An Official Journal of the American Heart Association BRIEF REVIEWS. Cardiac Transmembrane Potentials and Metabolism EDWARD CARMELIET

Circulation Research MAY 1978 VOL. 42 NO. 5 An Official Journal of the American Heart Association BRIEF REVIEWS Cardiac Transmembrane Potentials and Metabolism EDWARD CARMELIET METABOLIC INHIBITION induced by hypoxia or by pharmacological agents is accompanied by pronounced changes in the electrical activity of the heart. In contractile myocardial fibers, the first and most pronounced effect of hypoxia is a shortening of the action potential (Fig. 1 A); later the resting potential, as well as the amplitude of the action potential, is reduced and conduction is impaired.'" 1 The effects are frequency dependent.' 12 In Purkinje fibers, the effects of hypoxia are less pronounced and depend on the external K + concentration. At low external K + (2.7 mm), hypoxia results in a prolongation of the action potential; secondary slow depolarizations may interrupt the repolarization process (Fig. IB) and repolarization may fail completely, resulting in a depolarization to the plateau level, with or without oscillatory behavior.' At 5.4 miu [K + ] o, the disturbances of the repolarization process are less pronounced or absent and, after prolonged hypoxia, the action potential shortens (Fig. 1C). Metabolic inhibition by dinitrophenol (DNP), monoiodoacetate (MIA), azide, and cyanide causes changes similar to those observed during hypoxia. 5 In general, the shortening of the action potential is most pronounced with MIA. Dinitrophenol at a low concentration (2 x 10~ (i M), on the other hand, may prolong the action potential in the frog ventricle" and in sheep Purkinje fibers (Fig. 2, A and B). The effects of ischemia are more complex and less well documented. The electrophysiological analysis of ischemia is hampered by the absence of an in vitro model. Direct microelectrode recording from the center of a myocardial infarction, produced experimentally by ligation of a major coronary artery, shows a fall in resting potential, a decrease in overshoot, and a shortening of the action potential; excitability and conduction velocity are reduced. 7 The same changes can be simulated by superfusion of normal myocardial tissue with "ischemic" blood, obtained by cannulation of the vein draining the infarcted region. 8 The clinical correlate of these changes is found in a change of the S-T segment level and eventual T wave inversion. From the Laboratorium voor Fysiologie, Campus Gasthuisberg, Leuven, Belgium. Address for reprints: Dr. Edward Carmeliet, Laboratorium voor Fysiologie, Campus Gasthuisberg, B-3000, Leuven, Belgium. Examination of the electrical activity of excised myocardial tissue 10-24 hours after ligation of a coronary artery reveals that most of the myocardial fibers are electrically silent. Purkinje fibers are still active and show prolonged action potentials starting from a reduced membrane potential 9 ' 10 (Fig. 2C). No clear concept has been formulated concerning the relationship between electrical activity and metabolism. This is due to the fact that transmembrane potentials usually are explained as generated by passive ion movements, i.e., movements of ions down their electrochemical gradient. Metabolism, according to this simple view, affects electrical activity only indirectly via electroneutral ion pumps. During recent years it has become apparent that this picture should be changed in the following respects. (1) Active ion transport functioning as an electroneutral process is the exception rather than the rule; instead, active transport should be regarded as electrogenic, i.e., transferring net electrical charge across the membrane." (2) Passive ionic currents depend on the metabolic state of the cell. Metabolic inhibition not only changes the equilibrium potential of ions that carry current across the membrane, but also affects the permeability function (ion transfer function) and the kinetics of ionic currents. This regulatory function is exerted by determining the local concentration of ions and the renewal of organic molecules which form an essential part of the transporting channels. Intracellular Ca 2+, for instance, modulates the permeability for K ions. This was first shown for red cells from which the net loss of K ions during metabolic inhibition by MIA strongly depends on the presence or absence of Ca ions. 12 A similar effect of intracellular Ca 2+ on K + permeability also was demonstrated in nerve 13 and cardiac cells. 14 The external concentration of H ions affects the location of the activation and inactivation curve on the voltage axis for different ions, a result suggesting the existence of negatively charged groups with a pk of about 5.5. 15 Intracellular accumulation of Na ions blocks the K channel when the potential gradient forces Na ions to move outward. 16 Metabolism is further responsible for the synthesis of organic molecules that regulate the ion transfer mechanism and the kinetics of the membrane channels. In nerve,

578 CIRCULATION RESEARCH VOL. 42, No. 5, MAY 1978 I FIGURE 1 Effect of hypoxia on the cardiac action potential. A: Nineteen-day chick embryonic ventricle. A x, control; A it after 60 minutes of hypoxia. The recovery of the amplitude and of the (dv/dt) max is not delayed by hypoxia. 4 B: Secondary oscillations during hypoxia in a dog Purkinje fiber; [K + ] o 2.7 mm.' 04 C: Shortening of the action potential in a dog Purkinje fiber after 2 hours of hypoxia; [K + ] o 5.4 mwi. C,, control; C 2, 2 hours of hypoxia (Carmeliet, unpublished). Vertical calibrations: 50 mv and 80 V/sec. Horizontal calibration: 0.1 sec. inactivation of the Na + current can be eliminated by application of pronase to the inside of the fiber, indicating that the kinetic behavior of this current is controlled by a protein structure. 16 In heart muscle, the current carried by the slow channel is increased in the presence of catecholamines. 17 The hypothesis has been formulated that catecholamines favor phosphorylation of membrane sites via stimulation of cyclic adenosine 3', 5'-monophosphate (camp) formation. 17 - ie By decreasing the level of ATP and camp, metabolic inhibition results in a decrease of the number of channels available for passing Ca ions. 18 In young myocardial cells (chick embryo), the development of which has been arrested by in vitro culturing, incubation with messenger RNA caused the appearance of fast so- FIGURE 2 A: Prolongation and shortening of the action potential by dinitrophenol in a sheep Purkinje fiber. A x, control; A 2, after 5 minutes; and A 3, after 20 minutes in dinitrophenol, 0.2 mu (Carmeliet, unpublished). B: Extreme shortening of the action potential by monoiodoacetate, 0.2 mu, after 45 minutes. The control duration of the action potential (not shown) was 0.43 sec. Vertical calibrations: 50 mv and 400 V/sec. Horizontal calibration: 0.1 sec. (Carmeliet, unpublished). C: Decrease of the resting potential and prolongation of the action potential in dog Purkinje fibers after experimentally induced infarction. 1 "

