Dichloroacetic acid improves in vitro myocardial function following in vivo endotoxin administration

Dichloroacetic Acid Improves in Vitro Myocardial Function Following in Vivo Endotoxin Administration Alastair H. Burns, M a r y E, Giaimo, and Warren R. S u m m e r

Dichloroacetic acid (DCA), a stimulator of pyruvate dehydrogenase activity, ameliorates lactic acidosis end enhances myocardial function in several clinical shock states. DCA also improves survival in experimental lactic acidosis. Because of the key role played by the heart in the vascular collapse associated with fatal shock, we examined the effects of DCA on myocardial performance in vitro using the isolated working parfused heart preparation. Hearts were obtained from animals receiving a lethal dose for 50% of the group ILD60) six hours of E coil endotoxin or saline vehicle. The presence of DCA in the perfusate significantly elevated stroke work, cardiac output, and peak systolic pressure development in hearts from endotoxin-trsated rats at fitting pres-

A

NUMBER of recent clinical reports have highlighted the role 'of the heart in clinically encountered septic shock states. 1-5 However, the mechanisms by which these metabolic derangements influence myocardial function in septic shock arc poorly understood. Stacpoole and associates reported a palliative effect of dichloroacetic acid (DCA) on lactic acidemia. 1 They noted that the patient's response to DCA included improvement in both acid-base status and myocardial performance. Left unanswered was the question of whether the improvement in myocardial performance was a consequence of the improved acid-base status or the result of a direct metabolic effect of the drug on the heart. The present study was undertaken to determine if DCA had a direct inotropic effect on the metabolically depressed heart. For these studies we utilized a rat model of endotoxemia, which is characterized by low levels of ATP and depressed levels of mechanical function when the organs' intrinsic contractile properties are assessed in vitro using the working perfused heart3 In hearts from both normal and endotoxin-treated rats, DCA resulted in an increase in pyruvate dehydrogenase activity. DCA restored the diminished myocardial levels of ATP in hearts from endotoxin-injected rats and also augmented the reduced cardiac performance. The beneficial effects of DCA in this model were dependent upon the availability of glucose in the perfusate.

Journa/ of Critical Care, Vo! 1, No 1 (March), 1986: pp 11-17

sures from 7.5 to 25 cm of w a t e r . DCA had no significant effect on the mechanical performance of hearts obtained f r o m vehicle injected controls. In t h e absence of glucose, DCA produced a slight but significant reduction in myocardial performance. In both control end endotoxin treated rat hearts, DCA increased t h e activity of pyruvic dehydrogenase by about 200%. DCA did not produce any alteration in ATP levels in the |marts from control animals; h o w ever, after e n d o t o x i n t r e a t m e n t DCA increased myocardial ATP c o n t e n t t o control values. The data show t h a t DGA a u g m e n t s myocardial performRnce in hearts from endotoxin-injected rats, and it restores th,~) depressed ATP c o n c e n t r a t i o n to normal levels. © 1 9 8 6 b y G r i m e & S t r a i t e n , Inc.

MATERIALS A N D METHODS

Adult male Sprague-Dawley rats, each weighing about 300 g, were housed in group cages and had unlimited access to food and water. On the day of each experiment, rats were injected with either a LD~0six-hour ,Jose of E coli endot~xin (Lipopolysaceharid¢ B, from Difco Laboratories, Detroit, Mieh) or saline vehicle while under light ether anesthesia. The animals were returned to their cages and killed three hours later after anesthesia with Nembutol®. The hearts were excised, immersed in cold saline, and mounted by the stump of the aorta on a perfusion apparatus. Langendorff (retrograde aortic) pcrfusion of the coronary arteries began at once. Additional carnulae were placed in the left atrial appendage and the pulmonary artery. The perfusion buffer was KrebsRinger bicarbonate with 0.46 mmol/L palmitate complexed to 3% bovine serum albumin (BSA) (US Biochemical) and 5.5 mmol/L glucose present as substratesfl All buffer reagents were purchased from Sigma Chemical Co, St Louis. in the series of experiments to assess the efficacy of DCA in a glucose free perfusion, only the free fatty aeid/BSA complex was present. Hearts were perfused in the working configuration of Neely et al with myocardial work being increased by elevating preload (left atrial filling height) in increments from 7.5 to 25 cmJ Left atrial pressure was not routinely directly measured. Afterload was held constant. Variables monitored included pressure development and cardiac output (which was determined by simultaneously measuring aortic and coronary flows) and heart rate. Myocardial oxygen Front the Departments o f Physiology and Medicine, Louisiana State University Medical Center, New Orleans. Address reprint requests to Alastair H. Burns. PhD. Department of Physiology, LSU Medical Center, 1901 Perdido St, New Orleans. LA 70112. © 1986 by Grune & Stratton. Inc. 0883-9441/86/0101-0002505.00/0

