INTRODUCTION

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OXYGEN-DERIVED OXIDANTS (O₂ · , H₂O₂, and · OH) are produced in the heart extracellularly and intracellularly during ischemia, as well as during coronary artery reperfusion, as demonstrated by paramagnetic and histochemical studies (5, 9, 15, 31, 35, 38). Despite its relatively short half-life, H₂O₂ is rapidly converted into the highly reactive hydroxyl radical (· OH) in the presence of Fe²⁺; the iron-chelating agent deferoxamine blocks this iron-catalyzed · OH formation (16). Thus deferoxamine acts to prevent the effects that H₂O₂ exerts on the electrical and contractile properties of isolated cardiac tissue (13). It also reduces the effects that H₂O₂ induces on the electrical properties of isolated ventricular myocytes (7). The fact that reperfusion arrhythmias occur with less frequency in the presence of deferoxamine has been ascribed to the effects that this agent exerts on H₂O₂ formation (8, 9). Although oxygen-derived free radicals affect cardiomyocytes directly, they may also exert their effects in situ indirectly by modifying the intrinsic cardiac nervous system, which regulates cardiac function.

The function of the intrinsic cardiac nervous system in cardiac regulation has been studied in situ (2) and in vitro (1, 21). The intrinsic cardiac nervous system represents the final common pathway of sympathetic and parasympathetic efferent autonomic control of cardiomyocyte behavior (2). Intrinsic cardiac neurons are sensitive to a number of chemicals (2). It is also known that the activity generated by canine intrinsic cardiac neurons is modified by transient coronary occlusion (24). Whether these neurons are sensitive to oxygen-derived free radicals remains to be established.

Oxygen-derived free radicals produced by the myocardium during ischemia and reperfusion influence the activity generated by rat cardiac sensory neurites associated with afferent axons in vagal (33, 34) and sympathetic (11) nerves, as well as the sensory neurites associated with afferent axons in feline vagal (23) and sympathetic (22) nerves. Oxygen-derived free radicals have been implicated in the damage that hypoxia induces in rat hippocampal (28) and spinal cord (27) neurons. They also depress the propagation of electrical activity along frog sciatic nerves (10).

Modification of ATP-sensitive potassium (K_ATP) channels has been proposed to protect cardiomyocytes from damage incurred during ischemia and reperfusion, as K_ATP agonists exert cytoprotective effects in such a situation (for reviews of this subject, see Refs. 17, 19). Although K_ATP-channel agonists and antagonists have received considerable attention with respect to their effects on cardiomyocyte function in physiological and pathological states, their cardioprotective properties remain controversial (for a review of this subject, see Ref. 20). K_ATP channels have been described in several different types of neurons (6, 29, 32). Whether such channels exist in intrinsic cardiac neurons remains unknown. Whether they are involved in cardioprotection when the heart is exposed to oxygen-derived free radicals also remains unknown.

The purpose of the present study was to determine whether H₂O₂ can influence the activity generated by mammalian intrinsic cardiac neurons. Second, we sought to determine whether the Fe²⁺ chelator deferoxamine inhibits H₂O₂-induced intrinsic cardiac neuronal effects, as it has been shown to do with respect to cardiac afferent neurons (22, 23, 33). Third, we examined whether neuronal effects induced by oxygen-derived free radicals are modified by K_ATP-channel openers, since these agents protect the myocardium from ischemia-induced injury (19). In this manner we sought to determine whether intrinsic cardiac neurons are modified by the generation of oxygen-derived free radicals in situ.

	MATERIALS AND METHODS

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Adult mongrel dogs (n = 44) of either sex, weighing 16-24 kg, were used in this study. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996) and were approved by the animal care and use committee of Dalhousie University.

