Nitric oxide (NO) is a potent vasodilator that is released from a variety of cells including endothelial cells (1), smooth-muscle cells (2), polymorphonuclear leukocytes (3), and nerve endings (4). Its physiological role in the cardiovascular system is widespread and includes the local control of blood flow to vital organs (2), inhibition of platelet adhesion (5,6) and aggregation (7), and inhibition of leukocyte adhesion (8,9). Under pathophysiologic conditions such as myocardial ischaemia, exogenously administered NO (in the form of NO-donating drugs) has been shown to reduce myocardial infarct size and leukocyte accumulation (10) and to suppress occlusion- and reperfusion-induced ventricular arrhythmias (11-13).
Although the potential for exogenous NO to reduce arrhythmias has been well documented in some species, recent studies in rats have revealed conflicting results. For example, molsidomine (the parent compound of sin-1) has been shown to reduce both ventricular ectopic counts and the incidence of ventricular fibrillation during ischaemia (11) and reperfusion (12) in dogs, whereas pirsidomine had a less marked antiarrhythmic effect (i.e., no suppression of VF) in pigs (13). Studies performed in rats, however, have failed to demonstrate a beneficial effect of NO donors on arrhythmias. Glyceryl trinitrate was unable to prevent reperfusion-induced VF in anaesthetised rats (14), whereas neither sin-1 nor sodium nitroprusside modified arrhythmias during ischaemia (15).
In contrast to the preceding studies, there is evidence that endogenous NO may indeed play a protective role in rats. In a series of recent studies (16-18), Pabla and Curtis showed that, in rat isolated hearts, inhibition of NO synthesis increases the incidence of reperfusion-induced ventricular fibrillation after a prolonged (60-min) period of ischaemia but not after shorter (5- or 25-min) periods and improves postischaemic contractile dysfunction (19). However, although NO has been implicated as a potential mediator of the antiarrhythmic effect of preconditioning in dogs (20-22), there is evidence that it is not responsible for the protective effects of preconditioning against reperfusion arrhythmias in rats (23).
In addition to its potential beneficial effects, NO also has cytotoxic effects that may become evident under pathologic conditions. For example, NO is capable of interacting with superoxide, generated from a variety of sources during myocardial ischaemia and reperfusion, to produce peroxynitrite, which then decomposes to form the potentially damaging hydroxyl radical (24). In addition, NO binds avidly to iron sulphur-centred enzymes that are required for essential cellular metabiolic activity (25). Thus under conditions of ischaemia, in which mitochondrial respiration is already compromised, an excessive production of NO could be detrimental. Indeed, inhibition of NO production by NG-nitro-L-arginine methyl ester (L-NAME) has been shown to be protective against infarct size (26), myocardial reoxygenation injury (27), and necrosis after focal cerebral ischaemia (28).
Thus there is still wide disagreement regarding the potential for NO to act as a cardioprotectant. This may well stem from the fact that the studies described all used different species (dogs, pigs, rats) and models (isolated hearts vs. in vivo studies) and assessed different end points (ischaemic vs. reperfusion arrhythmias, infarct size). To consider the role of NO during myocardial ischaemia fully, we performed a series of experiments in one experimental model (coronary occlusion in anaesthetised rats) by using one end point (ischaemic arrhythmias) to assess (a) the effects of an NO donor on ischaemic arrhythmias, (b) whether endogenous NO is responsible for the marked attenuation of ischaemic arrhythmias by a short period of ischaemic preconditioning, and (c) whether endogenous NO plays a modulatory role in the genesis of ischaemic arrhythmias in the absence of preconditioning.
METHODS AND MATERIALS
Studies in an in vivo rat model of myocardial ischaemia
Surgical preparation. Male Sprague-Dawley rats (260-380 g) were anaesthetized with sodium pentobarbitone (60 mg/kg i.p.) and maintained under anaesthesia by injections of sodium pentobarbitone (6-mg i.v. bolus) as required. The left femoral artery was cannulated for measurement of arterial blood pressure (by using a Statham pressure transducer), and the left femoral vein was cannulated for the administration of anaesthetic and drugs. A tracheotomy was performed to allow artificial ventilation, and the core temperature was measured with a rectal thermometer and maintained within the normal range with a table lamp. The electrocardiogram (ECG) was recorded from standard limb leads.