11

12

BURNS ET AL

consumption was determined by measuring the oxygen partial pressure in the left atrial bubble trap and the pulmonary

acidified with perchloric acid, and the J4CO~ evolved trapped in Hyamine ® after the method o f Anderson and S n y d e r )

artery outflow with two Clarke-type oxygen electrodes connected to a Yellow Springs Oxygen Monitor (Model 53). Oxygen uptake was computed by subtracting these values and multiplying by the appropriate Bunsen Coefficient for oxygen at 37 °C and the coronary flow. Electrodes were calibrated against room air and a gas mixture of known oxygen content. Myocardial performance at different left atrial filling pressures was first estimated using DCA free buffer as perfusate. The hearts were then returned to the initial filling pressure, generally 7.5 cm of water, and switched to a perfusate containing dichloroacetate. Myocardial performance was then recstimated. Hearts were taken rrom either control (N = 5) or endotoxin injected (N ~ 8) rats. In addition, hearts from both control (N = 6) and endotoxin treated (N -~ 6) rats were also perfused with DCA and glucose free perfusate. In the protocols involving measurements of the oxidation of exogenous glucose, a tracer amount of ~4C-U-glucose (New England Nuclear) was also included in the perfusion buffer. Samples of the coronary effluent were obtained from the pulmonary artery cannula without exposure to room air. Five-milliliter aliquots were injectd into 25 mL Erlenmeyer flasks prefitted with a rubber Vacutainer ~ serum tube stopper holding a small plastic cup (Kontes®). The sample was

Glucose oxidation rates were obtained from this value and the specific activity of the perfusate glucose. In those experiments in which myocardial stores of ATP and pyruvate dehydrogenase activity were determined, the hearts were quick frozen between liquid.nitrogen.cooled tongs. The hearts were powdered in a similarly chilled mortar and pestle. Aliquots o f the frozen hearts were weighed and used for subsequent assays. ATP content was determined by the fluorometric procedure of Greengard, 9 and pyruvate dehydrogenase activity was determined according to the method of Mapes and Harris. t° Protein determinations were performed according to the methodology of Lowry) t The data between basal and DCA-treated hearts were statistically analyzed by the paired t-test. In addition, biochemical data for control and endotoxin-treated hearts were compared for statistical significance by means of the t-test. Significance was generally set at the P < .05 level,n

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When hearts from endotoxin-treated rats were perfused in the working configuration, their performance was severely depressed compared with that observed in hearts from vehicle-injected

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Fig 1. Effect of endotoxin and D C A on cardiac o u t p u t and peak systolic pressure d e v e l o p m e n t of isolated working rat hearts. T h e D C A t r e a t m e n t increased peak systolic pressure development (A} and cardiac o u t p u t (B) in t h e hearts from t h e e n d o t o x i n - t r e a t e d group, which was statistically significant at t h e P < .05 level at all filling pressures.

DICHLOROACETATE-INDUCED

INOTROPtSM

13

controls (Fig 1). The defective mechanical performance involved both vcntricular pressure development and cardiac output. The inclusion of4'mmol/L DCA in the perfusate resulted in an enhancement of both of these variables. Peak systolic pressure showed a 15% to 22% increase, which was significant at the P < .05 level at all preloads tested. Cardiac output was also positively affected by DCA, with an increase of 13.4% to 28.2% over the same range of filling pressures. This increase was also significant for each preload (P < .05). Calculated stroke work, the product of the above two variables, was increased from 28% to 50% over the range of preloads (Fig 2). Myocardial work in the presence of DCA, however, did not return to control levels in the hearts from endotoxin-treated rats. In contrast to the uniform stimulation produced by DCA in hearts from endotoxin-injected rats, hearts excised from vehicle-injected controls did not show any statistically significant changes in

any cardiodynamic mcasurements in thc presencc of DCA. The search for a mechanism to explain the inotropic effects of DCA centered on its wellknown primary effect on cellular enzyme systems, the activation of pyruvate dehydrogenase. Assessment of the activity of the enzyme from the hearts of endotoxin-trcated and control rats indicated that perfusion with DCA caused increases of 235% and 200%, respectively, above similar measurements made on hearts that had not been perfused with DCA (Fig 3). The increases in both groups of hearts were highly significant (P < .05). in the hearts from the endotoxin-treated rats, this increase in pyruvate dehydrogenase activity was accompanied by a nearly twofold increase in myocardial content of ATP (P < .05). This increase was large enough to raise levels of ATP in the endotoxin-treated group to levels not significantly different from that of the control. The

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Fig 2. The effect of in vivo e n d o t o x i n and in vitro D C A (4 retool/L) on t h e mechanical performance of isqlated working hearts. T h e presence of DCA in the perfusato increased s t r o k e w o r k at all filling pressures tested (P < .05).

