William A. Coetzee

Reduction of ischemic K+ loss and arrhythmias in rat hearts. Effect of glibenclamide, a sulfonylurea.

Glibenclamide, one of the antidiabetic sulfonylureas, is known to block ATP-dependent K+ channels. We used this drug to determine to what extent K+ loss from acutely ischemic myocardium is mediated via these channels. We also investigated whether glibenclamide would influence ischemic arrhythmias. Isolated rat hearts rendered globally ischemic showed no correlation between early lactate and K+ efflux rates. Cumulative K+ loss during 11 minutes of global ischemia (0.5 ml min-1 g-1) was reduced, from 3.2 +/- 0.3 to 2.5 +/- 0.1 mueq/g (p less than 0.025) by 1 microM glibenclamide and from 3.3 +/- 0.2 to 1.9 +/- 0.2 mueq/g (p less than 0.005) by 10 microM glibenclamide, while lactate efflux was unaltered by the drug. Glibenclamide also exhibited potent antifibrillatory activity, abolishing irreversible ventricular fibrillation during regional ischemia (0/6 vs. 5/6 controls; p less than 0.02) and during global ischemia (0/7 vs. 9/9 controls; p less than 0.01). Heart rate, coronary flow rate, peak systolic pressure, and myocardial oxygen consumption were unaltered by the drug (1 microM). Similarly, glibenclamide (1 microM) did not alter myocardial ATP, phosphocreatine or lactate content, or glucose utilization. Ventricular fibrillation threshold during normoxia was also unaltered by glibenclamide (1 microM). We conclude that K+ loss during acute myocardial ischemia is mediated partly by ATP-dependent K+ channels, and not by a tightly coupled co-efflux with anionic lactate.

Effects of proton buffering and of amiloride derivatives on reperfusion arrhythmias in isolated rat hearts. Possible evidence for an arrhythmogenic role of Na(+)-H+ exchange.

We investigated the hypothesis that an accelerated Na+o-H+i exchange on reperfusion may lead to a displacement of the 3[Na+] [Ca2+]i/o equilibrium in favor of an arrhythmogenic rise in cytosolic [Ca2+]. Supporting evidence was obtained by subjection of isolated rat hearts to 15 minutes of low-flow (5% of control) ischemia and 2 minutes of reperfusion in the presence of a Krebs-Henseleit HEPES buffer (pH 7.4) containing lactate (10 mM). At first, the [HEPES] was fixed at 5 mM; then, 2 minutes before reflow, either the [HEPES] was varied from 50 to 1 mM to slow H+o washout, or increasing concentrations of 5-(N,N-dimethyl)-amiloride (Ki 7 microM) or 5-(N,N-hexamethylene)-amiloride (Ki 0.2 microM) were added for inhibition of Na(+)-H+ exchange. In each case, reperfusion ventricular arrhythmias were reduced by 69-73% (p less than 0.001).

Role of calcium ions in reperfusion arrhythmias: Relevance to pharmacologic intervention

SummaryCalcium ions may play a role in reperfusion arrhythmias, as suggested by 1) evidence favoring excess internal recycling of calcium during the reperfusion period; 2) electrophysiologic studies in Purkinje fibers and guinea pig papillary muscle in which calcium-dependent delayed after-depolarizations (DADs) have been found; 3) identification of the transient inward current as the basic mechanism underlying DADs; 4) the influence of cyclic adenosine monophosphate (cAMP) in the ischemic period on reperfusion electrophysiologic abnormalities; and 5) calcium oscillations in reoxygenated myocytes. More direct evidence for the role of calcium lies in the concordance between the factors influencing DADs and those associated with reperfusion arrhythmias, as well as the role of an elevated extracellular Ca2+ in causing reperfusion ventricular fibrillation. However, a role for Ca2+ does not necessarily imply that calcium antagonist drugs will be antiarrhythmic in this situation; rather there is no good evidence that these agents are antiarrhythmic unless they have a protective effect in the ischemic period. The anti-arrhythmic role of alpha1-adrenergic blocking drugs remains controversial; in isolated hearts they work in high concentrations, not through specific receptor antagonism. Beta-blocking drugs have no established place in the therapy of reperfusion arrhythmias. The role of lidocaine and other sodium channel blockers is also controversial. In isolated preparations, lidocaine can be antiarrhythmic and can inhibit DADs. Mexiletine, another sodium channel blocker, can inhibit reoxygenation and reperfusion arrhythmias as well as DADs, all in therapeutic concentrations (10 μM). Such drugs may indirectly inhibit sodium-calcium exchange, which is one of the mechanisms underlying the formation of DADs and, hence, a potential site of pharmacologic inhibition of reperfusion arrhythmias.

