I5 min after coronleft anterior descending (LAD) coronary artery ary occlusion were assessed by means of radioactive was then dissected free for 0.5 cm. Subsequent microspheres. Measurements were made during an
429 Dichloroacetate and myocardial iscliaemic injury in dogs F I G . 1 Experimental procedures for assessing the effect of dichloroacetate (DCA) on the responseto subsequent coronary 4 occlusion. Four identical occlusions of 15 min duration were performed in each animal, two I I I I I I I in the absence of DCA and two I I I t TIME (mm) 0 20 40 60 80 100 I20 140 160 I80 200 in its presence. One of each pair of occlusions was performed during a continuous i v infusion of isoprenaline (0.2-0.3pg. PROCEDURE 2 kg-llmin), started 5 rnin before the occlusion. Recovery periods 20nhg 'k of 30 min were allowed between successive occlusions. Immediately bejore the release of each occlusion (indicated by verfical arrows) epicardial electrocarI I I I I diograms from 10-15 sites in the TIME ( m m ) 0 20 40 60 80 100 170 190 210 230 250 ischaemic arid non-ischaemic zones, mean aortic blood pressure and heart rate were recorded, and blood was sampled from a femoral artery and the coronary sinus. Sodium DCA was administered at the dose of 120 mglkg body weight by i v injection over 5 min in 10 ml 0.9% saline. Occln = coronary occlusion. Isopren = isoprenaline infusion. PROCEDURE
1
m
coronary sinus blood for measurement of plasma FFA radioactivity was sampled immediately before and 20 rnin after DCA injection.
Effects of DCA administration during established coronary occlusion
The severity of the acute myocardial ischaemic injury was assessed by epicardial ST-segment mapping (Maroko et al. 1971). Epicardial electrocardiograms were recorded 15 rnin after coronary occlusion from 10-1 5 anatomically recognizable sites supplied by the occluded artery and within the adjacent area, using a mobile cotton-wick electrode (Devices M4 recorder; paper speed, 25 mmls; sensitivity, 1 mV/mm deflection). The sum of the ST-segment elevations from all sites (XST) was used as an approximate index of the severity of ischaemic injury.
I n another four animals iv infusion of isoprenaline (0.2-0.3 pg.kg-'!min) was maintained for the duration of the study. Five minutes after its commencement control recordings of the epicardial electrocardiogram (ECG), KP, and H R were made before a branch of the LAD coronary artery was permanently occluded. Further ECG and haemodynamic measurements were made 5, 10, and 1 5 min following coronary occlusion. Sodium dichloroacetate (120 mgikg iv) was then infused over 2 min, and the ECG and haemodynamic recordings repeated 5, 10, 15, and 20min later. Arterial and coronary sinus blood was sampled immediately before and 20 rnin after the injection of DCA for measurement of plasma FFA and glucose concentrations. In the same animals the effect of DCA on the total myocardial uptake of radiolabelled palmitate was also examined. For this purpose a continuous iv infusion of albumin-bound (9.1 0 [nl-") palmitate (Radiochemical Centre, Amersham). Arterial and
Measurements Epicardial ST-segment mupping
Regional myocardial blood flow
Regional myocardial blood flow was measured with ' W e - and n5Sr-labelled radioactive microspheres (3M Riker Laboratories, Loughborough, UK; nominal diameter, 15 p) (Utley et al, 1974). Before injection 2.0-2.5 x lo6 microspheres (sufficient to give at least lo00 microspheresig of myocardium) were sonicated in 10% dextrose for 10 rnin to dispel aggregates (Dawe Sonicleaner, type 6441A), and drawn into a syringe containing 0.1 ml 5 %
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initial control occlusion, and then during a second occlusion performed 10 rnin after iv administration of sodium dichloroacetate ( 120 mgikg). The order of injection of the differently labelled microspheres was varied to avoid systematic errors due to possible differences in their behaviour.
430 M j m , Miller, Riemersnia, and Oliver Tween 30. The suspension was diluted with 10% Srutistics dextrose to a final concentration of Tween 80 of less Each dog served as its own control. Student's t test than 0.5 and then continuously agitated until for paired data was used to calculate probabilities. injected. Fifteen min after coronary occlusion blood P > 0.05 was regarded as not statistically significant. for estimation of reference blood flow (RBF) was withdrawn from a femoral artery into a weighed heparinised syringe at constant rate (approximately Results 9 mlimin) for 2 min. Immediately after the comEffects of DCA on the response to subsequent mencement of blood withdrawal, the microspheres were injected through a left atrial cannula over 10 s. coronary occlusion After each experiment the heart was excised, the The effects of DCA on the response to subsefree wall of the left ventricle dissected out, and visual quent coronary occlusion under basal conditions fat and large vessels removed. Between eight and 10 are summarized in Table I . Coronary occlusion full thickness tissue samples were taken and divided raised epicardial ST segments in all experiments. into epicardial and endocardial layers (wet weight In the absence of DCA, EST averaged 183,3 1.5-4.Og). Each layer was divided into 1&20 mV (mean f S E M ) 15 min after coronary blocks to produce constant geometrical factors for occlusion. Values for XST were significantly less measurements of radioactivity in a three channel when coronary occlusion was performed after gamma counter (GammaGuard, Tracerlab Ltd). the administration of DCA ( E S T 6 1 2 mV; radioactivity was Approximately 27 % of counted in the 141Ce channel. This factor was P