General methods. Dogs were tranquilized with Pentothal Sodium (20 mg/kg iv) and then anesthetized with Pentothal Sodium (10 mg/kg iv to effect every 10 min for the duration of the surgical procedures). After the surgery was completed, anesthesia was maintained using -chloralose (50 mg/kg iv), which was administered first as a bolus and then as repeat doses (15-20 mg/kg iv) every hour or less throughout the experiments, as required. Noxious stimuli were applied to a paw throughout the experiments to ascertain the adequacy of anesthesia. Heart rate was monitored continuously throughout the experiments via a lead II electrocardiogram (ECG). After intubation was completed, positive-pressure ventilation was initiated and maintained with a Bird Mark 7A ventilator using a gas mixture of 95% O₂-5% CO₂. The spontaneous activity generated by intrinsic cardiac neurons was transiently suppressed following bolus injections of -chloralose due to the neuronal depressor effects of this agent. Therefore, at least 10 min were allowed to elapse following repeat administration of -chloralose before recordings proceeded. Body temperature was maintained constant throughout the experiments by means of a heating pad.

Neuronal recording. The activity generated by ventral right atrial neurons was recorded by means of a tungsten microelectrode with a 10-µm diameter and an exposed tip of 50 µm (impedance of 9-11 M at 1,000 Hz), as has been described elsewhere (14). To minimize epicardial motion during each cardiac beat, a circular ring of heavy-gauge wire was gently placed around the epicardial fat of the ventral surface of the right atrium. The tungsten microelectrode, mounted on a micromanipulator, was used to explore the fat at varying depths ranging from the surface of the fat to regions adjacent to cardiac musculature. Proximity to cardiac musculature was indicated by increases in the amplitude of the ECG artifact. The reference electrode was attached to the pericardium.

Administration of chemicals. The chemicals, obtained from Sigma (St. Louis, MO) and Research Biochemicals International (Natick, MA), were administered in pharmacological doses. The following chemicals were studied: 1) H₂O₂ (600 µM at a rate of 3 ml/min for 20 min), 2) the free iron chelator deferoxamine (20 mg/kg iv), and 3) the selective K_ATP-channel opener cromakalim (20 µM at a rate of 3 ml/min for 20 min). H₂O₂ and cromakalim were administered individually into the regional arterial blood supply of right atrial neurons in a continuous fashion via a 50-ml syringe containing the chemicals. The syringe was placed in a Sage Instruments model 367 constant-infusion pump (Orion Research, Cambridge, MA) so that chemicals could be administered for prolonged periods of time. For this purpose, a PE-50 catheter was inserted into a branch of the right coronary artery, which arises immediately proximal to the root of the artery that supplies blood to neurons in the right atrial ganglionated plexus. The catheter was secured in place by ligatures, thereby ensuring that infused chemicals were delivered to the right atrial ganglionated plexus in a continuous fashion. Thereafter, the same doses of H₂O₂ and cromakalim were administered individually into the bloodstream of the descending aorta via a Cordis no. 7 catheter, which was attached to a 50-ml syringe placed in the constant-infusion pump described above.

Data analysis. Heart rate, left atrial chamber pressure, left ventricular chamber pressure, and aortic pressure, as well as left and right ventricular intramyocardial pressures, were measured for 10 consecutive cardiac cycles, and their means ± SE were calculated. Individual action potentials generated by right atrial neurons were analyzed for 30-s periods immediately before and during maximal responses elicited by each chemical. Data were grouped according to whether neuronal activity increased, decreased, or remained unchanged following administration of each chemical, depending on the population of neurons studied. Data obtained immediately before each administration were compared with data obtained at the point of maximum change after administration of a chemical using the two-tailed Student's t-test for paired data. Significance was assigned at the 0.05 and 0.01 levels.

	RESULTS

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Identification of active sites and injection of vehicle. Spontaneous activity generated by intrinsic cardiac neurons was recorded from one locus in each right atrial ganglionated plexus studied. Three to five spontaneously active units, as determined by the amplitudes and shapes of individual action potentials, were identified at each locus. When saline was administered into the local coronary artery, which supplied blood to the right atrial ganglionated plexus, neuronal activity and cardiac indexes were unaffected.