ENDOTOXIN

Fig 3. The effect of in vi t r o D C A on myocardial levels of pyruvete dehydrogenase activity and A T P content in hearts of vehicle and e n d o t o x i n - t r e a t e d rats. DCA t r e a t m e n t increased levels of PDH activity in h e a r t s f r o m both control and andotoxin-treated rats (P < .05). Similarly, ATP levels in t h e hearts from the e n d o t o x i n - t r e e t e d gr oup w e r e significantly elevated {P < .05). DCA did n o t significantly increase ATP levels in hoarts f r o m c o n t r o l animals.

14

BURNS ET AL Table 1. The E f f e c t o f D C A on E x o g e n o u s G l u c o s e O x i d a t i o n and M y o c a r d i a l W o r k by Isolated W o r k i n g Perfuaad H e a r t s F r o m C o n t r o l and E n d o t o x i n - T r e a t e d Rats Control Left Atrial

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% Increase in Myocardial Glucose Oxidation Caused by DCA

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4,647 5,553 6,194 6,154 6,514 5,974

-+ 4 6 9 ± 562 ± 597 ± 570 ± 866 ± 785

22.6 29,5 46.0 40.1 34.6 42.6 Endotoxin

-+ 4.8 ± 7.9 ± 9.3 ± 4.4 _+ 9.6 ± 7.1

4,411 _+ 3 4 8 79,3 _+ 1 . 6 t 4,573 ± 402 94.5 ± 6.1 t 4 . 9 3 7 _+ 699 108.7 ± 11.51" 4,761 ± 845 96.2 ± 7.6~ 4 , 6 2 8 _+ 8 7 2 121.4 _+ 13.O 1" 4 . 2 0 6 ± 860 111.1 ± 13.91" Endotoxin + DCA (4 mmol/L)

233 232 136 140 224 161

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1,429 1,352 1,335 1,433 1,465 1,477

~- 304 ± 198 ± 279 ± 279 ± 393 ± 320

31,2 22.7 28.6 45.5 29.9 34.6

± 7.6 ± 9.0 +- 7,9 ± 7.6 ~_ 6.2 +_. 7.3

2,293 2,528 2.485 2,599 2.369 2,409

184 329 229 117 193 178

± 273 ± 374 ± 397 + 434 +_ 3 7 0 ± 400

88.7 98.6 94.1 99.4 fl4.9 107.6

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"Cardiac oulput × peak systolic pressure. ~Significantly different by paired t-test from rates measured in the absence of DCA (P < .01),

provision of DCA did not alter ATP levels in the hearts from the control rats (Fig 3). The use of exogenous glucose as a substrate for oxidative metabolism increased after DCA treatment (Table 1). In control groups and endotoxin groups, the presence of DCA increased ~4COz production from 14C-U-glucose in excess of 200% over the entire range of preloads tested. In the hearts from the endotoxin-treated group, this increase was accompanied by a doubling of cardiac work as adjudged by the measurement of cardiac output times peak systolic pressure (Table I). By contrast, there was no consistent alteration in the mechanical performance of the control hearts associated with greater glucose substrate utilization. Thus, DCA shifted the

proportion of energy supplied by glucose and palmitate toward glucose. The presence of DCA did not change the slope of the line defined by the relationship between cardiac work (CO × PSP) and oxygen consumption in the control group (Fig 4). The work range of the hearts from the endotoxin-treated rats was significantly increased when DCA was added to the perfusate, but as previously mentioned, was still much less than that performed by hearts from sham injected rats. ln~terestingly, in the presence of DCA, the equations ~-epresenting the relationship of cardiac work and oxygen consumption from control hearts did not significantly differ, despite the overall reduced myocardial performance alter endotoxin.

