Effects of Glucose Metabolism on the Transmembrane Action Potential and Contraction of Human Papillary Muscle During Surgical Anoxia

Transcription

1 Effects of Glucose Metabolism on the Transmembrane Action Potential and Contraction of Human Papillary Muscle During Surgical Anoxia K. Prasad, M.D., and John C. Callaghan, M.D. A noxia has been reported to produce a shortening of the action potential duration (APD) and a decrease in the force of contraction of the guinea pig [ 171 and human [ 181 heart. Metabolic inhibitors (iodoacetate, 2,4-dinitrophenol, and sodium cyanide) and substrate-free medium have been reported to produce effects similar to anoxia on the APD and force of contraction of guinea pig [17] and human [18, 191 papillary muscles. Reduction in the APD due to anoxia and metabolic inhibitors has been reported to be caused by an increased efflux of potassium during activity [ 17, 241, because repolarization has been assumed to be caused by efflux of potassium [lo]. It seems that efflux of potassium and, hence, shortening of the APD are inversely related to the availability of the energy in the cells. Also, the contraction of the muscle is dependent upon the availability of energy. Myocardial ischemia [9, 221 and substrate-free Tyrode solution [16] have been reported to produce ventricular fibrillation in the dog and guinea pig, respectively. Efflux of potassium during ischemia has been implicated in the production of cardiac arrhythmias in dog [9, 221. During open-heart surgery, cardiac muscle is relatively hypoxic, and the patient is prone to develop cardiac arrhythmias. During this hypoxic state, the muscle activity will also be reduced. If an inverse relation between the shortening of the APD and, hence, the efflux of potassium and the energy production exists, high concentrations of glucose or insulin, or both, which increase glycolysis, should be able to counteract the effect of anoxia on the transmembrane action potential (AP) and contraction of the human papillary muscle. They should be able to From the Departments of Cardiovascular and Thoracic Surgery and Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada. Supported by Alberta Heart Foundation Grant No , and Canadian Heart Foundation Fellowship. Presented at the Fifth Annual Meeting of The Society of Thoracic Surgeons, San Diego, Calif., Jan , Address reprint requests to Dr. Callaghan, Division of Cardiovascular and Thoracic Surgery, University of Alberta, Edmonton, Alberta, Canada. VOL. 7, NO. 6, JUNE,

2 PRASAD AND CALLAGHAN prevent the occurrence of cardiac arrhythmias by preventing the shortening of the APD during anoxia. No study has been done on the electrical and mechanical activity of the human heart during anoxia in the absence or presence of glucose and insulin. The present paper deals with a study of the ability of various concentrations of glucose and insulin in counteracting the effects of anoxia on the transmembrane potential and contraction of human papillary muscle obtained from patients undergoing corrective open-heart surgery. MATERIALS AND METHODS The experiments were carried out on papillary muscles obtained from patients between 20 and 60 years of age undergoing corrective open-heart surgery. Immediately after excision, the muscle was transferred to a chilled Krebs-Ringer solution and removed to the laboratory for experiment. The muscle strips (3 to 4 mm. long, 1 to 2 mm. wide, and 0.5 to 0.75 mm. thick) were prepared in such a way that the fiber bundle would run parallel to its length. The muscles were fixed horizontally at a resting tension of 2 to 2.5 gm. in jacketed 100 ml. constant-temperature bath (37 C.) containing Krebs-Ringer solution of the following composition (milliequivalents per liter): Na, 138.5; K, 4.16; Ca, 4.9; Mg, 2.3; HC03, 21.91; PO.,, 3.48; C1, 125; and glucose, 20 mm. Henceforth, this solution will be called normal solution. The solution was equilibrated with 95% oxygen and 5% carbon dioxide. The muscles were stimulated with a stimulus of 2 to 10 volts of 5 msec. duration at the rate of 60 per minute by a Grass stimulator through platinum electrodes placed at one end of the muscle. Simultaneous recording of the transmembrane action potential (AP) and contraction were made on an Offner Dynograph, displayed on a 502A oscilloscope, and recorded on Polaroid film. Single-cell electrical