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Anaerobic Performance and Metabolism of the Hyperthyroid Heart

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Anaerobic Performance and Metabolism of the Hyperthyroid Heart Anaerobic Performance and Metabolism of the Hyperthyroid Heart Rum A. ALTSCHULD, ALAN WEISS, FRED A. KRUGER, and ARNOLD M. WEISSLER From the Department of Medicine, Ohio State University College of Medicine, Columbus, Ohio 43210 A B S T R A C T Anaerobically perfused hearts from rats all consequences of hyperthyroilism (7-9). These with experimentally induced hyperthyroidism exhibited changes are accompanied by increased cardiac oxygen accelerated deterioration of pacemaker activity and ven- consumption and free fatty acid utilization, whereas tricular performance. The diminished anaerobic per- glucose uptake and oxidation are decreased (10, 11). formance of hyperthyroid hearts was associated with In view of the recent demonstration in our laboratory decreased adenosine triphosphate (ATP) levels and a of the importance of glucose metabolism in preserving reduced rate of anaerobic glycolysis as reflected in de- structure and function of the anoxic heart (12) and the creased lactic acid production during 30 min of anoxic reported inhibition of glucose metabolism in hyperthy- perfusion. roid hearts (6), it seemed desirable to determine whether Studies on whole heart homogenates demonstrated in- anaerobic metabolism is altered in the intact hearts hibition at the phosphofructokinase (PFK) step of the from hyperthyroid animals and to ascertain what effects glycolytic pathway. Such inhibition was not demonstrated these metabolic alterations have on anoxic performance in the hyperthyroid heart cytosol. It is postulated that of the heart. an inhibitor of PFK which resides dominantly in the particulate fraction is probably responsible for the di- METHODS minished anaerobic glvcolvsis and performance of the Male Wistar rats fed ad lib. with Purina laboratory chow hyperthyroid heart. were selected for study. The experiments were so designed to permit matching of both heart weight and body weight of euthyroid and hyperthyroid animals. In one series (matched INTRODUCTION heart weight) rats weighing 160-180 g were made hyper- The effect of thyroxine administration on oxidative me- thyroid with seven daily injections of 0.2 mg of sodium L-thyroxine dissolved in 0.25 ml of 0.01 N NaOH. At the tabolism has received considerable attention in recent time of sacrifice, mean body weight was 180 g (range 149- years. Many metabolic consequences of hyperthyroidism 205 g), while the mean body weight of the euthyroid control have been attributed to such alterations in oxidative animals was 250 g (220-280 g). The heart weights of the metabolism as reduced mitochondrial respiratory con- two groups did not differ significantly. Mean wet heart weight of the control rats was 1.20 ±0.15 g (range 0.94-1.56 trol (1, 2) and increased levels of several oxidative g) compared with 1.11 +0.15 g (0.82-1.47 g) for the hyper- enzymes (3, 4). Less attention has been paid to the thyroid animals. These hearts were used in both perfusion and effects of hyperthyroidism on anaerobic glycolytic me- homogenate studies. tabolism. Glock, McLean, and Whitehead have reported In a second series (body weight matched) rats weighing that liver is 125-150 g were injected daily for 7 days with 0.25 ml of glycolysis stimulated by thyroxine treat- 0.01 N NaOH, while rats weighing 135-160 g were injected ment (5), while Bressler and WNittles have reported de- daily with 0.2 mg of sodium L-thyroxine dissolved in 0.25 ml creased aerobic glycolysis in homogenates of hyper- of 0.01 N NaOH. At the time of sacrifice mean body weight thyroid guinea pig hearts (6). of the sodium hydroxide-injected group was 154 +5.1 g heart is extremely sensitive to thy- (SEM) compared with 155 +5.8 g for the thyroxine injected The mammalian group. Mean heart weight of the sodium hydroxide-injected roxine administration. Myocardial hypertrophy, increased group was 0.73 ±0.025 g (SEM) compared with 0.99 ± 0.029 g heart rate, and increased mvocardial contractility are for the thyroxine injected animals. These hearts were used only in the studies on homogenate glycolysis. Received for publication '8 April 1969 and in revised Perfused hearts. In the studies on the perfused hearts, forin 13 June 1969. rats from the first series (matched heart weights) were The Journal of Clinical Investigation Volume 48 1969 1905 used. In this manner, constancy of perfusion per unit weight mg of homogenate protein or 0.5 mg of cytosol protein per ml. of tissue was maintained. Hearts were perfused at constant Each tissue preparation was incubated in duplicate with rate in a modified Langendorf apparatus which permitted four different substrate preparations; glucose, glucose plus constant monitoring of the electrocardiogram, left ventricular 0.1 mg/ml of type III yeast hexokinase, glucose-6-phosphate, pressure (LVP), the first derivative of left ventricular pres- and fructose-1,6-diphosphate. sure (LV dp/dt), and oxygen consumption (QO2) (12). Samples were withdrawn at 0, 15, and 30 min of incubation The maximum rate of left ventricular rise was calculated and immediately deproteinized. Samples for determination of from the recording of LV dp/dt. The hearts performed lactic acid or phosphorylated compounds were added to an isovolumically. The perfusion fluid was 5% bovine serum equal volume of chilled perchloric acid (0.6 mole/liter), albumin (BSA) which had been dialyzed against and diluted mixed, and centrifuged at 3000 g for 15 mm in a refrigerated with Krebs-Ringer bicarbonate buffer (KRB) (13). The centrifuge. KRB was modified to contain 5.0 mg of calcium per 100 ml. Homogenate ATPase activity was measured continuously Coronary flow was maintained at 10 ml/min, and the with a Gilford recorder attached to a Beckman DU temperature was 320C. spectrophotometer. The composition of the assay mixture Aerobic control hearts. The first group of hearts (eight was 0.5 M Tris-acetate, pH 7.4, 3 mm MgC12, 5 mM phospho- euthyroid and seven hyperthyroid) was perfused for 60 min enolpyruvic acid, 5 mm adenosine triphosphate (ATP), and with 5%o BSA in KRB which had been gassed with 96%o 02, 0.15 mM nicotinamide adenine dinucleotide, reduced form 4%o CO2 (pH 7.40). The aerobic perfusion fluid contained no (NADH) (17). The assay mixture also contained 2 jsg/ml glucose. These hearts were rinsed for 1 min with oxygenated of lactic dehydrogenase and 10 ,ig/ml of pyruvic kinase. The KRB after the 60 min perfusion period and were clamped reaction was started by adding 50 pi of homogenate. Hydroly- between liquid nitrogen-cooled Wollenberger tongs (14). sis of ATP generated ADP which was then available to Anoxic hearts. The second group of hearts (six euthyroid react with phosphoenolpyruvic acid in the presence of and six hyperthyroid) was perfused aerobically without pyruvic kinase regenerating ATP and liberating pyruvic glucose for 1 hr and then anaerobically with fluid containing acid. The pyruvate was converted to lactate, and the NADH 200 mg of glucose per 100 ml (11.1 mm glucose) for 30 min. consumed was proportional to the amount of ATP hydro- The anaerobic perfusion fluid was gassed with 96% N2, 4% lyzed. The disappearance of NADH was measured by CO2. After the anaerobic perfusion period the hearts were recording the change in optical density at 340 mA. rinsed for 1 min with anaerobic KRB containing 200 mg Anialysis of metabolic intermediates. Each frozen heart of glucose per 100 ml and were clamped between liquid was ground to a fine powder under liquid nitrogen in a nitrogen-cooled tongs. nitrogen-cooled mortar and pestle. The frozen powder was Recovery hearts. The third group of hearts (six euthyroid gradually added to a tared tube containing 2.5 ml of 0.6 M and six hyperthyroid) was perfused aerobically without glu- perchloric acid. The tubes were well mixed after each addi- cose for 1 hr, anaerobically with glucose for 30 min, and tion of approximately 50 mg of heart so that each particle then aerobically without glucose for an additional 30 min. was in contact with the perchloric acid as it thawed. The The hearts were rinsed for 1 min with oxygenated KRB tubes were then weighed, and sufficient perchloric acid was and clamped between liquid nitrogen-cooled tongs. The added to give a ratio of volume of extract to tissue weight of frozen hearts were stored in liquid nitrogen until analysis of 4: 1. (Total perchloric acid = 3.25 X tissue weight in grams, heart metabolic intermediates. assuming the water content of heart tissue is 75%o (18). The Lactic acid content of the perfusing fluid was determined samples were well mixed and centrifuged at 5000 g for by the enzymatic technique of Scholz, Schmidt, Bitcher, and 10 min at 2°C. Lampen (15). Total lactate production was calculated from Equal volumes of perchloric acid extract (whole heart the concentration and volume of the perfusing medium. or homogenate) and 0.4 M triethanolamine buffer, pH 7.6, Homogenate studies. Three hyperthyroid and three euthy- to which sufficient K2CO3 had been added to bring the final roid rats were used for each of the 25 heart homogenate concentration to 0.55 M K2CO3 were combined, mixed studies. The animals were killed by decapitation, and the and allowed to stand in the cold for 10 min. Aliquots of the thoracic cavity was opened and immediately flooded with clear supernatant fluid were used immediately for the deter- iced 0.154 M KCl. The hearts were removed to iced KCl and mination of metabolic intermediates. Glucose-6-phosphate and flushed via the aortic stump with KCl to remove blood. The fructose-6-phosphate were measured enzymatically according hearts were then blotted, weighed and forced through a to the method of Hohorst (19). Fructose-1,6-diphosphate and stainless steel tissue press.
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