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Despite the explosion of new information on nitric oxide (NO), important questions about its role in regulating cardiac electrophysiology remain unanswered. Recent in vitro and in vivo animal studies have discovered a number of new electrophysiological properties of NO, some of which may contribute to a reduction in fatal arrhythmias induced by acute myocardial ischemia. This review summarizes the influences of NO on heart rate, atrioventricular conduction, ventricular repolarization and the development of ventricular arrhythmias during acute myocardial ischemia.
Nitric oxide (NO) was initially known as endothelium-derived relaxing factor (1,2). The physiological and pathophysiological roles of NO within the cardiovascular system have been actively investigated, and a tremendous amount of new information has been reported. The endothelial l-arginine-NO pathway is a well known vasodilation mechanism that influences peripheral vascular resistance and systemic blood pressure. NO also plays an important part in regulating contractility of myocytes and the responsiveness of myocytes to inotropic stimuli (3). Investigations on the electrophysiological actions of NO are gathering pace, and a number of new electrophysiological properties of this compound have now been discovered. Some of these newly identified electrophysiological characteristics may have significant clinical implications, such as prophylaxis of cardiac arrhythmias and sudden cardiac death. This review summarizes the developments in this area, with a view to stimulating further debate and introducing new areas of research.
At physiological conditions an impulse is initiated by the sinus node and then passes successively through the atrial myocardium to the atrioventricular (AV) node, the His bundle, the right and the left bundle branches, and the Purkinje fibres in the ventricular myocardium. A basal heart rate is set by this intrinsic conduction system and is regulated by the autonomic (sympathetic and parasympathetic) innervation of the heart.
The effects of NO on the heart rate are complex. Exogenous and endogenous NO seem to have different actions on the basal heart rate. In the isolated animal heart, NO appears to have a dose-dependent chronotropic effect on the sinus node. Sodium nitroprusside, a NO donor, has a biphasic chronotropic effect; it increases the beating rate of the guinea pig sinus node at a low concentration and decreases the heart rate at a high concentration (4). The action of NO on the sinus node is believed to result from the stimulation of the hyperpolarization-activated inward current, If, in the atrial and sinus tissues (4). In the isolated rat heart, sodium nitroprusside (10 μmol/L) increases the average heart rate by 56 beats/min (5). Another NO donor, 3-morpholinosydonimine, had little effect on the beating rate of the isolated rat right atrium at low concentrations but reduced the rate at very high concentrations (6).
In the intact animal heart, the NO donors molsidomine and sodium nitroprusside have been found to elicit a linear increase in heart rate in anesthetized rabbit after cardiac autonomic denervation and beta-adrenergic blockade (7). In healthy subjects, sodium nitroprusside (2 μg/kg/min) causes some 12% increase in heart rate in the absence of changes in brachial and aortic blood pressure (8).
The effect of endogenous NO on the intrinsic heart rate is controversial. Manning et al (9) showed that long term blockade of NO release with Nω-nitro-l-arginine methyl ester (L-NAME) in dogs can cause hypertension and bradycardia. Heart rate variability, measured as a 24 h standard deviation, was significantly diminished during chronic L-NAME administration. Hypertension, but not bradycardia, was reversed by l-arginine, a NO precursor (9). One may argue that the alteration in heart rate in the above study is difficult to interpret because of the presence of the baroreflex, which may itself influence the basal heart rate following L-NAME administration and the subsequent increase in blood pressure. However, the changes in heart rate following endogenous NO suppression may be explained by the alteration of baroreflex sensitivity, and reversal of hypertension with L-NAME was not accompanied by recovery of bradycardia (9). Also, chronic NO synthase (NOS) inhibition does not change baroreflex sensitivity (10). Therefore, bradycardia during long term L-NAME treatment is likely the result of NO inhibition.
In pentobarbital-anesthetized dogs, in which cardiac innervation and baroreflex were blocked by atropine and propranolol, infusion of a NOS inhibitor, NG-monomethyl-l-arginine (L-NMMA), into the sinus artery had no appreciable effect on the basic heart rate (11). In pentobarbital anesthetized sheep, an increase in endogenous NO synthesis during high pressure intracoronary perfusion had no significant effect on heart rate but prolonged ventricular repolarization without changing the heart rate (12).
NO does not appear to play a critical part in regulating AV nodal conduction in intact animals at physiological conditions. Inhibition of basal NO production following infusion of L-NMMA into dog AV nodal artery did not alter the A-H interval, a measurement of AV nodal conductivity (11). In anesthetized sheep, intravenous infusion of l-arginine did not cause appreciable changes in the PR interval on body surface electrocardiogram (13). However, NO seems to modulate the AV node’s responses to autonomic stimulation. In isolated rabbit AV nodal cells, NO was found to mediate adenosine’s suppressive effect on an isoprenaline-induced increase in the inward calcium current through a cGMP pathway (14,15). In intact dog heart, infusion of L-NMMA into the AV nodal artery attenuated the negative chronotropic and dromotropic responses of these tissues to vagal nerve stimulation (11). Intravenous administration of l-arginine reversed these responses toward the control values. In addition, NO inhibition with L-NMMA enhanced the effects of sympathetic stimulation or isoproterenol infusion on AV conduction (11), indicating that basal NO suppresses the stimulative effect of sympathetic nerves on the heart.