CARDIAC TRANSMEMBRANE POTENTIALS AND METABOLISM/Camie/je/ 579 dium channels; the induction was blocked by cycloheximide, indicating dependence on protein synthesis. 19 In the same cells cultured by the cell reaggregation technique, cycloheximide also blocked the appearance of tetrodotoxin-insensitive slow channels. 20 In this brief review the following topics will be discussed: (1) metabolism, active transport, and changes in ion concentration, and (2) metabolism and transmembrane potential. Metabolism and Active Transport In living cells, cations and anions are unequally distributed between the intra- and extracellular medium. For many ions this distribution is not in equilibrium with the membrane potential. To maintain a steady state, energy is needed to move ions uphill against their electrochemical gradient. Na + -K + Distribution A TP Hydrolysis and Na + -K + -A TPase In heart muscle as in other cells, intracellular Na + concentration is held low by an ATP-driven pump. Uncoupling of oxidative phosphorylation by DNP reduces the efflux of radioactive Na ions in isolated heart muscle preparations. 21 Supervision with an oxygen-deficient, glucose-free solution results in a fall in intracellular ATP concentration, concomitant with an accumulation of intracellular Na + and a decrease in intracellular K +. 3 Excess glucose may prevent partly the fall in ATP and the changes in ion concentrations. Ischemia produced by ligation of a major coronary artery of the in situ heart or by clamping of the aorta results in similar changes in the infarcted area. 22 Active Na + efflux is mediated through a Na + -K + ATPase system. The idea of a sodium pump driven by ATP hydrolysis received strong support from J.C. Skou's discovery of an enzyme system in crab nerve membrane that produces a net hydrolysis of ATP (see Ref. 23). Since then, an ATPase complex, assumed to be an integral part of the Na pump, has been isolated from many other membrane systems, including heart cell membranes. Actual information about the characteristics of this enzyme system can be summarized as follows: 23 (1) The ATPase activity is stimulated by Na + and K +, and cross-competition exists between both ions. The enzyme system contains two active sites an internal site with high affinity for Na + and an external site with high affinity for K +. (2) Activation by Na + and K + is modified by several conditions. The external K + site can be blocked by cardiac glycosides. Ca 2+ competes with Na + for the internal site but reduces the concentration of K + required for half-maximal activation of the enzyme. The enzyme activity is dependent on ph; an optimal ph of 7.2-7.5 yields a very high Na + -K. + - ATPase activity. (3) ATP splitting with simultaneous phosphorylation of the enzyme occurs at the internal site and is dependent on Na + and Mg 2+. Dephosphorylation is dependent on the interaction of K + with the external site. In contrast to the blocking effect of ouabain on the Na + - K + -ATPase, Godfraind and Ghysel-Burton 24 found a stimulation of 42 K uptake (human heart slices and guinea pig atria) and a net uptake of K, net loss of Na (guinea pig atria) at low concentrations of ouabain (1-3 x 10~ 9 M). Stimulation of the Na + -K + pump was also assumed to explain the shift in reversal potential for the pacemaker current in cardiac Purkinje fibers. 25 An unequivocal demonstration of stimulation of the Na + -K + -ATPase at these low concentrations of ouabain has not been given. 26 Electrogenic Na + Pump vs. Active K + Transport Early information obtained from experiments with the squid giant axon and red cells suggested active transport of Na + and K + with tight coupling between Na + outward and K + inward transport. A one-for-one exchange for Na + and K +, however, seems to be rather the exception, and more recent information 23 indicates that per mol ATP, 3 mol Na + are pumped out while less than 3 mol K + are pumped in, so that the overall reaction might be written as follows: 3 Na + (in) + 2 K + (out) + ATP" 4 + H 2 O 3 Na + (out) + 2 ADP~ 3 H + This stoichiometry suggests that the total pump reaction induces an outward current, making the cell interior negative. A direct contribution of outward Na + transport to the membrane potential has been demonstrated in many cell types." In heart Purkinje fibers, a block of the electrogenic Na + pump has been assumed to explain the drop in membrane potential during rapid cooling. 27 Hyperpolarizations beyond the equilibrium potential of K + (E k ) were described following abrupt rewarming, when [Na^ was first increased by prolonged incubation at low temperature." Electrogenic Na + transport also contributes to the membrane potential under more physiological conditions. This is suggested by studies on beating guinea pig atria 28 and on Purkinje fibers. 29 ' 30 In Purkinje fibers, stimulation at a high frequency is followed by a temporary block of spontaneous activity. This phenomenon is called overdrive suppression and can be inhibited by preincubating- the tissue with Tyrode's solution containing DNP, Li +, or ouabain, all of which block active outward Na + transport. 24 McDonald and MacLeod 3 have claimed that electrogenic Na + transport is responsible for the high resting potential (-75 mv) in guinea pig ventricle after prolonged (8-hour) exposure to an oxygen-free solution; the equilibrium potential for K + was only -45 mv under those conditions. Although electrogenic Na + transport certainly can hyperpolarize the membrane to more negative potentials than E K, it should be realized that this phenomenon is only temporary. Because of the high permeability to K ions, K + will redistribute and tend to an equilibrium. Thus, the low intracellular K + concentration remains unexplained, and the evidence that an electrogenic Na pump is responsible for the high membrane potential under those conditions remains incomplete. Active Na + transport is regulated by external K + and internal Na + concentration. In cow Purkinje fibers, the efflux of radioactive Na ions is relatively insensitive to [K + ] o between 0.54 and 16 mm, but an increase of about