The rats were prepared for ligation of the left main coronary artery by the method described by Clark et al. (29). In brief, a left thoracotomy was performed by sectioning the fourth and fifth ribs, 4-5 mm from the sternum, and the animals were immediately ventilated on room air by using a Harvard respirator (Harvard Apparatus, South Natick, MA, U.S.A.) at a tidal volume of 2 ml/100 g and a rate of 54 strokes/min. This maintained blood gas parameters within the normal range (PO2, >85 mm Hg; PCO2, 35-40 mm Hg; pH, 7.40-7.45). The pericardium was opened and the heart exteriorized by gentle downward pressure on the ribs. A 6-0 silk suture on a curved taper needle (Mersilk; Ethicon, Edinburgh, Scotland) was passed underneath the left main coronary artery just below the tip of the left atrial appendage, 2-3 mm from its origin. The heart was then repositicned within the thorax.
Regional myocardial ischaemia was achieved by tying the ligature with a single knot. In animals that were subjected to a preconditioning protocol, the ends of suture were threaded through the hole of a small plastic button and a length of polyethylene tube. The ligature was then pulled tight and fixed by inserting another narrower tube. Reperfusion could then be allowed by releasing this snare. All animals were allowed to stabilize for 15 min after the surgical procedure. Any animal that developed spontaneous ventricular arrhythmias or showed a sustained decrease in mean arterial blood pressure to <70 mm Hg during this period was automatically excluded from the study.
Haemodynamic parameters and arrhythmia analysis. Systolic, diastolic and mean arterial blood pressures were measured from the arterial pressure trace. Heart rate was calculated from the ECG. The ECG and blood pressure were continuously recorded on a Grass polygraph (Model 7D; Grass Instruments, Quincy, MA, U.S.A.). Ventricular arrhythmias were analysed under the guidelines of the Lambeth Conventions for the analysis of experimental arrhythmias (30). These were identified as single ventricular premature beats (VPBs), salvos (couplets and triplets), and ventricular tachycardia (defined as a run of four or more consecutive ectopic beats). Bigeminy was identified as repeated salvos of couplets. The total number of ventricular arrhythmias was calculated as the sum of these three types of arrhythmia. The percentage incidence of ventricular tachycardia (VT), reversible and irreversible ventricular fibrillation (VF), and mortality were determined for each group.
- To determine the effect of the NO donor C87-3754, on ischaemia-induced arrhythmias
- After a 15-min equilibration period following surgical preparation, 22 rats were given either saline (as control; n = 10) or C87-3754 (1 mg/kg; n = 12). The injection volume for both groups was 0.2 ml. Ten minutes after drug or saline administration, the coronary artery was occluded for 30 min. The subsequent ventricular arrhythmias were then analysed as described previously.
- To determine the role of the NO pathway as a mediator of the antiarrhythmic effects of preconditioning
- The role of the endogenous NO pathway as a possible modulator of ischaemic preconditioning was studied by determining the effects of two NO pathway blockers, NG-nitro-L-arginine methyl ester (L-NAME, a NO synthesis inhibitor) and methylene blue (a guanylate cyclase inhibitor), on the reduction in the incidence and severity of ischaemia-induced arrhythmias by preconditioning. The preconditioning protocol used in this study involved a 3-min occlusion, followed by 10 min of reperfusion, before a second period of 30 min of sustained coronary occlusion. Rats were given an intravenous infusion of either L-NAME (10 mg/kg, n = 9; 50 mg/kg, n = 9; 100 mg/kg, n = 9), methylene blue (10 mg/kg, n = 9; 50 mg/kg, n = 9), or saline (controls, n = 9) a rate of 0.1 ml/min for 10 min before the preconditioning protocol was started. The incidence and severity of the arrhythmias during the second period of coronary occlusion were analysed as described previously.