Under physiological conditions, basal NO does not appear to have a significant influence on ventricular repolarization. In pentobarbital-anesthetized, open chest sheep, intravenous infusion of NG-nitro-l-arginine did not change the epicardial activation-recovery interval, a well established measure of ventricular repolarization in the in vivo heart, or the QT interval on body surface electrocardiogram, another indicator of ventricular repolarization (13). In dogs, NO inhibition with pericardial application of L-NMMA exerted little change in the ventricular effective refractory period (16).
Although basal NO has little effect on ventricular repolarization, increased NO synthesis or release prolongs it. In anesthetized sheep, high pressure intracoronary perfusion, which inevitably increases NO production, led to a small but significant prolongation in mean ventricular fibrillation intervals and activation-recovery intervals (12,13). NO also mediates the changes in ventricular repolarization during sympathetic stimulation. In the intact dog heart, pericardial l-arginine administration and a subsequent increase in the systemic content of NO reduced the shortening of ventricular effective refractory periods during sympathetic stimulation (16). This effect was completely abolished by simultaneous administration of L-NMMA (16).
Acute myocardial ischemia or reperfusion of the ischemic myocardium results in fatal ventricular arrhythmias such as ventricular tachycardia or fibrillation. NO has two actions that may potentially prevent ventricular arrhythmias induced by myocardial ischemia or reperfusion. It dilates coronary arteries, which may reduce the extent of ischemia, and suppresses platelet adhesion and aggregation (17), which may prevent thrombosis within the coronary arteries and therefore the recurrence of ischemia. In addition, NO has some favourable effects on ventricular repolarization (16), all of which may interrupt the formation of ventricular arrhythmias.
However, recent reports on the potential antiarrhythmic effect of NO have been inconsistent and sometimes contradictory. Pabla and Curtis (5) showed that NO behaves as an endogenous antifibrillatory factor in the isolated rat heart during reperfusion following sustained ischemia. L-NAME, which blocks NOS, increased the incidence of reperfusion-induced ventricular fibrillation from 5% in the control condition to 35% after 60 min of ischemia (5). The profibrillatory effect of L-NAME was prevented in hearts co-perfused with l-arginine (5). Furthermore, the proarrhythmic effect of L-NAME was prevented by pretreatment with a NO donor, sodium nitroprusside (5).
The above beneficial effects of NO on isolated rat heart have not been reproduced in the intact rat heart. In pentobarbital-anesthetized rats, sodium nitroprusside had no prophylactic effects on ventricular tachycardia, ventricular fibrillation or mortality resulting from 25 min myocardial ischemia (18). Another NO donor, 3-morpholinosydnonimine-N-ethylcarbamide, did not suppress the incidence or severity of ischemia-induced arrhythmias either (18). The authors of this study concluded that NO donors do not prevent arrhythmias induced by acute coronary artery occlusion or reperfusion in anesthetized rats. In a recent study, Sun and Wainwright (19) showed that C87-3754, also a NO donor, caused a significant reduction in arterial blood pressure in anesthetized rats without reducing the incidence or severity of ventricular arrhythmias during a 30 min myocardial ischemia.
Recent studies of intact rat hearts have shown that NO may mediate the antiarrhythmic effect of certain chemical compounds. Resveratrol, an antioxidant in red wines, reduces the incidence and duration of ventricular tachycardia and fibrillation induced by ischemia and reperfusion (20). This antiarrhythmic effect is associated with an increase in plasma NO concentration in these animals (20). Suppression of NO synthesis with L-NAME has been found to abolish the antiarrhythmic effects of a newly discovered compound, honokiol, and its isomer, magnolol, in rats with 30 min coronary ligation (21).
Other evidence indicates that instead of being antiarrhythmic, NO may actually facilitate the occurrence of arrhythmias during myocardial ischemia in rat hearts. Naseem et al (22) reported that NO inhibition with NG-nitro-l-arginine, a NOS inhibitor, improves the left ventricular function and reduces the incidence of ventricular arrhythmias induced by ischemia or reperfusion. The duration of the sinus rhythm, or the arrhythmia-free period, in the NG-nitro-l-arginine-treated group was also prolonged (22). Ferdinandy et al (23) showed that in the isolated rat heart, pretreatment with tetrakis-2-pyridylmethyl-ethylenediamine, a potent metal chelator, reduced basal cardiac NO content and prevented the accumulation of NO during ischemia-reperfusion. The incidence of ischemia- and reperfusion-induced ventricular fibrillation and ventricular tachycardia was significantly reduced following NO inhibition (23). In line with this study, Liu et al (24) found that, in the in situ rat heart, myocardial ischemia and reperfusion-elicited arrhythmias were accompanied by a 90% increase in the NO content in the ischemic tissues, along with a sixfold rise in inducible NOS activity in the same tissue.