580 CIRCULATION RESEARCH VOL. 42, No. 5, MAY 1978 50% is observed when [K + ] o is increased from zero to 5.4 or 16.2 mm. 31 Similar results were obtained by Haas et al. 21 in frog atrium. Na + efflux was little affected by variations in [K + ] o between 1.35 and 27 mm. In a recent paper, Glitsch et al. 32 found active Na + efflux (calculated as the sum of Na + influx and net outward Na + transport) to be dependent on [Na + ]i and [K + ] o. Maximal active Na + transport was about 30 pmol/ cm 2 -sec. Half-maximal activation was obtained at about 22 mm for [Na + ], and 0.2 mm for [K + ] o. The authors concluded that active Na + efflux is regulated mainly by alteration in [Na + ]i under normal conditions. In cat papillary muscle, net Na + efflux in Na + -free medium is also highly dependent on [Na + ], and is proportional to the second power of [Na + ],. 33 The dependence of Na + efflux on [K + ] o does not necessarily mean that the active outward transport of Na + is coupled to an active inward K + transport. According to the analysis of Page, 34 in cat papillary muscle, the distribution of K + is entirely passive. This conclusion is based on experiments in which K + influx and efflux were unaltered when external NaCl was removed, or when ouabain was applied to Na + -depleted preparations; the temperature dependence of K + exchange also was barely affected by removing Na +. According to this hypothesis, Na + transport would be entirely electrogenic. External K + is necessary only to activate the external site of the ATPase system, but K + is not transported by the enzyme. The large decrease in K + influx by ouabain in the presence of Na + is then explained 35 by the depolarization resulting from the block of the electrogenic pump. The difference between the membrane potential measured by intracellular microelectrodes and E K, calculated from flame-photometer determinations of K +, usually is considered to be an indication of active K + inward transport. These calculations do not take into account the intracellular activity, 36 " 38 which yields lower values than the concentrations. The activity coefficient for K + in the intracellular medium might be lower than in the extracellular medium. 37 When activities instead of concentrations are used, the membrane potential is close to the equilibrium potential for [K] o > 5 mm. At lower [K] o, the resting potential deviates from the theoretical line for a K + electrode. Under those conditions, passive fluxes generate a net outward K + movement, and maintenance of the steady state requires the existence of an active inward K + transport. From the experimental evidence available at the present time, one may thus conclude that, depending on the conditions, the Na + pump may operate as a coupled or an electrogenic mechanism. Ca 2+ Distribution Although the intracellular Ca 2+ concentration calculated from the total intracellular Ca 2+ content is about the same as the extracellular concentration, it generally is assumed that the concentration of free intracellular Ca 2+ is of the order of 10~ 6 M or less. This low concentration is a requisite to explain the low values for resting tension; at Ca 2+ concentrations above 10~ 6 M, the actomyosin system of the muscle becomes activated. The cell incorporates three major mechanisms to keep the Ca 2+ concentration in the cytoplasm at this low level. 39 1. Ca ions are taken up actively in mitochondria. This "uphill" transport is linked to the respiratory chain and is accompanied by a release of H ions into the cytoplasm. 2. A second and faster mechanism for removing Ca 2+ from the cytoplasm is present in the sarcoplasmic reticulum. Ca 2+ not only binds to the membrane surface, but also is taken up in the interior of the sarcoplasmic reticulum by a transport mechanism that operates via a Mg 2+ - Ca 2+ -ATPase. This enzyme system differs from the Na + - K + -ATPase in the outer cell membrane in that it is not blocked by ouabain and is activated selectively by Ca 2+. 3. Since there is a continuous passive inflow of Ca ions into the cell, especially in the heart, by activation of the slow inward current during the action potential, net outward movement of Ca 2+ across the outer cell membrane is required for cell survival. A Ca 2+ extrusion mechanism, operative at the level of the cell membrane, has been found coupled indirectly to metabolism. In the guinea pig, radioactive Ca 2+ efflux, for instance, is barely affected by DNP in resting conditions and shows a low temperature sensitivity. 40 Ca 2+ efflux can be separated into a Ca 2+ - and a Na + -activated component, the relative magnitude of the components being determined by the ratio lca 2+ ]/[Na + ] 2. The results suggest a carrier system, with an affinity for both Na + and Ca 2+ on two sites. The affinity for Ca 2+ is higher than for Na + at the inner site, whereas the reverse is true at the outer site. Under these conditions, Na + flows downhill into the cell, providing the necessary energy for the uphill transport of Ca 2+ out of the cell. For this mechanism to continue functioning, metabolic energy has to be put into the active outward transport of Na ions via the Na + -K + -ATPase system. When metabolism is blocked, intracellular Na + will increase, followed by a rise in intracellular Ca 2+. A rise in [Ca 2+ ], in heart muscle during perfusion with hypoxic solutions has not been measured by direct methods but is suggested by the increase in resting tension 41 ' 42 and concomitant rise in Ca 2+ efflux. 42 In the squid axon, 45 Ca 2+ efflux also increases, secondary to metabolic blockade by DNP. In this case, the rise in Ca 2+ efflux has been demonstrated to be due to a rise in [Ca 2+ ]i, since light emission in the presence of aequorin was markedly increased. 39 Dramatic increases (up to 10 times) in intracellular Ca 2+ in heart muscle have been found after prolonged obstruction of a coronary artery, even when normal perfusion was resumed. These changes, however, are irreversible and signify cell death, 43 Active Regulation of Intracellular H + Concentration The intracellular ph of most animal cells is approximately 7 under normal conditions in vivo. 44 This means that H ions are not passively distributed across the cell membrane. Control of intracellular ph can be compared with that of intracellular Ca 2+. It is regulated by two important mechanisms: (1) active transport of H + (or OH~ or HCO~ 3 ) across the cell membrane and (2) intracellular buffering. The intracellular buffering power will