- To determine the modulatory role of endogenous NO in arrhythmogenesis during ischaemia
- The potential effects of the inhibitors L-NAME and methylene blue on ischaemic arrhythmias were assessed on the incidence and severity of ischaemia-induced arrhythmias. Rats were given a 10-min i.v. infusion of L-NAME (10, 50, or 100 mg/kg; n = 9 for each group), methylene blue (1, 10, or 50 mg/kg; n = 9 for each group), or saline (n = 9) before being subjected to a single 30-min coronary occlusion.
- The experimental protocols used in the three studies are summarized in Fig. 1.
Studies in rat isolated Langendorff-perfused hearts
To demonstrate the ability of L-NAME to block NO synthesis in rat hearts, the following additional experiments were performed. Twenty-seven rats (300-350 g) were anaesthetized with sodium pentobarbitone (60 mg/kg, i.p) containing heparin (500 IU/kg). The hearts were excised and transferred rapidly (i.e., within 1 min to avoid triggering of preconditioning) to a Langendorff apparatus, where they were perfused at constant flow, the rate of which was determined by the body weight of the animal, as described previously (31). The hearts were perfused with a modified Krebs-Henseleit buffer (31), which was composed of NaCl (118.0 mM), KCl (4.7 mM), CaCl2 (2.52 mM), MgSO4 (1.66 mM), NaHCO3 (24.88 mM), KH2PO4 (1.18 mM), glucose (5.55 mM), and Na-Pyruvate (2.0 mM), gassed with 5% CO2 + 95% O2 and equilibrated to a pH value of 7.4. Perfusion pressure was measured via a side-port on the aortic cannula, and heart rate was calculated from a surface ECG, recorded via platinum electrodes (Grass Type E2) placed on the apex and right atrium.
After 15 min of stabilisation perfusion with buffer, the hearts were switched to perfusion medium supplemented with either L-arginine (10 μM; n = 9), L-NAME (100 μM; n = 9), or both compounds (n = 9) for 20 min. The subsequent changes in perfusion pressure and heart rate from baseline values were assessed at regular intervals after the start of drug perfusion.
All results are expressed as means ± SEM or as the percentage incidence of events. To compare the means of multiple groups, in which the numbers of groups in a study were >2, an overall test based on analysis of variance (ANOVA) was first performed to give a single test statistic for differences between all groups. For normally distributed data (A-type data), such as mean blood pressure and heart rate, one-way ANOVA was used, whereas for nonparametric data (B-type data), such as the number of VPBs, the Kruskal-Wallis test was used. If, and only if, these tests indicated the significance of differences among groups, the following further tests were used to detect the source of difference. For A-type data, the Tukey's test was applied to the case for which all pairs comparisons were of interest, whereas Dunnett's test was used for comparing the control group with each of the other groups. For the B-type data, the Dunn's multiple-comparison was used. If the comparison was only between two groups, an unpaired t test (for A-type data) or Mann-Whitney U test (for B-type data) was used. To compare the incidence of events, the Fisher's exact test was used. The statistical significance was taken as p < 0.05.
Haemodynamic responses to C87-3754 before and during ischaemia
The changes in blood pressure and heart rate after intravenous administration of C87-3754 before and during coronary ligation are shown in Table 1. C87-3754 markedly lowered the mean arterial blood pressure immediately after injection. In an additional group of rats not subjected to coronary occlusion, the maximal response to C87-3754 was observed during the first 5-20 min, after which the blood pressure gradually returned to the predrug level by 60 min (data not shown). Coronary artery occlusion in control rats resulted in a transient decrease in blood pressure. In rats given C87-3754, in which blood pressure was markedly reduced before ischaemia, this effect of coronary artery occlusion was less marked. Furthermore, the depressor effect of C87-3754 was fully reversed by 20 min after occlusion (30 min after C87-3754 injection). C87-3754 had no sustained effect on heart rate.