Neuronal and cardiac effects elicited by local arterial administration of H₂O₂. H₂O₂, when infused into the local coronary arterial blood supply at a concentration of 600 µM (3 ml/min, 20 min), modified neurons in every animal tested within 5-7 min of constant exposure (Table 1; see also Table 3). H₂O₂ increased the activity generated by right atrial neurons (15.7 ± 3.7 to 30.1 ± 5.8 impulses/min, P < 0.01) in 16 of 36 animals by 92% (Fig. 1, Table 1). H₂O₂ decreased the activity generated by identified neurons in the remaining 20 animals by 61% (19.5 ± 2.7 to 7.7 ± 2.2 impulses/min, P < 0.01). Neuronal activity returned to baseline values 15 min after administration of H₂O₂ was discontinued, even when H₂O₂ had been administered for periods of time lasting as long as 30 min. When H₂O₂ enhanced neuronal activity (n = 16 dogs), there was a slight (~1%, P < 0.05) but consistent increase in left ventricular chamber systolic pressure (Table 1). Repeat administration of H₂O₂ into the regional arterial blood supply of right atrial neurons induced neuronal responses that were similar to those induced during their first exposure.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   H2O2 (600 µM arrow at bottom), when administered into arterial blood supply of right atrial ganglionated plexus, activated previously quiescent right atrial neurons (bottom trace). Heart rate, as determined from electrocardiogram (ECG, top), was unchanged. Left ventricular chamber systolic pressure (LVP, middle) was fluctuating between 128 and 118 mmHg in a cyclic fashion.

Neuronal and cardiac effects elicited in the presence of deferoxamine. Deferoxamine, when administered as a bolus into the systemic circulation (20 mg/kg iv) of 10 animals, reduced the activity generated by right atrial neurons by 57% (44.6 ± 15.6 to 19.0 ± 9.4 impulses/min, P < 0.05) in 8 of 10 animals tested (Fig. 2). Deferoxamine did not change neuronal activity in the remaining two animals [29.8 ± 11.9 to 49.8 ± 16.7 impulses/min; not significant (NS)]. Cardiac variables were unaffected overall by deferoxamine. H₂O₂ elicited no changes in neuronal activity when administered in the presence of deferoxamine. For instance, in the six dogs in which H₂O₂ decreased neuronal activity when administered alone (18.8 ± 3.8 to 13.2 ± 4.3 impulses/min, P < 0.05), H₂O₂ elicited no change in activity (25.4 ± 9.1 to 27.4 ± 13.9 impulses/min, NS) when readministered in the presence of deferoxamine. Recorded cardiac variables did not change when H₂O₂ was administered in the presence of deferoxamine.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Effects of anti-oxidant, iron-chelating agent deferoxamine (20 mg/kg iv) on spontaneous activity generated by right atrial neurons. After 2 min of deferoxamine administration (arrow between panels), ongoing activity generated by right atrial neurons was nearly eliminated. LV IMP, left ventricular intramyocardial systolic pressure.

Neuronal and cardiac effects elicited in the presence of cromakalim. Continuous administration of the K_ATP-channel opener cromakalim (20 µM, 3 ml/min) to right atrial neurons via their local artery blood supply resulted in a reduction (54%) in the activity generated by neurons in 15 of 21 animals tested. Neuronal activity changes were induced without alterations in monitored cardiac indexes (Table 2). The activity generated by neurons in 16 of these animals did not change when H₂O₂ was administered in the presence of cromakalim. This lack of neuronal responsiveness occurred whether H₂O₂ had previously enhanced or suppressed the activity generated by different populations of intrinsic cardiac neurons (Table 3). H₂O₂ caused a 9% reduction in left ventricular systolic pressure in nine of these animals when administered in the presence of cromakalim (Table 3).