CARDIAC TRANSMEMBRANE POTENTIALS AND METABOLISM/Ca/7?ie/(«581 determine immediate responses of ph, to the addition of acid or base, while active transport is responsible for the longer-term response. 44 In snail neurons, squid axons, and cardiac Purkinje fibers, 45-4 " time constants of about 5 minutes have been found for the longer-term response. There is no direct information available on the mechanism of the active pump, but it has been speculated that H ions are transported via the Na-K pump. Time courses for changes in [Na + ], or [H + ], are similar when Na + or H + is injected into cells. 4S An increase in internal H + concentration (produced by increasing Pco 2 ) changes the kinetics of Na transport from third order to second order in frog muscle, 47 in agreement with the hypothesis that H ions can replace Na + in the outward limb of the Na + -K + exchange pump. In spite of an increase in lactate production in hearts perfused with oxygen-deficient solutions, 48 anoxia without reduction of perfusion rate does not result in dramatic changes in intracellular ph (estimated by the DMO method), the ph decreases only slightly and temporarily. 49 Ischemia, on the other hand, is accompanied by a lowering of both intra- and extracellular ph. In totally ischemic rat hearts, [ph], and lph] 0 dropped from 7.25 to 6.8. 49 After ligation of a coronary artery, the ph of tissue homogenates and the venous ph also decrease. 50 Metabolism and Transmembrane Potential Resting Membrane Potential Metabolic blockade is accompanied by a fall in resting potential. In the case in which the active Na + pump is functioning as an electroneutral mechanism, the depolarir^ition will be the result of a decrease in equilibrium pc ential for K ions. In the case in which Na + outward transport is functioning as an electrogenic pump, with a passive distribution for K +, metabolic inhibition will result in the disappearance of an outward current carried by Na ions. The extent of the immediate depolarization will depend on the permeability ratio for K and Na ions. When the permeability for K + is high relative to that for Na +, an important net outward movement of K ions will counteract the inward current of Na ions, and the membrane potential will remain close to E K, the equilibrium potential for K ions. As the cell loses more and more K +, however, the membrane potential will steadily decrease. The fall in E K during metabolic inhibition is due essentially to a loss of intracellular K +, but eventually also to accumulation of extracellular K +. This may occur when diffusion in the extracellular space is restricted by the narrow clefts between the cells. The fall in intracellular K + and the accumulation of extracellular K + are amplified if the preparation is stimulated at a high rate. Accumulation of extracellular K + has been measured by K + -sensitive electrodes during a single action potential in the frog ventricle; r ' the effect showed summation when the preparation was stimulated at a high frequency. It is evident that accumulation may play an important role in ischemia where exchange between the vascular space and the interstitial space has been reduced or completely blocked. Increases of K + concentration in the venous effluent have been measured after release of an occluded coronary artery. 52 Secondary changes during hypoxia and ischemia may modulate the passive leak of ions. A rise in intracellular Ca 2+ will enhance 12 and a fall in ph will decrease the passive leak of K ions. 53 The effect of lactate accumulation is less clear; depolarization, 54 as well as hyperpolarization, 55 ' 56 has been described. In severe cases of hypoxia, the membrane may become so leaky that intracellular enzymes are released in large amounts. 52 These secondary changes also affect the activity of the active Na + pump. Competition of intracellular Ca 2+ and H + ions for the site that normally transports Na + will reduce Na + outward transport. A shift to acid ph decreases the Na + -K + -ATPase activity. 23 In line with this observation, lowering the ph reduces the efflux of radioactive Na ions from cow Purkinje fibers 58 and causes a net loss of K from cat heart 35 and from the perfused rat heart. 59 Under acidotic conditions, an increase in extracellular K + activity has been measured by K + -sensitive electrodes in the rabbit atrium. 38 In contrast to these findings, a rise in intracellular K + has been described for the perfused rabbit septum in respiratory or metabolic acidosis; 42 K + efflux in this preparation was not changed by decreasing HCO 3 at constant Pco 2 (ph change from 7.4 to6.12). 53 Upstroke of the Action Potential The Fast Na + Current A direct measurement of the fast inward Na + current by the voltage clamp technique is, at present, difficult in heart muscle. In those preparations (e.g., frog atrium) in which an estimation has been made, the effect of metabolic inhibition by DNP on the fast inward current is controversial; the current has been reported to be suppressed 60 or unchanged. 61 Indirect measurements such as (dv/dt) max indicate that the Na + current is reduced by anoxia, even in the absence of a decrease in maximum diastolic membrane potential. 4 The reasons for this reduction in (dv/dt) max during anoxia probably are multiple and complex in origin. Increases in [Na + ] b [Ca 2+ ]t, and [H + ] probably play a role in this process. (1) A rise in [Na + ] will decrease the inward current by lowering the gradient for Na ions; it also may shift the inactivation curve to more negative potentials. Such a shift, secondary to a rise in [Na + ] h was proposed as a possible explanation for the reduction of the inward current in cooled Purkinje fibers. 62 (2) Acidification has been found to decrease (dv/ dt) max in Purkinje fibers 63 and to lower the inward current in frog atria, estimated by the voltage clamp technique. 64 A shift in the inactivation curve was not observed. 63 (3) A rise in intracellular Ca ions, which bind to negatively charged groups on the internal side of the cell membrane, might result in a shift of the inactivation curve to more negative membrane potentials. However, taking into account the high intracellular Mg 2+ concentration, such an effect is highly unlikely. In contrast to the reduction of (dv/dt) max, removal of inactivation does not seem to be slowed by anoxia as long