Effects of C87-3754 on ischaemia-induced ventricular arrhythmias
The number and pattern of ventricular arrhythmias over the 30-min occlusion period in control and treated rats are shown in Fig. 2. In controls, >60% of the total arrhythmia count was due to VT, with the remaining arrhythmias being seen as single VPBs, bigeminy, and salvos. C87-3754 had no significant effects on either the total number of arrhythmias or any of the different arrhythmia types, compared with controls. C87-3754 had no effect on either the incidence of VT (100% in both groups) or VF (60% in controls vs, 58% in drug-treated rats) or on mortality (40 and 42%) during the 30-min period of ischaemia.
Haemodynamic response to L-NAME and methylene blue before and during myocardial ischaemia
Over the dose range of 10-100 mg/kg, L-NAME induced a significant increase in blood pressure (Fig. 3). This effect was rapid in onset for all doses tested and reached a plateau within 4-6 min. Furthermore, the maximal effects observed with each dose were similar. The pressor effects of all doses of L-NAME in rats not subjected to ischaemia lasted for >30 min (examined in an extra group of rats; data not shown). However, this pressor effect appeared to be lost rapidly after the induction of sustained ischaemia, irrespective of whether or not the rats were preconditioned. Methylene blue (10 mg/kg) had no effect on blood pressure, whereas the higher dose of 50 mg/kg produced a marked hypotension (Fig. 4). Coronary occlusion had no further effects on the blood pressure in the presence of either inhibitor.
Effects of L-NAME and methylene blue on the antiarrhythmic effect of ischaemic preconditioning
The ventricular arrhythmias that occurred during the 30-min sustained coronary occlusion in controls are shown in Fig. 5. Sustained occlusion without preconditioning resulted in severe ventricular arrhythmias with a high incidence of VF (67%), with 44% of the rats dying of sustained VF. However, when the sustained ischaemia was preceded by a brief (3-min) coronary occlusion, the severity of arrhythmias was dramatically reduced. The total VPB count was reduced, and the incidence of VF declined to 9%. None of the preconditioned rats died of sustained VF. None of the doses of L-NAME or methylene blue attenuated this profound antiarrhythmic effect of preconditioning. Indeed, L-NAME enhanced the antiarrhythmic effect of preconditioning at a dose of 50 mg/kg, and methylene blue produced a further reduction in total number of VPBs and the incidence of VT in preconditioned rats (Fig. 5).
Effects of L-NAME and methylene blue on ischaemia-induced arrhythmias without preconditioning
L-NAME (10 mg/kg) given to rats before coronary occlusion without preconditioning caused a significant reduction in the total number of VPBs (Fig. 5). A higher dose of 50 mg/kg also reduced arrhythmia count to the same degree (data not shown). There was also a reduction in the incidence of VF with L-NAME (from 67% in nonpreconditioned controls to 33% and 45% with 10 and 50 mg/kg, respectively), although this was not statistically significant. Methylene blue reduced arrhythmia count (not significant because of spread of data) and the incidence of VF (significant to p < 0.05 with 50 mg/kg; Fig. 5) in a dose-dependent manner.
Effects of L-NAME and L-arginine on coronary perfusion pressure in rat isolated hearts
In agreement with the results seen in the in vivo experiments, L-NAME induced a marked vasoconstrictor effect in isolated rat hearts (Fig. 6). Perfusion pressure was gradually increased and reached a maximum after 15-20 min. This effect of L-NAME was significantly inhibited when the hearts were coperfused with L-NAME and L-arginine, although complete inhibition was not achieved. L-Arginine alone caused a slight decrease in perfusion pressure. Interestingly, when hearts were washed with drug-free medium after L-arginine + L-NAME perfusion, the perfusion pressure did not recover to the baseline but showed a level similar as that seen in the presence of L-NAME. Heart rate was only slightly affected (not significant) by both L-NAME and L-arginine (Fig. 6).
This study aimed to assess the ability of both endogenous and exogenous NO to modify ischaemia-induced arrhythmias in rat hearts in vivo and to determine whether NO plays a role in the antiarrhythmic effects of ischaemic preconditioning in this species. The results of our investigation showed that an NO donor is unable to suppress ischaemic arrhythmias and that endogenous NO is unlikely to act as a mediator of the protective effect of ischaemic preconditioning against these arrhythmias. Interestingly, we also found that inhibition of NO synthesis (with L-NAME) and guanylate cyclase (with methylene blue) both suppressed ischaemic arrhythmias, albeit at relatively high doses.