Effects of oxygen free radicals on isolated cardiac myocytes from guinea-pig ventricle: Electrophysiological studies

Free oxygen radicals are formed during early reperfusion and are thought to contribute to some types of reperfusion abnormalities, including arrhythmias and myocardial stunning. The purpose of this study was to investigate electrophysiological effects of oxygen free radicals using voltage clamped single ventricular myocytes from guinea-pig hearts. Oxygen free radicals were produced enzymatically by the direct addition of xanthine oxidase (XOD, 0.04 U/ml) in the experimental chamber to a solution containing hypoxanthine (0.96 mM). The generation of oxygen radicals was confirmed by the formation of adrenochrome from adrenaline. Oxygen radicals caused automaticity of isolated myocytes within 20-30 min, followed by later hypercontracture. The percentage of rod-shaped cells declined sigmoidally as a function of time, with a half maximal value at 40.9 +/- 1.6 min, and a Hill slope of -0.10 +/- 0.01 (n = 26). These effects were prevented by a combination of superoxide dismutase (10(5) U/L) plus catalase (10(6) U/L). The rate at which cells underwent morphological shape changes was unchanged by ryanodine (0.5 microM) which is thought to act on the sarcoplasmic reticulum or by the Ca2+ channel blockers nisoldipine (1 microM) or Cd2+ (30 microM). Cellular automaticity and hypercontracture were delayed by variable degrees, and sometimes completely prevented, by zero (1 mM EGTA) extracellular Ca2+, MnCl2 (2 mM) and LaCl3 (50 microM), and amiloride (1 mM). On the other hand, in the presence of a low extracellular Na+ (30 mM) or caffeine (10 mM), hypercontracture occurred at a faster time scale. Whole cell voltage clamping revealed a decrease of the inward rectifying K+ current (IK1), and a decrease of the peak of the L-type Ca2+ current (ICa,L). The total ICa,L during the clamp step was increased, mainly because of an increased time constant of inactivation (47.6 +/- 4.7 ms to 72.7 +/- 15.5 ms after 30 min, n = 4, P less than 0.05). We conclude that oxygen radicals cause automaticity and hypercontracture of isolated myocytes, that these effects may be due to an increased intracellular Ca2+ concentration ([Ca2+]i), and despite an increased ICa,L, that the enhanced Ca2+ influx may occur predominantly via the Na/Ca exchange.

A Potential Role of Calcium Ions in Early Ischemic and Reperfusion Arrhythmias

The genesis of early ventricular ischemic arrhythmias is still not well understood. Among the factors thought to explain such arrhythmias is the loss of potassium from intracellular to extracellular sites in the ischemic zone, thereby causing conduction slowing and predisposing to reentrant arrhythmias. More recently, abnormal control of intracellular calcium ion movements has been proposed as also playing a role in the development of injury current in the genesis of ischemic ventricular arrhythmias.’”’3s63 We have systematically explored the role of Ca2+ by (1) using pharmacological probes to achieve inhibition of Ca2+ movements both a t cell entry level and a t the level of the sarcoplasmic reticulum; and (2) studying factors provoking or inhibiting calciumrelated electrophysiological abnormalities such as delayed afterdepolarizations (DADs) and the transient inward current (iti), slow responses, and conduction slowing. Our electrophysiological data lead us to suggest that the effects of Ca2+ can be most clearly linked to a specific electrophysiological abnormality, namely, the provocation of DADs, in conditions where there is an adequate supply of energy available in the form of ATP. Thus abnormalities of intracellular Ca2+ control are more likely to be linked to reperfusion rather than to ischemic arrhythmias.

Characterization of NGP 1-01, an aromatic polycyclic amine, as a calcium antagonist

Primary pharmacological and electrophysiological screening procedures were carried out on the aromatic polycyclic amine 3-hydroxy-4-benzyl-4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8, 11]- dodecane (NGP 1-01). Effects observed in the isolated guinea-pig atria and ileum were, respectively, reversal of CaCl2 induced positive chronotropy and inotropy and papaverine-like depression of the dose-response curve induced by acetyl choline. Electrophysiological studies using isolated guinea-pig papillary muscle showed a reduction in the action potential (AP) plateau potential and an increase in AP duration with accompanying negative inotropic effects. The calcium-mediated (slow response) AP was completely, but reversibly blocked by NGP 1-01 at concentrations of 5 X 10(-5) M and higher. Experiments on sheep Purkinje fibres strongly supported the finding that NGP 1-01 blocks the slow inward (calcium dependent) current Isi, but also suggested a decrease brought about in an outward current, most probably the background potassium current, IK1.