582 CIRCULATION RESEARCH VOL. 42, NO. 5, MAY 1978 as the maximum diastolic membrane potential is not affected. After 1 hour of anoxia, the recovery of (dv/ dt) max and the overshoot are not retarded with respect to the end of the action potential 4 (Fig. 1A). If depolarization occurs after prolonged hypoxia or during ischemia (intracellular K + loss and extracellular K + accumulation), recovery from inactivation of the Na + current will be slowed. Such slowing at depolarized levels has been demonstrated to occur in the presence of elevated K + concentration; 65 under those conditions, time constants for recovery of the fast Na + current are of the same order as those for the slow inward current. 65 Such modifications may explain the pronounced changes in refractory period and excitability threshold seen in experimental myocardial infarction. 52 The Slow Inward Current Activation of the slow inward current determines to a large extent the late part of the upstroke in many auricular and ventricular muscle fibers and plays an exclusive role in the upstroke of the action potential in the sinus and atrioventricular nodes. 66 Although the channel is often called a Ca channel, it should be realized that Ca 2+ is not the only current carrier and that the contribution of other ions may be important. 67 The extent to which Ca 2+ and Na + contribute differs among atrial, ventricular, and Purkinje preparations, and varies from one animal to the other. An important contribution of Na + to the slow inward current has been described for Purkinje fibers, muscle fibers of guinea pigs, cows, rats, and frogs. 67 ' 68 In the frog, the relative contribution of Na is controlled by the extracellular Mg 2+ concentration. 64 Voltage clamp measurements in cat ventricle 69 show that inhibition by cyanide (100 mg/liter) and dinitrophenol (40 mg/liter) reduces the slow inward current by 25.4% and 46%, respectively. Inactivation is slowed slightly, but recovery from inactivation is unaffected. A reduction of the slow inward current by DNP also has been described for the frog atrium. 61 As a possible explanation, the authors propose a rise in intracellular Ca 2+, but since Na + might be an important contributor to the total current, the increase in [Na + ] t during metabolic inhibition should not be neglected. Metabolism, however, also may affect the ion transfer mechanism, g Cn. The hypothesis has been put forward that the number of slow current channels is controlled by phosphorylation of specific sites in the membrane, via camp. 17> l8 Catecholamines enhance the slow inward current by increasing g 17 Ca The effect of metabolic inhibition would be to decrease g Ca. In this way the high sensitivity of Ca 2+ -mediated action potentials to hypoxia and DNP 70 and their restoration by ATP 18 can be explained. In the bullfrog ventricle, exogenous ATP also prolongs the action potential. 71 The slow inward current is increased under those conditions, an effect which the authors compared to that of adrenaline. 71 Changes in ph also may play a role in the reduction of the slow inward current during hypoxia. Lowering the ph of the perfusion solution diminishes the slow inward current in frog atria 64 and in cat papillary muscle. 72 In the latter preparation, the time constants for inactivation and recovery from inactivation are increased and steady state inactivation is not changed. Plateau and Action Potential Duration From the existence of a threshold for repolarization and its shift to less negative potentials during the action potential 73174 it has been deduced that repolarization during the plateau is brought about by one or more timedependent conductance changes (for discussion, see Ref. 75). The phenomenon of all-or-nothing repolarization disappears at the end of the plateau. 73 From that time on, repolarization is due essentially to the passive charging of the membrane capacity by an outward current which is dependent only on membrane potential. 76 Time-dependent conductance changes responsible for repolarization during the plateau are the slow inward current (i sl ) and the slow outward current (i x or i K ). The extent to which these two currents contribute to the repolarization process differs depending on cell type. In Purkinje fibers and in frog atrial muscle, repolarization is due essentially to the activation of outward current; in many mammalian ventricle preparations, it is due to the inactivation of the slow inward current; in some preparations (e.g., cat, guinea pig), inactivation of inward current and activation of outward current are both important. 75-76 Although time-dependent currents are essential in explaining the repolarization, a change in one of these currents is not the only mechanism by which the action potential duration can be modified. Since the net outward current during the plateau and the final repolarization are due to the interplay between time-dependent background currents and time-dependent conductances, a change in any of these currents will modify the repolarization process. In myocardial (contractile) fibers, metabolic inhibition results in a shortening of the action potential. In Purkinje fibers, prolongation, as well as shortening of the action potential, has been described (Figs. 1 and 2). Shortening of the Action Potential The following changes in ion currents may play a role in the shortening of the action potential in myocardial fibers: (1) a reduction in time-dependent slow inward current, due to a decrease in driving force (accumulation of [Ca 2+ ] and [Na + ]i and/or a decrease in the maximal conductance of the channel (see preceding section); (2) a shift of the background current in the outward direction. Such a shift in the outward direction may be due to an increase in the time-independent K + outward current (i Kl ) or to a decrease in inward background current. Application of cyanide in combination with a reduction of external Na + results in a displacement of the steady state current-voltage relationship in the outward direction (mammalian ventricle). The result is explained as an increase in i Kl secondary to a rise in the intracellular Ca 2+ concentration. 77 In Purkinje fibers, an injection of Ca 2+ in the cell causes the membrane to hyperpolarize, shortens the action potential, and shifts the steady state currentvoltage relation in the outward direction. 14 42 K efflux is increased by hypoxia in a variety of

