Faculty Bibliography

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.

There is evidence that the "ATP-sensitive" potassium channel opens, at least during the early stages of myocardial ischemia, despite relatively high ATP levels. Thus, channel opening may partially contribute to potassium efflux and accumulation of extracellular potassium, but probably much more profoundly to electrical abnormalities associated with ischemia, including the development of lethal arrhythmias. Several factors are discussed that may promote a significant open-channel probability of the channel, in spite of relatively high levels of ATP. It is argued that, even with a very low open probability, the magnitude of total membrane current carried by these channels may be substantial (comparable to other potassium currents) because of the high density and conductance of the ATP-sensitive potassium channel. Finally, it is shown how the ATP-sensitive potassium channel may play a role in various tissue types, ranging from the physiological to the pathophysiological. This potassium channel is therefore increasingly targeted for drug development and research.

Hypothetically, certain ischemic and reperfusion arrhythmias may result from the activity of the calcium-dependent transient inward current. The effects of the angiotensin converting enzyme inhibitor, perindoprilat, on the transient inward current of guinea pig ventricular myocytes were studied. The transient inward current was evoked by superfusing the cell with a modified Tyrode's solution (5.4 mM CaCl2 and 0.54 mM KCl). Repetitive voltage clamp steps from a holding potential of -55 to +20 mV (1,000 ms, 0.1 Hz) were applied while dialyzing the cell internally. When administered simultaneously with the change over to the low K+ high Ca2+ solution, perindoprilat (1 microM) decreased the transient inward current from -9.55 +/- 0.31 to -3.24 +/- 0.24 microA/cm2 (p less than 0.05). A similar decrease was observed when perindoprilat was administered after first inducing the transient inward current. Perindoprilat also protected from the effects of norepinephrine (0.01 and 0.1 microM), which increased the amplitude of the transient inward current from -9.76 +/- 0.17 and -9.99 +/- 0.32 microA/cm2 at the end of the 15-min control period to -11.13 +/- 0.67 and -12.67 +/- 0.49 microA/cm2, respectively (p less than 0.05). The effects of perindoprilat were independent of angiotensin II, which in this preparation decreased the transient inward current. Based on our results, we conclude that perindoprilat decreases the transient inward current and prevents the action of norepinephrine on the transient inward current. The direct effect of the angiotensin converting enzyme inhibitor observed on the transient inward current might explain why angiotensin converting enzyme inhibitors reduce calcium-dependent ouabain-induced or reperfusion arrhythmias.

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).

STUDY OBJECTI

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.

Although the Ca++ channel blockers can reduce early ischemic ventricular arrhythmias, the mechanisms are unclarified. The antiarrhythmic action of Ca++ antagonists may either be due to vasodilation and negative chronotropism or to trans-sarcolemmal Ca++ influx inhibition. In these studies we investigated the possible individual and additive effects of coronary flow, heart rate and Ca++ antagonism on ventricular arrythmia development in isolated, paced, globally underperfused guinea pig hearts. When the coronary flow during ischemia was raised from 5 to 7% of control and/or the stimulation frequency was decreased from 6 to 4 Hz, ATP and creatine phosphate levels were conserved and intraventricular conduction slowing leading to ventricular tachycardia (VT) was delayed. In contrast, when the coronary flow and pacing rates were fixed at 7% and 6 Hz and diltiazem (10(-6) M) was included in the perfusion medium, there was no effect on tissue high-energy phosphate depletion and development of VT. Even when the breakdown of ATP and the onset of VT were accelerated by isoprenaline (10(-6) M), diltiazem was not antiarrhythmic at this flow rate. Only when the coronary flow was reduced to 5% of control, in the absence and presence of isoprenaline, did diltiazem delay ventricular arrhythmias through a mechanism that was independent of changes in coronary flow and heart rate.