Previous studies from our laboratory in anaesthetised pigs showed that the sydnonimine NO donor pirsidomine exerts a moderate suppressant effect against ischaemiainduced arrhythmias, in that there was a reduction in the total count of VPBs but no reduction in the incidence of VF (13). In that study, pirsidomine exerted a marked, but slowly developing, hypotension, which was proposed as the main mechanism by which it reduced arrhythmias (by decreasing afterload on the heart and thus reducing the severity of ischaemia). In this study in rats, we used the metabolite of pirsidomine, C87-375432, because we found that pirsidomine, given in a dose similar to that used in the pig study, did not have any effect on blood pressure. However, despite the fact that C87 3754 produced a hypotension, it failed to reduce arrhythmias resulting from coronary occlusion. Although this finding is in contrast to studies with sydnonimine NO donors in pigs (13) and dogs (11,12), it does agree with the results from a study of ischaemia-induced arrhythmias in rats (15) in which doses of sin-1 (the metabolite of molsidomine) and sodium nitroprusside sufficient to cause a marked systemic hypotension did not reduce ischaemic arrhythmias. The effects of systemic hypotension do not appear to have the same impact on ischaemic arrhythmias in rats as they do in larger animals. In dogs, NO donors are seen to decrease the incidence of VF (11,12), where it could be argued that the hypotension is indicative of coronary vasodilation, which would improve collateral flow to the ischaemic tissue and thus reduce ischaemia severity. This would not necessarily act as an antiarrhythmic mechanism in rats because of the low level of collateral blood flow (33). Pigs also have a low collateral reserve (34), which might explain why pirsidomine was able to reduce only the less severe arrhythmias (i.e., the number of VPBs) in this model rather than suppressing VF.
The second aim of this study was to determine whether, as has been suggested from studies in dogs, endogenous NO may be responsible for the profound antiarrhythmic effect of preconditioning. We found that neither preventing NO synthesis with L-NAME nor blocking the increase in cGMP with methylene blue had any effect on the ability of a short period of ischaemia to exert a marked antiarrhythmic effect against ischaemic arrhythmias. This is in contrast to a series of studies in mongrel dogs (20-22), which showed that inhibition of NO synthesis can attenuate both the antiarrhythmic effects of preconditioning and those of intracoronary bradykinin. We previously showed that, in in vivo rats, bradykinin does not reduce ischaemic arrhythmias nor does the bradykinin B2 antagonist prevent the protection conferred by preconditioning (35). Clear differences appear to exist between rats and other species with respect to the possible roles of NO/bradykinin in the antiarrhythmic effects of preconditioning and indeed in the potential roles of other endogenous mediators of the different facets of preconditioning (recently reviewed in 36). Our results agree with those of Lu et al. (23), who failed to demonstrate any reversal of the protective effects of preconditioning against reperfusion-induced arrhythmias in rats with L-NAME or NG-monomethyl-L-arginine (L-NMMA). It could be argued that the reason for the differences between rats and other species is an absence of NO generation in rat hearts. However, our observation that L-arginine reversed the vasoconstriction by L-NAME in isolated perfused hearts confirms previous evidence that NO contributes to vasodilator tone in this species (37). Furthermore, we measured NO synthase (NOS) activity in the hearts of rats by measuring the conversion of L-arginine to L-citrulline and found that whereas the activity is relatively low (11,436 ± 145 pmol L-Cit g/min for Ca2+-dependent and 34,832 ± 112 pmol L-Cit g/min for Ca2+-independent isoforms) in comparison with other tissues, it is nevertheless there.