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the relationship between s t r o k e w o r k and m y o c a r d i a l oxygen consumption during d i f f e r e n t conditions. T h e line d e f i n e d by t h e e n d o t o x i n data p o i n t s differs f r o m t h e o t h e r s w i t h r e g a r d t o both slope and i n t e r c e p t {P < .O5|. A l t h o u g h substantially s h i f t e d t o t h e left, t h e line d e s c r i b e d by e n d o t o x i n plus D C A does n o t d i f f e r f r o m those of control experiments.

D!CHLOROACETA'I'E-INDUCED )NOTROPISM

15

In a final series of experiments, we wished to determine whether the palliative effect of DCA on the hearts from the endotoxin-shocked animals depended on the availability of glucose in the perfusate. For these experiments, the only substrate present in the buffer was 0.46 mm palmitate complexed to 3% bovine serum albumin, As the data in Fig 5 indicate, in a glucosefree perfusate, the presence of 4 m m o l / L DCA had no stimulatory effect on myocardial mechanical performance as adjudged by the product of cardiac output times peak systolic pressure. In fact, in the absence of glucose, DCA had a tendency to further depress myocardial performance, suggesting glucose utilization might be central to the DCA effect. Marked increases in glucose concentration in the perfusate (up to 20 mmol/L) with DCA had only limited additional effects on cardiac performance. DISCUSS'ION

Classically, circulatory shock has been viewed as being primarily a perfusion deficiency. The accumulation of certain metabolites, principally lactate, has been viewed as mere)y a consequence of this inadequate perfusion. In part, because of the accumulation of lactate in the blood, the normal patterns of myocardial substrate oxidation, eg, preference of free fatty acids over carbohydrates, is deranged? J'14 in shock models of a number of differing causes, the heart uses more ET ÷ D C A

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carbohydrates than do control hearts. At least a portion of this enhanced utilization is probably due to increased availability of carbohydrates, primarily lactate. 14 In addition, it takes a smaller initial energy investment to metabolize simple substrates, and their preferential utilization may be energy efficient. The heart normally derives only about onethird of its oxidative requirements from carbohydrate sources. ~s'~ The use and pattern of carbohydrate catabolism are regulated by a number of steps. Kobayashi and Neely, using extremely high concentrations of glucose and insulin in their perfusate, concluded that at near maximal work loads, c a r b o h y d r a t e oxidation could account for about 80% of the heart's oxidative metabolic needs, t7 They concluded that the ultimate rate limiting step in carbohydrate catabolism was the ability of the mitochondrial shuttle systems, principally the malate/aspartate cycle, to remove cytosolic N A D H and restore N A D levels to concentrations suMcient to sustain the production of pyruvate for the Krebs cycle. ~7 These data are consistent with measurements of the maximal rates of activity of the cycle made by Safer and Williamson ~ and Digerness and Reddy. 19 At lower work loads and at physiologic levels of hormones and substrates, other regulatory steps assume prominence, including phosp'flofructokinase, glyceraldehyde 3, phosphate dehydrogenase, which is N A D dependent, and pyruvate dehydrogenase,15 Pyruvate dehydrogenase (PDH) is regulated by a phosphorylation/dephosphorylation cycle catalized by a kinase and a phosphatase, respectively, which are an integral part of the PDH complex. The activity of the enzyme is influenced by a number of factors, such as the redox state of the cell, substrate type, and availability, cellular calcium concentration, and catecholamine stimulationfl ° Dichloroacetate activates PDH by inhibiting the activity of the kinase. 2°'2~Thus, the primary site of action of the DCA is the mitochondrial matrix. A considerable volume of literature chronicles changes in the mitochondrial structure and function during shock, z2 Occurring concurrently, and probably caused by these mitochondrial aberrations, are a series of metabolic abnormalities, including decreased rates of long chain fatly acid oxidation, accumulation of long chain fatty acid esters within the celt, and a metabolic block that inhibits the oxidative degra-