CARDIAC TRANSMEMBRANE POTENTIALS AND METABOLISM/Cawje/Ze/ 583 preparations from different species. 42 Concomitant with this increase in K + outward movement, resting tension and Ca efflux are enhanced, suggesting an increase in [Ca z+ ]i. In agreement with this hypothesis cell-to-cell resistance was found to increase. 41 Furthermore, measurements of slope resistance at different levels of membrane potential show that the increase in slope resistance, characteristic for depolarizations between -50 mv and 0 mv, is absent in hypoxia. This result suggests that the normal inward-going rectification disappears during hypoxia and is consistent with the hypothesis of a displacement of the steady state current-voltage relationship in the outward direction and an increase in i Kl. Slope resistance and K efflux normalize when extra glucose is added to the hypoxic preparation; it is known that under these conditions the action potential duration and plateau also revert to normal values. + All these data suggest that the background K conductance (i Kl ) is increased in conditions of metabolic inhibition and that the underlying mechanism is a rise in [Ca]. Another factor which might affect the background K + current is an eventual increase in extracellular K +. Since metabolic inhibition results in a net loss of K + from the cells, K + may accumulate in the narrow extracellular clefts. In experiments on turtle ventricle in which an extra amount of K + was injected into the coronary circulation during the action potential, Weidmann 29 showed that an increase in extracellular K + shortens the action potential. The result was explained as being due to an increase in K + conductance, favoring K + outward movement in spite of a decrease in chemical gradient. In agreement with this explanation, the instantaneous current-voltage relation is shifted in the outward direction in sheep and calf ventricular muscle" 0 and in frog atria. 81 In these preparations, a moderate increase in external K + shortens the action potential. The effect of [K + ] o, however, is far from general. In the cat and guinea pig ventricle, 82 ' 83 an increase in [K + ],, between 2.7 and 8.1 ITIM does not affect the action potential duration; in the cat, the action potential may even be slightly prolonged at higher K concentrations (in Cl-free media). In agreement with the absence of a shortening effect, the instantaneous current-voltage relation in the cat is not affected by a rise in [K],, in the potential range corresponding to the plateau level (W. Trautwein, personal cummunication). From a purely theoretical point of view, a rise in outward background current also could be due to a block of an electrogenic active inward transport. Inhibition of an active electrogenic K + pump has been invoked to explain the marked shortening of the action potential and increase in outward current by DNP in frog atrium. 60 The experimental evidence for such a mechanism is still incomplete. The main argument in favor of this explanation is that the rise in K + outward movement, as measured by radioactive K + in the presence of DNP, is smaller than the rise in outward current, measured during voltage clamp. The matter is still more complicated because Nargeot 61 found a decrease in delayed outward current in the presence of 10~ 4 M DNP, instead of the increase described by Haas at a higher concentration of DNP. In connection with these divergent results, it should be remembered that DNP can prolong as well as shorten the action potential 6 (Fig. 2A). Prolongation of the Purkinje Fiber Action Potential As mentioned in the introductory paragraphs, hypoxia or addition of metabolic inhibitors in the presence of a low external K + concentration results in a prolongation of the action potential and, eventually, in a complete failure of the repolarization process. Depolarization to the plateau level with oscillatory behavior was also described after experimental myocardial infarction. 9 - l0 A block of the electrogenic Na + pump current seems to be of primary importance in explaining this disturbance of the repolarization process. In sheep Purkinje fibers, a block of the active Na + pump by dihydro-ouabain or low concentrations of Li + (4 ITIM) leads to a pronounced depolarization and a shift of the steady state currentvoltage relation in the inward direction. 30 Such effects are not seen in myocardial contractile fibers, suggesting that the contribution of a Na + electrogenic pump current to the genesis of the membrane potential is more important in Purkinje fibers. Inhibition of the electrogenic Na + pump also may be the explanation for depolarization to the plateau level in acid ph,' but direct experimental evidence still is lacking. Extrapolating from data on ph effects in frog auricle, Coraboeiif et al. suggest that a decrease in delayed outward current (i x ) plays a role in the failure of the repolarization process. It should be realized that phenomena leading to the shortening of the action potential in muscle are less important in Purkinje than in muscle fibers. The slow inward current, for instance, is of small amplitude in Purkinje cells. The eventual reduction of this component by metabolic blockade will thus have little effect on the repolarization process, and an increase in i Kl, secondary to inflow of Ca 2+ through the slow channel, will be less pronounced. Pacemaker Activity Pacemaker activity is characterized by a slow diastolic depolarization following the repolarization phase. Under normal conditions, heart rate is determined by the pacemaker activity in the sinus node. Pacemaker activity, however, is also present in Purkinje fibers and has been described in muscle fibers of the mitral valve. 66 Under abnormal conditions, such as low intra- and/or extracellular K, mechanical elongation, and presence of short-circuit currents, pacemaker activity can be induced in myocardial fibers of the atrium and ventricle. 66 Pacemaker Activity in the Sinoatrial Node Information obtained by voltage clamp analysis in the sinus node suggests that slow diastolic depolarization is due to the deactivation of an outward current and the simultaneous activation of an inward current, probably the slow inward current. 85 ' 86 In agreement with this hypothesis, the diastolic depolarization is blocked by Mn, 87 " 89 Ni, 88 or Co 88 ions and by verapamil. 88-66 According to Brown et al., 90 (frog atrial fibers) the main factor in determining the rate of diastolic depolarization is the deactivation of the outward current, because activation of