The final aim of our study was to determine the effects of interfering with endogenous NO production (by blocking NOS with L-NAME) and responses to NO (by blocking guanylate cyclase with methylene blue) on ischaemic arrhythmias, without preconditioning, to see if NO plays a modulatory role during ischaemia. Our results were somewhat surprising in that, rather than seeing an increase in arrhythmias, both of these interventions had an antiarrhythmic effect. Furthermore, at the highest doses, both L-NAME and methylene blue augmented the antiarrhythmic effects of preconditioning. These results contrast with those from two studies by Pabla and Curtis, who found that both inhibition of guanylate cyclase (with methylene blue) and blockade of neuronal NOS (with 7-nitro-indazole) in rat isolated hearts subjected to ischaemia and reperfusion caused an increase in the incidence of reperfusion-induced VF (16,18). However, in the studies by Pabla et al. (16), the protective antifibrillatory effect of NO was found to be evident only after an antecedent period of 60 min of ischaemia but not after periods of 5 or 35 min of ischaemia. Because in our experiments the arrhythmias we observe take place in the first 30 min of a sustained occlusion, this may be too early to see any antiarrhythmic effect. Ischaemic arrhythmias in rats occur in two phases (29), the first (phase 1) appearing between 6 and 25 min after occlusion and the second (phase 2), 1.5-4 h after occlusion. The electrophysiologic bases of these two phases of arrhythmias are different, the first phase being largely due to reentry (and featuring VF as a common occurrence) and the later phase due to an increased automaticity (comprising mainly single premature beats, salvos, and some VT; 38). Furthermore, it is probable that the electrophysiologic basis of reperfusion-induced arrhythmias is quite different from that of ischaemia-induced arrhythmias (39). Thus agents that are protective against one type of arrhythmia would not necessarily protect against another. With respect to ischaemic arrhythmias, why NO appears to be so effective in dogs but not in rats is not immediately clear. Once again, an effect of NO (both generated locally and delivered by a NO donor) on collateral blood vessels to improve blood flow to the ischaemic myocardium may be the underlying mechanism in dogs.
In support of our findings with L-NAME and methylene blue on ischaemic arrhythmias, some studies showed a cardioprotective effect of NOS inhibition with L-NAME against myocardial infarct size in rabbit hearts (26) and in piglets (27). Furthermore, a recent study by Naseem et al. (40) demonstrated that prolonged inhibition of NO production could improve myocardial function and reduce arrhythmias after ischaemia/reperfusion in rat isolated perfused hearts, an effect that was reversible with L-arginine. In a further study, Yellon's group (41) attributed the effects of L-NAME on myocardial infarct size in rabbits to an increase in endogenous adenosine levels, and it has been shown that a reduced NO production results in an increased adenosine production (42). Because previous studies from our laboratory have shown that endogenous adenosine can act as a very powerful antiarrhythmic agent in a range of species (43-45), including rats, this could explain our findings with L-NAME. One final explanation for the antiarrhythmic effect of L-NAME could be based on actions other than NOS inhibition. It has been reported that L-NAME can also block muscarinic receptors (46), although it is difficult to reconcile this effect with a protective effect because acetylcholine has been shown to reduce myocardial injury in rats (47).
It is also possible that the protective effect seen with methylene blue against ischaemic arrhythmias may not necessarily be related to inhibition of guanylate cyclase, but rather may be directly related to an effect on the metabolic status of the ischaemic cell. Methylene blue can accept electrons and transfer electron flow through the respiratory chain (48), just as molecular oxygen does in normally functioning mitochondria. Under ischaemic conditions, methylene blue could, at least partially, take the place of oxygen, thus serving as as electron carrier to maintain both the function of the mitochondria and the energy balance of the myocardial cell. This would have a beneficial effect on the electrophysiology of the cardiac cell, decreasing the predisposition to arrhythmias.
In summary, the results of this study have failed to show any protective effect of either exogenously administered NO or of endogenously generated NO against ischaemia-induced arrhythmias in rats. There is also no evidence that, in this species, NO is responsible for the marked antiarrhythmic effect of ischaemic preconditioning. The finding that inhibitors of NOS and guanylate cyclase exert an antiarrhythmic rather than a proarrhythmic effect is somewhat surprising. However, these effects may be related to actions unrelated to interference with the NO-cGMP pathway.
Acknowledgment: W. Sun was supported by an ORS Award and a University of Strathclyde PhD Studentship.
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