16

dation of pyruvate. 2 This inhibition is reflected by the lower levels of PDH activity measured in the hearts from endotoxin-treated groups. The provision of DCA increased the oxidation of exogenous glucose by a factor of over 3. This finding suggests that in the heart, at least a portion of the restriction of pyruvate use is in the PDH complex activity, and this inhibition can be reversed by DCA. Calcium is a known stimulant of pyruvate dehydrogenase activity, and a decrease intracellular calcium availability could be expected to lower the activity of the enzyme. Recent investigations by Carli et al using a neonatal cardiac myocyte culture system have implicated an endotoxin-induced defect in the myocardial handling of calcium as the basis for some of endotoxin's effects on the heart. 23 The provision of DCA raised A T P levels in the myocardium for the endotoxin-treated animals to levels not significantly different from that of control. Concurrently, the mechanical performance of these hearts improved considerably. A T P depletion is not, however, the entire explanation for the depression caused by endotoxin administration; both cardiac output and ventricular pressure development still were depressed compared with control hearts at similar filling pressures. Thus, even with ATP levels restored to normal by DCA, myocardial mechanical function was still diminished. in addition to its ability to activate pyruvate dehydrogenase, DCA has several other actions within the myocardial cell, including an inhibition of free fatty acid oxidation by a mechanism that appears to be independent of the effect of the compound on pyruvate dehydrogenase activity. 24 In the absence of an alternative substrate, eg glucose, it would be logical to expect that this decrease in oxidative metabolism would be accompanied by a further deterioration in myocardial performance. This was the case in the present studies. Whether other carbohydrate moieties would substitute for glucose was not investigated. If the primary basis of the palliative effect is due to an enhanced oxidation of pyruvate, it would be logical to expect that other metabolites further down the glycolytic pathway, especially pyruvate, could substitute for glucose and might even help cardiac function if additional rate limiting steps are induced by endotoxin. Increased pyruvate concentration has been reported, however, to cause a significant increase in PDH activity that was not linked to inotropism

BURNS ET AL

or myocardial energy utilization, suggesting yet another unexplained effect of DCA on the heart. 24 The effects of DCA may be secondary to improved intraceilular pH as suggested by Stacpoole et al. I Our isolated heart, however, is perfused with a pH neutral solution and should not have persistently high concentrations of intracellular hydrogen ions or lactate. DCA has been used on a trial basis in a number of human pathologic conditions, such as diabetes mellitus, where it has been shown to lower blood sugar, hyperlipidemia of both diabetic and hereditary origins (homozygous familial hypercholesterolemia), and hyperlactatemia. It has also been used in vivo in canine myocardial ischemia studies. 24 In these experiments, DCA reduced the degree of epicardial S-T segment elevation and lactate from the ischemic zone. The effects of the drug in the heart, as in other tissues, would appear to be largely in pyruvate dehydrogenase. Similarly, its effects in iactatemia presumably reside in the activation of the enzyme. In their patient report, Stacpoole et al' observed that cardiac performance improved in minutes after the administration of DCA, prior to the improvement in acid-base status. The authors hypothesized that although acidosis can depress cardiac function, the rapid response in blood pressure suggested an independent inotropic effect reflecting possible improved myocardial energy metabolism, which precedes measurable changes in total body acid-base balance. Park and Arieff found that DCA produced a similar improvement in depressed cardiac indices in two experimental models of lactic acidosis that they believed was not the result of correction in pH. 25 The present report suggests a myocardial metabolic explanation for the improvement in cardiac performance and acid-base status following DCA. DCA directly improves cardiac substrate utilization and A T P production, which then improves myocardial stroke work. The improved pumping capacity of the heart augments perfusion of peripheral organs, particularly skeletal muscle, and enhances oxygen consumption. As a consequence, anaerobic metabolism decreases, the peripheral production of lactate decreases, and acid-base status improves. In addition, DCA works directly on peripheral cells to stimulate lactate removal (oxidation) with secondary increases in bicarbonate formation and a rise in arterial pH. The improve-

DtCHLOROACETATE-IND UCED INOTROPtSM

17

ment in the clinical picture is likely to be an amalgam of direct cellular effects in both cardiac and peripheral tissue and their interaction. The present report of the inotropic action of DCA raises some interesting p6ssibilities for future basic studies as well as possible clinical applications. Normally, the heart relies on free fatty acids for about 70% of its oxidative metabolic substrates. Carbohydrates make up most of the remaining 30%. in spite of this quantitative discrepancy, glucose appears to occupy a special, if as yet undetermined, place within the metabolic schema of the heart. 26 Historically, Bayliss et al noted that their isolated heart-lung preparation was stable for longer periods of time and more mechanically etficient with glucose present

as a substrate. 27 The recent report of improved cardiac function following glucose/insulin/ potassium infusion in some patients with septic shock may be a manifestation of this improved energy availability. However, blood lactate was increased in these studies, suggestiflg a rate limiting step further down the glycolytic pathway. 28 In our studies, DCA augments the utilization of carbohydrate in both the normal and metabolically stressed hearts. From numerous other studies it can be assumed that lactate utilization should also increase at the cellular level. Accompanying this switch to a carbohydrate source based metabolism is an enhancement in myocardial ATP stores and mechanical performance in metabolically depressed hearts.