584 CIRCULATION RESEARCH VOL. 42, No. 5, MAY 1978 the fast and slow inward current is too fast in this range of potentials. In the presence of adrenaline, however, the slow inward current has to be taken into account to explain the increase in frequency. 75 The relation between metabolism and pacemaker activity in the sinus node has not been studied intensively. Hypoxia alone results in an initial increase in frequency followed by depression. 91 Application of metabolic inhibitors leads to similar effects. In the presence of DNP (0.2 ITIM), the firing rate increases during the first minutes (1-2 minutes) but decreases afterward. The action potential is shortened and the preparation is finally arrested in a depolarized state. 92 The excitatory period is longer (10-20 minutes) during application of azide (10~ 3 M) 92 but is not seen during application of MIA (2 ITIM). 93 Occlusion of the sinus node artery leads to a slowing of frequency, 94 an effect which is not due to local or reflex cholinergic activity or to distension of the arterial wall. Direct information on the changes in ionic currents under conditions of metabolic blockade is lacking, and any proposed explanation therefore remains speculative. As a working hypothesis, one might propose that a block of the electrogenic Na + pump is responsible for the initial increase in frequency by hypoxia. The reverse phenomenon, i.e., hyperpolarization and stabilization of the membrane potential, has been demonstrated when the pump was stimulated by a rise in temperature 95 or electrical pacing (overdrive suppression). 87 The secondary slowing of the firing rate and eventual arrest of spontaneous activity can be explained by a reduction in the slow inward current (responsible for the diastolic depolarization and the upstroke) and an increase in K + background current. An increase in extracellular K + as a consequence of metabolic inhibition is probably of no importance in the explanation of the decrease in frequency. Sinus node activity can persist with external K + concentrations as high as 20 mm. 9HI This resistance to high external K has to be related to the high ratio of P Na+ /P K + in the sinus 85 97> 98 ' and, possibly, also to the existence of an important sympathetic tonus." Pacemaker Activity in Purkinje Fibers In Purkinje fibers, pacemaker activity can be present at two different levels of membrane potential. At the high potential level (-90 to -60 mv), the diastolic depolarization is due to deactivation of a K outward current, i K2, in the presence of an inward background current. 100 The steady state background current has to be regarded as the balance between inward currents (Na +, Cl~, Ca 2+ ) and outward currents (K +, electrogenic Na + transport). Changes in diastolic depolarization thus can be due to changes in the time-dependent i K2 current but, also, to changes in the background current. At reduced membrane potentials, the potential of Purkinje fibers can oscillate between -50 and 0 mv. This oscillatory behavior seems to be generated by time-dependent variations in i x, 101 an outward current normally responsible for initiating repolarization during an action potential. Thus, the appearance of oscillations represents a failure of the normal repolarization process, probably as a consequence of a small change of the background current in the inward direction. Since the oscillations appear at potential levels at which the slow inward current is activated, a regenerative increase in inward current also probably is important. In agreement with this explanation, it was found that the amplitude and the frequency of the oscillation increase on addition of catecholamines (A. Pappano, unpublished observations). The relation between metabolism and pacemaker activity in Purkinje fibers has been studied by reducing oxygen tension, increasing CO 2 tension, or addition of metabolic inhibitors. The primary effect of hypoxia is an increase in automaticity. From the published records of the electrical activity, the effect is due less to a change in the slope of the diastolic depolarization than to a gradual decline in maximum diastolic potential. 1 Prolonged exposure to hypoxia shifts the pacemaker activity from the high potential level to the low level; the action potential first lengthens, and repolarization is incomplete with secondary slow depolarizations (Fig. IB), leading to an increase in the overall rate. Finally, the preparation fails to repolarize and stays depolarized at the plateau level, where the potential may continue to oscillate between 50 and 0 rav. 1 A decrease in maximum diastolic potential and oscillatory behavior at the plateau level are also observed in the presence of elevated CO 2 tensions 56 (Fig. 3B) and in Purkinje fibers excised from ischemic areas produced by coronary ligation 9-10 (Fig. 3C). Somewhat different results were reported by Scherlag et al. 102 During hypoxia, the spontaneous frequency of isolated Purkinje fibers decreased, an effect due to a fall B 0 100 msec 1sec r j * FIGURE 3 Pacemaker activity at reduced levels of membrane potential in dog Purkinje fibers. A: In the presence of dinitrophenol; lm B: in the presence of elevated CO 2 tension /ph] 0 6.6; M C: after experimental infarction. 9