activity was recorded by means of glass microelectrodes filled with 3M KCl, using the floating electrode technique of Woodbury and Brady [251. Potential measurements were made through a Medistor negative capacitance electronmeter, monitored on a Tektronix 502 oscilloscope, and recorded on an Offner Dynograph. Force of contraction was recorded by means of a Statham force displacement transducer. The muscles were equilibrated in normal Krebs-Ringer solution for three to four hours before the normal electrical and mechanical activity were recorded. Muscles were made anoxic by bubbling a gas mixture of 95% N2 and 5% CO, through the solution in the bath containing the muscle. Action potential duration at 50% level of repolarization and contraction of the muscles were measured as shown in Figure 1. R ES UL TS EFFECTS OF ANOXIA IN GLUCOSE-FREE SOLUTION After three hours of incubation in oxygenated normal Krebs-Ringer solution, 10 muscle strips were exposed to glucose-free Krebs-Ringer solution equilibrated with 95% N, : 5% C02 for a period of 90 minutes. Figures 2 and 3 show the changes in the APD and contraction of the muscles produced by anoxia in glucose-free solution. The most consistent early change in all the muscles was a decrease in the force of contraction, which appeared within 10 to 15 minutes after their exposure to anoxia and glucose-free solution. Fifteen to 20 minutes later, that is, 25 to 35 minutes after exposure to anoxia and glucose-free solution, the changes in the transmembrane action potential were also observed. The decrease in the 572 THE ANNALS OF THORACIC SURGERY

3 Glucose Metabolism During Surgical Anoxia miec FIG. 1. The transmembrane action potential (lower record) and contraction (upper record) of human papillary muscle. 0 = zero leuel of resting potential. Calibrations for magnitude of action potential in milliuolts (mu.), action potential duration (APD) in millisecond (msec.), and muscle tension in gram (g) are indicated in the figure. The measurement of APD at 50% leuel of repolarization is shown. A R 30 min - 100mrec ' CONTROL GLUCOSE - FREE + ANOXIA FIG. 2. Efltfect of glucose-free Krebs-Ringer solution and anoxia on the simultaneously recorded transmembrane action potential and contraction of human papillary muscle. (A) Control in oxygenated Krebs-Ringer solution containing 20 mm. glucose. Arrow marks the start of the exposure of the muscle to glucose-free solution and anoxia. The subsequent tracings (B, C, D) are in glucose-free solution and anoxia at interuals shown at the top of each tracing. Calibration is indicated. Note the shortening of the APD and contraction in glucose-free solution and anoxia. contractile force progressed with time until 90 minutes (time p-riod of observation), when it was reduced to 5 2 7% of the control value in oxygenated Krebs- Ringer solution containing 20 mm. glucose. The consistent early change in the transmembrane action potential was a shortening of the APD. This shortening of the APD progressed slowly with time, and at the end of 90 minutes it was reduced to % of the control value in oxygenated normal solution. The shortening of the APD was not associated with any reduction in the overshoot or resting potential for approximately 40 to 50 minutes. Later on, a reduction in the overshoot and resting potential were also observed (Fig. 2C and 2D). It was observed that shortening of the APD was associated with a decrease in the force of contraction. The decrease in the force of contraction was always greater than the shortening of the APD. EFFECTS OF ANOXIA IN PRESENCE OF VARIOUS CONCENTRATIONS OF GLUCOSE Papillary muscle strips, after equilibration in oxygenated normal Krebs- Ringer solution, were exposed to Krebs-Ringer solution containing 0, 5, 20, or VOL. 7, NO. 6, JCWE,