The reports on the antiarrhythmic effect of NO in large animals are less controversial than those in small animal models such as rats. In anesthetized dogs, inhibition of basal NO resulted in a reduction in the antifibrillatory effect elicited by two ‘preconditioning’ occlusions of the left anterior descending coronary artery, implicating NO as an endogenous mediator of preconditioning against ventricular fibrillation (25). Also in anesthetized dogs, aminoguanidine, a NOS inhibitor, attenuated the cardiac protection against ischemic arrhythmias afforded by cardiac pacing (26). In chloralose-anesthetized pigs, intravenous administration of pirsidomine (1 mg/kg), a NO donor, reduced the incidence of ventricular ectopic beats following occlusion of the left anterior descending coronary artery (27). Although the incidence of ventricular fibrillation was unaffected by pirsidomine, the time to onset of this arrhythmia was significantly prolonged by this intervention (25). In open chest dogs, pericardial application of l-arginine reduced the severity of ventricular arrhythmias induced by sympathetic stimulation on top of coronary occlusion (16). Pretreatment with the NO donors nicorandil and isosorbide-2-mononitrate reduced the incidence and severity of ventricular arrhythmias following occlusion of the left descending coronary artery in anesthetized dogs (28,29).
NO seems to mediate some drug-induced arrhythmias. NOS enzymes are essential to the control of NO synthesis. A constitutive form of NOS, eNOS, is expressed in myocytes and regulates the synthesis of NO in these cells. A higher rate of ouabain-induced ventricular arrhythmias was found in ventricular myocytes isolated from mice lacking a functional eNOS gene (30). Application of a NO donor, S-nitrosoacetylcysteine, diminished the drug-induced arrhythmias in these preparations (30). Furthermore, in isolated guinea pig heart, pretreatment with sodium nitroprusside and l-arginine reduced the incidence of digoxin-induced ventricular arrhythmia (31). Sodium nitroprusside, but not l-arginine, was also found to suppress digoxin-induced ventricular arrhythmias in intact guinea pig heart (31).
So far the electrophysiological properties of NO have mostly been studied using NO donors or NOS inhibitors. The actions of NO in the myocardium are extremely complex, with myocytes or cardiac neurons being the potential sites of action. It is almost impossible, at this stage, to show the exact mechanisms of action in the myocardium.
NO has been shown to mediate intracellular production of cyclic GMP through guanylyl cyclase (32), which may account for many of the observed electrophysiological actions of NO. NO has also been shown to inhibit slow inward calcium currents through cyclic GMP-dependent protein kinase (33). How these molecular effects may contribute to the observed electrophysiological effects of NO is still unclear.
Because NO dilates coronary arteries and suppresses platelet aggregation (17), a NO-mediated decrease in ventricular arrhythmias following myocardial ischemia may be due to improvement in myocardial perfusion in addition to the favourable changes in ventricular repolarization. Several studies have shown that l-arginine and NO donors diminish ischemia-reperfusion injury and improve postischemic mechanical function, whereas NOS inhibitors are associated with a poor recovery from the ischemia-reperfusion damage (34–37).
Studies addressing the role of NO in regulating cardiac electrophysiology have rapidly evolved from early experiments in small and large animal models. Some of the electrophysiological properties appear to be species specific. Endogenous NO does not have a significant effect on basal heart rate, but exogenous NO from NO donors increases the heart rate in vitro and in vivo. In large animals, NO prolongs ventricular repolarization under certain circumstances, such as during sympathetic stimulation or high-pressure intra-coronary perfusion. NO deficiency seems to mediate cardiac glycoside-induced ventricular arrhythmias in intact and isolated ventricular preparations. Data from large animal experiments have shown that NO reduces the severity and incidence of ventricular arrhythmias induced by sympathetic stimulation or acute ischemia. However, this antiarrhythmic effect has not been consistently reproduced in small animals such as rats.
The cellular mechanisms of the above electrophysiological actions of NO are largely unknown. Evidence for the direct effect of NO on cellular electrophysiology, such as action potential duration of myocytes, has not yet emerged. Further studies at both the systemic and the cellular level are warranted to clarify the role of NO in the regulation of cardiac electrophysiology, particularly its prophylactic effect on ischemia-induced ventricular arrhythmias.