CARDIAC TRANSMEMBRANE POTENTIALS AND METABOLISM/Ca/we/ie/ 585 in the rate of diastolic depolarization. The difference in results probably reflects differences in experimental conditions, such as external K +. At [K + ] o > 4 ITIM, the action potential also does not lengthen, but, rather, shortens during hypoxia (Fig. 1C). The initial effect of DNP (0.2 ITIM) is to accelerate the diastolic depolarization 29 ' 103i 104 and to decrease maximum diastolic potential. The action potential prolongs and repolarization may fail altogether, with oscillatory behavior at the plateau level (Fig. 3A). Up to this stage, which lasts only a few minutes, the changes in electrical behavior are comparable to those obtained in hypoxia. Following this excitatory period, spontaneous activity stops. The fiber remains electrically excitable and even may hyperpolarize from -50 to -75 mv. 104 The action potential now shortens and the slope of diastolic depolarization falls below the control level.' 105 To a certain extent, the addition of MIA causes similar u3> l0(i changes. In the presence of [K + ] o, 5.4 ITIM, the excitatory period is absent, the diastolic depolarization completely disappears, and the action potential shortens to such an extent that it resembles the action potential of nerve and skeletal muscle (Fig. 2B). Experimental analysis in terms of changes in ionic currents is very fragmentary. The initial excitatory period does not seem to be related to changes in i Ka current. In the presence of DNP, Aronson et al. 105 found a reduction in maximal activation of i K2, an effect which would tend to slow spontaneous depolarization. The shift of the activation curve for i Ka to more negative potentials in acidosis 107 ' 108 also does not provide an explanation for the increase in automaticity. Reduction of the delayed outward plateau current has been proposed by Coraboeuf et al. 5 " as in analogy with the results obtained in acidosis in the frog auricle. Inhibition of the electrogenic Na + pump is probably the most important mechanism leading to an increase in automaticity of Purkinje fibers during metabolic inhibition. The existence of an electrogenic Na + pump has been demonstrated by the use of dihydro-ouabain. 30 The changes in pacemaker activity induced by ouabain, however, should not be equated with those induced by hypoxia. In digitalis intoxication, the maximum diastolic potential is reduced while the rate of diastolic depolarization is enhanced. 109 In a quiescent preparation, stimulation results in action potentials followed by a transient depolarization. This transient depolarization is faster and greater, the higher the frequency of stimulation." 0 - "' The characteristic of being triggered by previous electrical stimulation is not restricted to ouabain-induced pacemaker activity. Triggered sustained rhythmic activity has also been described in sinoatrial fibers, in embryonic fibers, in Purkinje fibers, and in muscle fibers of the mitral valve. The transient depolarization induced by ouabain corresponds to a transient inward current measured in voltage clamp conditions. This transient inward current is activated by a depolarizing pulse positive to 40 mv; in comparison with the slow inward current, it is activated more slowly and along a sigmoidal time course." The ionic nature of this current still is poorly understood. The transient depolarization is more pronounced in high [Ca 2+ ] 0 and is blocked by Mn 2+," 3 suggesting a participation of Ca ions, but it is equally well inhibited by local anesthetics such as lidocaine (personal observation). According to Lederer and Tsien," 2 the pacemaker current i Ka is not modified, but Aronson et al." 4 described a marked reduction of this current by ouabain. The reason for the difference in pacemaker activity between hypoxia and ouabain is not clear. Intracellular Ca 2+ is probably increased under both conditions; the concentrations of both ATP and ph are decreased in hypoxia but are probably normal in the presence of ouabain. Arrhythmias in Myocardial Ischemia Arrhythmias in myocardial infarction are of complex origin; both pacemaker activity and reentry play a role. 52 Apart from the mechanisms described in the preceding section, additional factors will favor the appearance of pacemaker activity in ischemia. Purkinje fibers overlying the central, mechanically inactive area of the infarct will be stretched passively during contraction of the heart. Mechanical elongation of Purkinje fibers elicits pacemaker activity. At the border zone, the contact between normal and depolarized tissue will create short-circuit currents. By passing depolarizing current or by creating a K + concentration gradient," 5 it is possible to elicit pacemaker activity. Release of catecholamines" is an additional factor favoring rhythmic activity. In myocardial ischemia, conditions also are present which allow the appearance of reentrant excitation; the refractory period, especially in contractile fibers, is shortened and conduction is impaired (slowing and unidirectional block). During metabolic inhibition, conduction of the action potential will be impaired not only by a decrease in amplitude and (dv/dt) max of the upstroke but, also, by an increase in longitudinal resistance. 41 This effect of hypoxia on longitudinal resistance is enhanced by catecholamines and can be reduced by excess glucose. The basis for this increase in longitudinal resistance probably is a rise in intracellular Ca 2+. Intracellular injection of Ca 2+ in Purkinje fibers has been shown to reduce cell communication and to cause cell uncoupling." 7 Uncoupling also results from injection of Na ions" 8 and from blocking the Na pump by ouabain," 8 ' l19 probably by increasing intracellular Ca 2+ through the Na + /Ca 2+ exchange mechanism. Metabolism thus exerts a controlling effect on conduction of the action potential via the Na pump. To a certain extent, cell uncoupling can be regarded as a protective mechanism in severe hypoxia or ischemia. By electrically isolating the cells in the center of an infarct from the surrounding cells, this mechanism reduces short-circuit currents, which normally would lead to depolarization and automatic activity. References 1. Trautwein W, Gottstein U, Dudel J: Der Aktionsstrom der Myokardfaser im Sauerstoffmangel. Pfluegers Arch 260: 40-60, 1954 2. Coraboeuf E, GargouH YM, Laplaud J, Desplaces A: Action de 1'anoxie sur les potentiels electriques des cellules cardiaques de Mammiferes actives et inertes (tissu ventriculaire isole de cobaye). Cr Acad Sci (Paris) 246: 3100-3103, 1958 3. McDonald TF, MacLeod DP: Metabolism and the electrical activity of anoxic ventricular muscle. J Physiol (Lond) 229: 559-582, 1973

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