4 PRASAD AND CALLAGHAN %CHANGE I MINUTES FIG. 3. The effects of uarious concentrations of glucose in the presence of anoxia on the action potential duration (APD) and contraction of human papillary muscle with respect to time. The values are expressed as percentage change from the control in 20 mm. glucose, with 0, taken as 100%. Note the marked reduction in the APD and contraction in the absence of glucose, as compared to less marked changes in the presence of glucose. Vertical bars represent standard error. 30 mm. glucose equilibrated in 95% N, : 5% CO, for a period of 90 minutes. Effects of each concentration of glucose were investigated in 10 muscle strips. Figure 3 shows the changes in the APD and contraction produced by such treatments. The changes in the APD and contraction with various concentrations of glucose were qualitatively similar to those observed in 0 mm. glucose, but there were quantitative differences. It will be evident from Figure 3 that there was a direct relation between the decrease in the force of contraction and APD on the one hand and the decrease in the glucose concentration in the solution on the other. It will also be observed that in 30 mm. glucose, although there was a moderate decrease in the force of contraction, there was an insignificant shortening of the APD at the end of 90 minutes. EFFECTS OF GLUCOSE ON THE ANOXIA-INDUCED CHANGES IN THE APD AND THE CONTRACTION IN GLUCOSE-FREE SOLUTION After equilibration in oxygenated normal Krebs-Ringer solution, muscle strips were exposed to glucose-free Krebs-Ringer solution and equilibrated with 95% N, : 5% CO, for 90 minutes. Glucose solution was then added to the bath to make the final bath concentration of glucose 20 or 30 mm., and the effects on action potential and contraction were observed for 30 minutes. A representative recording of the changes in the action potential and contraction from a single muscle with such treatments is shown in Figure 4. Anoxia in glucose-free Krebs-Ringer 574 THE ANNALS OF THORACIC SURGERY

5 Glucose Metabolism During Surgical Anoxia A R 90 min 30min n F 90 min F 30 mtn GLUCOSE -FREE GLUCOSE (30mM) CONTROL +ANOXIA +ANOXIA FIG. 4. Etfects of various concentrations of glucose on the simultaneously recorded transmembrane action potential and contraction of human papillary muscle from a single experiment. (A, D) Control in oxygenated Krebs-Ringer solution containing 20 mm. glucose; (B, E) after 90 minutes in glucose-free solution and anoxia; (C, F) after 30 minutes in 20 and 30 mm. glucose, respectively, under anoxia. Note the reversal of the effect of glucose-free solution and anoxia on the APD and contraction by glucose. Ca!ibration is indicated. solution produced a marked shortening of the action potential duration and a marked decrease in the force of contraction at the end of 90 minutes. There was a small decrease in the overshoot and resting potential, also. Addition of glucose (20 mm.) in the bath produced a marked increase in the APD and contraction in 6 muscles. Addition of 30 mm. glucose in the bath produced increases in APD and contraction in 6 muscles similar to but greater than those produced by 20 mm. glucose. These concentrations of glucose increased the overshoot and resting potential, also. It was also observed that APD and contraction in 20 mm. glucose and oxygen (Fig. 4A) were greater than those in 20 mm. glucose in the absence of oxygen (Fig. 4C). EFFECTS OF INSULIN Experiments were carried out in which the muscle strips, after incubation in oxygenated normal Krebs-Ringer solution, were exposed to g;ucose-free Krebs- Ringer solution equilibrated with 95% N, : 5% CO, for a period of 90 minutes. Insulin was then added to the bath in concentrations of 0.2, 1, 2, and 5 mu. per milliliter and the effects observed for 30 minutes. Five experiments were conducted with each concentration of insulin. It was observed that none of the concentrations of insulin was able to increase the anoxia-induced reduction in the APD and contraction in glucose-free solution. A representative recording of the changes in the APD and contraction from a single muscle with such treatments is shown in Figure 5. Some experiments were conducted in which the effects of insulin in the presence of glucose (20 mm.) were studied. After 90 minutes of exposure of the muscles to anoxia and glucose-free solution, the muscles were exposed to Krebs- Ringer solution containing 20 mm. glucose and anoxia for 30 minutes. Insulin was VOL. 7, NO. 6, JUNE,