REFERENCES

1. Stacpoole PW, Harmer EM, Curry SH, et al: Treatment of lactic acidosis with dichloroaeetate. N Engl J Med 309:390-396. 1983 2. Mizock B: Septic shock: A metabolic perspective. Arch Intern Med 144:579-585, 1984 3. Ozier Y, Gueret P, Jardin F, et al: Two dimensional eehocardiographic demonstration of acute myocardial depression in septic shock. Crit Care Med 12:596-599, 1984 4. Parker MM, Shelhamer JH, Bacharach SL, et al: Profound but reversible myocardial depression in patients with septic shock. Ann Intern Med 100:483-490, 1984 5. Carmona RH, Tsao T, Dac M, et al: Myocardial dysfunction in septic shock. Arch Surg 120:30-35, 1985 6. Romanosky AJ, Burns AH, Shepherd RE: In vitro myocardial performance following in vivo administration of E. coil endotoxin. Fed Proc 42:608, 1983 (abstr) 7. Neely JR, Liebermeister H, Battersby EJ, et al: Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 212:804-814, 1967 8. Anderson RE: Snider F: Quantitative collection of t+CO, in the presence of labelled short-chain acids. Anal Biochem 27:211-214, 1969 9. Greengard P: Adenosine 5' triphosphate, in Bergmeyer HU (ed): Methods of Enzymatic Analysis. Orlando, Fla, Academic, 1963, pp 539-55 I I0. Mapes JP, Harris RA: Regulatory functions of pyrurate dehydrogenase and the mitochondrion in lipogenesis. Lipids 10:757-764, 1975 11. Lowry OH, Rosebrough N J, Farr AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275, 1951 12. Steel RGD, Torrie JH: Principles and Procedures of Statistics. New York, McGraw.Hill, 1960 13. Sptizer J J: Myocardial substrate utilization in anaphyiactlc shock. Proc Soe Exp Biol Med 151:28-3 I, 1976 14. Spitzer J J, Bechtel AA, Archer LT, et al: Myocardial substrate utilization of carbohydrate and lipids. Am J Physiol 27:132-136, 1974 15. Neely JR, Morgan HE: Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Annu Rev Physlol 36:413--459, 1974

16+ Neely JR, Rovetto MJ, Oram JF: Myocardial utilization of carbohydrate and lipids. Prog Cardlovasc Dis 15:289329, 1972 17. Kobayashi K, Neely JR: Control of maximum rates of glycotysis in rat cardiac muscle. Cire Res 44: t 66-175, 1979 18. Safer B, Williamson JR: Mitochondrial-cytosolie interaction in perfused rat heart. J Biol Chem 248:25792580, 1973 19. Digerness SB, Reddy WJ: The malate aspartate cycle in heart mitochondria. J Mol Cell Cardiol 8:779-785, 1976 20. Whitehouse S, Cooper RH, Randle PJ: Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids. Biochem J 141:761-774, 1974 21. Mela L, l~inshaw LB, Coalson J J: Correlation of cardiac performance, ultrastructure, morphology and mitochondrial function in endotoxin shock in the dog+ Circ Shock 1:265-272, 1974 22. Carli A, Auclair MC, Verninmen C, et al: Reversal by calcium of rat heart cell dysfunction induced by sera in septic shock. Circ Shock 5:147-157, 1979 23. Latipaa PM, Hiltunen JK, Hassinen IE: Regulation of fatty acid oxidation heart muscle: Effects of pyruvate and dichloroacetate. Biochim Biophys Acta 752:162-171, 1983 24. Mjos OD, Miller NE, Riemersma RA, et al: Effects of dichloroaeetate on myocardial substrate extraction, epicardial ST-segment elevation and ventricular blood flow following coronary artery occlusion in dogs. Cardiovasc Res 10:427-436, 1976 25. Park R, Arieff AI: Treatment of lactate acidosis with dichloroaeetate. J Clin Invest 70:853-862, 1982 26. Opie LH: The glucose hypothesis: Relation to acute myocardial ischemia. J Mol Cell Cardiol 1:107-115, 1970 27. Bayliss LE, Muller EA, Starling EH: The action of insulin and sugar on the respiratory quotient and metabolism of the heart-lung preparation. J Physiol 65:33-47, 1928 28. Bronsveld W, van den Bos GC, Thijs LG: Use of glucose-insulin-potassium (G IK) in human septic shock. Crit Care Med 13:566-570, 1985