Inhibition by simulated ischemia or hypoxia of delayed afterdepolarizations provoked by cyclic AMP: Significance for ischemic and reperfusion arrhythmias

Controversy exists about the role of an increased level of tissue cyclic adenosine 3-5 monophosphate (cAMP) in the genesis of early ischemic ventricular arrhythmias. Evidence for an arrhythmogenic role for cAMP was proposed by Podzuweit et al. (1978) and Opie et al. (1979) who argued that ischemic ventricular fibrillation was associated with increased levels of tissue cAMP in the ischemic zone. Lubbe et al. (1978) found that infusion of dibutyryl (dBcAMP), or the beta-adrenergic stimulant epinephrine, or the phosphodiesterase inhibitor theophylline, all produced a marked fall in the ventricular fibrillation threshold and an increase in the duration of the vulnerable period of the isolated perfused rat heart. In contrast, Muller et al. (1986) recently showed that prevention of ventricular fibrillation by beta-adrenergic blockade is not directly associated with decreased levels of cAMP, while Manning et al. (1985) used forskolin to stimulate adenylate cyclase and found that the markedly elevated tissue cAMP levels in the rat heart did not promote ischemic or reperfusion arrhythmias. Some of these contradictions could be resolved if the electrophysiological mechanisms by which increased levels of cAMP might predispose to arrhythmias were better understood. It is known that intracellular injection of cAMP into cardiac myocytes can enhance delayed afterdepolarizations (DADs; Matsuda et al. 1982) and that DADs may explain certain arrhythmias such as those evoked by digitalis toxicity (Ferrier, 1977) or reperfusion (Ferrier et al. 1985).(ABSTRACT TRUNCATED AT 250 WORDS)

Proposed role of energy supply in the genesis of delayed afterdepolarizations--implications for ischemic or reperfusion arrhythmias.

Delayed afterdepolarizations (DADs) are Ca++-dependent electrophysiological abnormalities, which are evoked by a variety of conditions that induce intracellular Ca++ overload, including fast pacing, isoproterenol, dibutyryl cyclic AMP, and intracellular injection of Ca++. Since Ca++ overload is suspected of playing a role in both ischemic and reperfusion cellular damage, a reasonable hypothesis would be that DADs could play a role in ischemic or reperfusion arrhythmias. No direct proof has, however, been obtained for such a role for DADs. We propose that DADs could be associated with arrhythmias in which there is Ca++ overload of sufficient magnitude to cause an increased oscillatory release of Ca++ from the sarcoplasmic reticulum (SR), provided energy is available in the form of ATP. A sustained increase of Ca++ is likely to reflect energy depletion and therefore exclude a significant contribution of DADs to arrhythmia development. Thus, DADs are more likely to play a role in: (i) reperfusion arrhythmias and (ii) arrhythmias arising in moderately ischemic tissue, than in severe ischemia with marked energy depletion.

Calcium channel blockers and early ischemic ventricular arrhythmias: electrophysiological versus anti-ischemic effects.

Calcium antagonists are able to reduce the incidence of early ischemic arrhythmias when given as pretreatment in a variety of models of acute myocardial regional ischemia. Two possible modes of action are electrophysiologic and anti-ischemic. First, a direct electrophysiologic effect of the calcium antagonist agents on the inward calcium current could inhibit four calcium-dependent phenomena, each of which has been linked to the development of ischemic arrhythmias: the slow response, delayed afterdepolarizations (DADs) and calcium-dependent automaticity, enhanced membrane depolarization and conduction slowing. The special circumstances in which each of these could occur are delineated. An anti-ischemic effect of calcium antagonists must also be considered in some models, because of the negative inotropic, negative chronotropic and coronary and peripheral vasodilator properties. Thus far a specific role for the calcium current in early ischemic arrhythmias has only been shown in a few models and not tested in man.

Trimetazidine: Effects on delayed afterdepolarizations (DADs) and upstroke velocity of the action potential

Delayed afterdepolarizations have been described to occur during reperfusion after ischemia; it is the oscillatory uptake and release of Ca 2+ from the sarcoplasmic reticulum that underlies the phenomenon [1-2]; oscillations of intracellular Ca z+ concentration are accompanied by fluctuation of membrane current and mechanical activity. The tested hypothesis was that trimetazidine might be antiarrhythmic by