6 PRASAD AND CALLAGHAN A B 90 min c 30 min mec 7 GLUCOSE -FREE ' GLUCOSE-FREE + ANOXIA CONTROL + ANOXIA * + INSULIN (ImU/mi) FIG. 5. Etfect of insulin on the simultaneously recorded transmembrane potential and contraction of muscle in the absence of glucose and anoxia (from a single experiment). (A) Control in oxygenated Krebs-Ringer solution containing 20 mm. glucose; (B) after 90 minutes in glucose-free solution and anoxia; (C) after 30 minutes in insulin (I mu. per milliliter) in the presence of glucose-free solution and anoxia. Note that insulin was unable to increase the APD and contraction. Calibration is shown. A p 90 nln r 30 nln - 1 GLUCOSE- FREE t GLUCOSE (20rnMI t GLUCOSE l20rnwi I00 nsw I +ANOXIA 2 tanoxi4 3 +4NOXI4+ INSULIN1 ImU/mll CONTROL FIG. 6. The eflect of insulin on APD and contraction of the muscle in the presence of glucose and anoxia. (A) Control tracing in oxygenated Krebs-Ringer solution containing 20 mm. glucose (at arrow 1, the muscle was exposed to glucosefree solution and anoxia); (B) 90 minutes later (at arrow 2, glucose was added in the bath to make a concentration of 20 mm.); (C) 30 minutes later (at arrow 3, insulin was added in the bath); (D) 30 minutes later. Note the increase in APD and contraction with insulin in the presence of glucose and anoxia. then added in the bath to make the final concentration of 0.5, 1, or 2 mu. per milliliter, and the effects were observed for 30 minutes. Five experiments were carried out with each concentration of insulin. Insulin in all the concentrations increased the force of contraction and APD in the presence of glucose. A representative of the tracing with such treatments is shown in Figure 6. COMMENT Our results have indicated that during anoxia the maintenance of transmembrane potential and contraction of human papillary muscle near normal levels depends upon the glycolysis, because glucose or glucose and insulin were necessary for the maintenance of these activities. There is only one available study in the literature regarding the shortening of the action potential duration and decrease in the force of contraction of human papillary muscle during anoxia in the absence or presence of low glucose [18]. However, the literature regarding such 576 THE ANNALS OF THORACIC SURGERY

7 Glucose Metabolism During Surgical Anoxia effects of anoxia on the heart muscle of various other animal species in substrate-free or low glucose media is plentiful [ll, 17, 20, 24, 261. Our results show that glucose could prevent or abolish the effects of anoxia on the APD and contraction of human papillary muscles under anoxic conditions. Although insulin in the presence of glucose was effective in increasing the action potential duration and contraction of the anoxic papillary muscles, it was ineffective in the absence of glucose. No literature is available regarding the effects of glucose or glucose and insulin on the simultaneously recorded transmembrane potential and contraction of human papillary muscles under anoxic conditions. However, there are some reports regarding the effect of glucose on the transmembrane potential and contraction of guinea pig and cat papillary muscles under anoxic conditions [ 1 1, 17,201. These reports indicate that the anoxia-induced reduction in action potential duration and contraction could be reversed or prevented by increasing the glucose concentrations in the medium. They reported, however, that glucose was less effective in preventing or reversing the effect of anoxia on the contraction, as compared to its effects on APD. Effects of insulin on the transmembrane potential and contraction of anoxic guinea pig heart have been reported to be variable [17]. Sometimes there was an increase and other times either there was no effect or a slight decrease in the APD and contraction. In our preparation there was always an increase in the APD and contraction. These variations in results might be due to differences in the animal species. The shortening of the action potential duration and decrease in the force of contraction in glucose-free or low glucose solution under anoxia can be explained by the reduced availability of aerobic energy. Under anoxic conditions the energy required for the action potential and contraction must come from stored glycogen, from adenosine triphosphate (ATP), or from the anaerobic metabolism of glucose. It has been reported that during myocardial ischemia there is a decrease in the glycogen, ATP, and creatine phosphate (CP) content of the heart [5, 7, 121. It is known that during anoxia there is an increase in the phosphorylase activity [2], which, in turn, increases the conversion of glycogen to hexose substrate for glycolysis. In the absence of glucose, the energy is derived mainly from glycogen during anoxia. This amount of energy may not be sufficient for maintaining the normal transmembrane potential and contraction of the heart for long periods. Under anaerobic conditions at low pressure-volume work loads, the heart uses glycogen stores rather than exogenous glucose to provide hexose substrate for glycolysis. Increase of work load has been reported to be associated with the utilization of exogenous glucose or, in the absence of glucose, with mechanical failure of the heart [2 11. During anaerobic glucose metabolism, four molecules of ATP are formed from one mole- VCL. 7, NO. 6, JUNE,

8 PRASAD AND CALLAGHAN cule of glucose, and since two molecules of ATP are used up during glycolysis, the net yield of ATP is only two molecules. This ATP formation accounts for an energy liberation in glycolysis of only 9% of the energy content of the glucose. Thus, it seems that under anoxic conditions, when glycogen and ATP stores are depleted, low glucose is not providing enough energy to maintain the APD and contraction at normal or near normal. Hypoxia is known to produce a net loss of potassium in the heart muscle [3, 4, 223. Lack of substrate might also increase the loss of potassium from the cell because substrate is required to supply the energy for the reentry of the potassium into the cell [23]. Thus, it seems that during anoxia or lack of substrates, the shortening of the action potential duration is related to an increased efflux of potassium. The excitability of the heart might increase during anoxia due to a decrease in the absolute refractory period of the muscle, since the absolute refractory period is dependent upon the action potential duration [8]. An increased excitability of the heart might lead to a ventricular ectopic beat and ventricular fibrillation. In our experiments, glucose not only prevented but also abolished the effects of anoxia on the APD and contraction of the muscle strips, and these effects of glucose were concentration dependent. This result is expected, because the uptake of glucose in the heart is a function of extracellular glucose [13] and an increased uptake of glucose will provide an increased amount of energy through glycolysis. Also, certain enzymes of the glycolytic pathway of glucose metabolism are stimulated during anoxia [14]. These factors will lead to production of enough energy to maintain APD by decreasing the efflux of potassium and to maintain contraction during anoxia, provided that enough glucose is present in the extracellular fluid. Insulin in the absence of glucose was unable to increase the anoxiainduced reduction in APD and contraction, but in the presence of glucose it was quite effective. These findings can be explained as follows. Insulin is known to increase the transport of glucose, synthesis of glycogen, and glycolysis [l, 61. Ineffectiveness of insulin in the absence of glucose might be due to lack of glucose-substrate, because glycolysis and synthesis of glycogen are dependent upon glucose-substrate. Effectiveness of insulin in the presence of glucose might be due to (1) an increased glycogen synthesis as a result of insulin and anoxia-mediated increased glucose uptake, and as a result of stimulation of enzymes of glycogen synthesis (increased glycogen, in turn, will provide substrate for glycolysis); (2) an increased glycolysis as a result of increased glucose uptake and stimulation of certain glycolytic enzymes by insulin itself [l]. Thus, it seems that during anoxia, glucose or glucose and insulin might provide enough energy to reduce the efflux of potassium during 578 THE ANNALS OF THORACIC SURGERY

9 Glucose Metabolism During Surgical Anoxia activity and thus maintain the normal APD. A decreased efflux of potassium from the heart during hyperglycemia under anoxic conditions has been reported by Regan et al. [22]. The increase in the APD or the maintenance of the normal APD during anoxia would increase the refractory period and hence reduce the chances of cardiac arrhythmias. In support of this are the results by Penna and associates [16], who found that arrhythmias produced by lack of substrates can be abolished by administration of glucose. However, Penna et al. [15] reported that ATP was not quite effective in abolishing the arrhythmia produced by lack of substrate. This might be due to inability of ATP to enter the cell, and ATP cannot supply energy when it is outside the cell. On the basis of these results in the human heart, it is suggested that addition of sufficient glucose or glucose and insulin to the priming fluid used in the cardiopulmonary bypass and coronary arteries perfusion might prevent or reduce the chances of occurrence of cardiac arrhythmias and might maintain the functional integrity of the myocardial cell during open-heart surgery. SUMMARY Studies of glucose (0, 5, 20, 30 mm.) and insulin (0.5 to 5 mu. per milliliter) on the anoxia-induced changes on the simultaneously recorded transmembrane potential and contraction of human papillary muscle excised during open-heart surgery were made. Anoxia produced a marked reduction in the action potential duration and force of contraction in 0 mm. glucose. Glucose or glucose and insulin prevented and abolished the effects of anoxia on the action potential duration and contraction. These effects of glucose were concentration dependent. It is suggested that addition of sufficient glucose or glucose and insulin to the priming fluid might prevent the occurrence of cardiac arrhythmias and maintain the integrity of the cardiac muscle during open-heart surgery. REFERENCES Bessman, S. P. A molecular basis of the mechanism of insulin action. Amer. J. Med. 40:740, Buis, B., and Lacroix, E. The phosphorylase activity of the heart in acute hypoxia. Arch. Znt. Physiol. 73:387, Cherbakoff, A., Toyama, S., and Hamilton, W. F. Relation between coronary sinus plasma potassium and cardiac arrhythmia. Circ. Res. 5:517, Conn, H. L., Jr. Effects of digitalis and hypoxia on potassium transfer and distribution in the dog heart. Amer. J. Physiol. 184:548, Danforth, W. H., Naegle, S., and Bing, R. J. Effect of ischemia and reoxygeneration on glycolytic reactions and adenosinetriphosphate in heart muscle. Circ. Res. 8:965, Ellis, S. The physiological interactions of epinephrine and insulin on metabolism. Zsrael J. Med. Sci. 2:673, VOL. 7, NO. 6, JUNE,

10 PRASAD AND CALLAGHAN 7. Furchgott, R. F., and de Gubareff, T. The high energy phosphate content of cardiac muscle under various experimental conditions which alter contractile strength. J. Pharmacol. Exp. Ther. 124:205, Ganong, W. F. Review of Medical Physiology (2nd ed.). Los Altos, Calif.: Lange, P Harris, A. S., Bisteni, A., Russel, R. A., Brigham, J. C., and Firestone, J. E. Excitatory factors in ventricular tachycardia resulting from myocardial ischemia. Potassium a major excitant. Science 119:200, Hoffman, B. F., and Cranefield, P. F. Electrophysiology of the Heart. New York: McGraw-Hill, P MacLeod, D. P., and Daniel, E. E. Influence of glucose on the transmembrane action potential of anoxic papillary muscle. J. Gen. Physiol. 48:887, Michal, G., Naegle, S., Danforth, W. H., Ballard, F. B., and Bing, R. J. Metabolic changes in heart muscle during anoxia. Amer. J. Physiol. 197: 1147, Morgan, H. E., Neely, J. R., Wood, R. E., Liebecq, C., Liebermeister, H., and Park, C. R. Factors affecting glucose transport in heart muscle and erythrocytes. Fed. Proc. 24: 1040, Morgan, H. E., and Randle, P. J. Regulation of glucose uptake by muscle: 111. Effects of insulin, anoxia, salicylate, 2,4-dinitrophenol on membrane transport and intracellular phosphorylation of glucose in the isolated rat heart. Biochem. J. 73:573, Penna, M., Illanes, A., and Pupkin, M. Effects of adenosinetriphosphate and potassium chloride on ventricular fibrillation induced by lack of substrates. Circ. Res. 10:642, Penna, M., Illanes, A., Rivera, J., and Mardones, J. Electrogram changes induced by the lack of metabolites on the isolated guinea pig heart. AC~Q Physiol. Lat. Amer. 7: 110, Prasad, K. Glucose Metabolism and Transmembrane Electrical Activity of Cardiac Ventricular Muscle. University of Alberta Ph.D. Thesis, Prasad, K. Substrate-dependent effects of ouabain on the transmembrane action potentials and contractions of human heart in-vitro. Pharmacologist 10: 186, Prasad, K. Further studies on the substrate-dependent effects of ouabain on the transmembrane action potentials and contractions of human heart invitro. Proc. Canad. Cardiovasc. SOC. 21:6, Prasad, K., and MacLeod, D. P. Agents affecting the action of glucose on the electrical activity of anoxic papillary muscle. Proc. Canad. Fed. Biol. SOC. 9:44, Reeves, R. B. Control of glycogen utilization and glucose uptake in the anaerobic turtle heart. Amer. J. Physiol. 205:23, Regan, T. J., Harman, M. A., Lehan, P. H., Burke, W. M., and Oldewurtel, H. A. Ventricular arrhythmias and K+ transfer during myocardial ischemia and intervention with procaine amide, insulin or glucose solution. J. Clin. Invest. 46:1657, Skou, J. C. Relationship of ATP metabolism to ion transport. Proceedings of the International Congress of Physiological Sciences. IV, 578, Trautwein, W., and Dudel, J. Aktionpotential und Kontraktion des Herzmuskels im Sauerstoffmangel. Pflueger. Arch. Ges. Physiol. 263:23, Woodbury, J. W., and Brady, A. J. Intracellular recording from moving tissues with a flexibly mounted ultramicroelectrode. Science 123: 100, Yang, W. C. Anaerobic functional activity of isolated rabbit atria. Amer. J. Physiol. 205:781, THE ANNALS OF THORACIC SURGERY

11 Glucose Metabolism During Surgical Anoxia DISCUSSION DR. A. GERSON GREENBURG (Chicago, Ill.): This excellent paper is a fine example of how biochemical and physiological information can answer clinical problems and problems with technique. The authors have shown that human capillary muscle during anoxia exhibits loss of contractility and shortening of action potential. They also suggest a mechanism by which the arrhythmias and loss of ventricular function seen by many of us after anoxia can be explained by a very sophisticated and highly accurate method. The results achieved with this method confirm results obtained in our laboratory in 1965 using an in vivo preparation in which dogs on cardiopulmonary bypass were given coronary perfusates with high levels of glucose and then rendered ischemic at varying temperatures. Ventricular function studies, contractility studies using the Walton-Brody strain gauge, and biochemical studies were performed. These contractilities showed that without glucose 30 minutes of ischemia gave about 9% of control values, closely paralleling Dr. Prasads 5% of contractility in human papillary muscles. Ventricular function studies showed similar protection with high glucose perfusate. In addition, coronary sinus blood studies revealed very marked increases in potassium loss without protection of glucose, corroborating the efflux of potassium during anoxia. Our in vivo experiments, using much less accurate measurements than the present essayists, showed that perfusion of the coronaries with glucose was associated with better myocardial performance at all temperatures studied. In addition to these data I would like to offer other evidence that ventricular function is protected with glucose in the incubation medium. Gord and Fredrickson showed, in a very excellent paper, that when glucose is presented to the heart in high concentration, if glucose is preferentially metabolized, free fatty acids and other lactic acids are not used. They also showed that when the heart is incubated anaerobically without glucose, the myocardium subsequently utilizes glucose impartially under either aerobic or anaerobic conditions. There is thus considerable evidence indicating that glucose should be present when the heart is rendered ischemic, and I think the evidence presented by the essayists does much to support this conclusion. Their paper is significant on a clinical as well as a physiological basis. DR. HERMAN FIELD FROEB (San Diego, Calif.): I found this paper most interesting, and I wish to ask a few questions of Dr. Prasad. If these muscles were studied in a completely anaerobic condition with complete lack of oxygen, were there any studies done in which similar membrane potential and contraction of the muscle were studied under hypoxic conditions? In other words, did you study any critical level of hypoxia or just complete anoxia of the media in which you made your experiments? DR. PRASAD (Alberta, Canada): I have done about a hundred experiments or more giving different pressures of oxygen-30% oxygen and 70% nitrogen and, say, 40% oxygen and 60% nitrogen, 50% oxygen and 50% nitrogen-concentrations of that type, but not in the human heart. I have done such experiments in the guinea pig, and even in a concentration of 40% oxygen and 60% nitrogen there is a reduction in the action potential and, of course, in the contraction. But this takes time, and I have found that maybe after two hours the action potential duration is reduced to about 50% of the control, giving 40% oxygen and 60% nitrogen. VOL. 7, NO. 6, JUNE,