	DISCUSSION

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The activity generated by intrinsic cardiac neurons changed in every animal investigated when H₂O₂ was infused into its local arterial blood supply. As has been found to be true with regard to many other chemicals (2), H₂O₂ increased the activity generated by one population of investigated intrinsic cardiac neurons in situ (Fig. 1) and decreased the activity generated by another population. The results of this study indicate that the relative size of these two subpopulations of intrinsic cardiac neurons is approximately equal (Table 1). The variability of neuronal responses elicited by H₂O₂ could have been due, in part, to the variability of the blood supply to an intrinsic cardiac ganglionated plexus among dogs (24). It could also have been due to the fact that H₂O₂ might have exerted direct effects on regional coronary arteries that perfused the investigated neurons. Although we could not test local coronary effects of this agent in situ, if indeed H₂O₂ modifies the flow in coronary arteries, then this would represent yet another manner by which locally liberated H₂O₂ modifies the intrinsic cardiac nervous system. That the neuronal responses induced by H₂O₂ were reversible indicates that H₂O₂ did not induce any long-term deleterious effects on their function or, for that matter, on other regional tissues.

The effects that H₂O₂ exerted on intrinsic cardiac neurons were no longer induced in the presence of the iron-chelating agent deferoxamine, an agent that blocks iron-catalyzed · OH formation from H₂O₂ (16). Thus it appears that H₂O₂-induced effects on intrinsic cardiac neurons are due, in part at least, to the generation of the · OH radical. Because neuronal responses occurred without alterations in recorded cardiac indexes overall, it is unlikely that the responsiveness of intrinsic cardiac neurons to the generation of · OH radicals was secondary to cardiodynamic changes. That deferoxamine influenced ongoing activity generated by a population of intrinsic cardiac neurons (Fig. 2) suggests that some intrinsic cardiac neurons may be influenced in a tonic fashion via oxygen-derived free radicals in situ. The finding that H₂O₂ no longer induced neuronal responses in the presence of an agent that attenuates · OH formation supports the contention that a population of intrinsic cardiac neurons is sensitive to oxygen-derived free radicals.

Local arterial administration of the K_ATP-channel opener cromakalim altered the ongoing activity generated by intrinsic cardiac neurons (Fig. 3). These data indicate that the activity generated by such neurons in situ also depends, in part, on their having K_ATP channels. Cromakalim also reduced the capacity of H₂O₂ to affect intrinsic cardiac neurons (Table 3), which suggests that K_ATP channels associated with intrinsic cardiac neurons may be involved in the effects that H₂O₂ exerts on such neurons in situ.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Administration of ATP-sensitive potassium (KATP) channel agonist cromakalim (20 µM) into local arterial blood supply of right atrial neurons (arrow between panels) suppressed the activity generated by such neurons. Data on right were obtained 2 min after the local infusion of cromakalim commenced. Cardiac variables remained unchanged.

Myocardial ischemia-reperfusion generates oxygen-derived free radicals locally (5, 9, 15, 31, 35, 38). The activity generated by canine intrinsic cardiac neurons in situ is influenced by interrupting their local coronary arterial blood supply (24). Local ischemia-reperfusion induces varied intrinsic cardiac neuronal responses in situ (24), as does exposing intrinsic cardiac neurons to exogenous H₂O₂ (Table 1). The fact that intrinsic cardiac neurons generate varied responses when exposed to local ischemia-reperfusion has been attributed to the presence of several populations of neurons within the intrinsic cardiac nervous system (2, 21, 24, 37).

It has been suggested that iron participates in free radical reactions (18). Support for the oxyradical hypothesis for postischemic injury has been provided by studies using spin-trapping techniques. Free radical generation during postischemic myocardial dysfunction (myocardial stunning) in vivo can be prevented by administering the iron chelator deferoxamine before coronary artery flow is reestablished (9). The protective role of deferoxamine against H₂O₂ injury has also been demonstrated in single cardiomyocytes (7). Thus iron-catalyzed reactions that produce the highly reactive · OH radical are thought to be important in the genesis of ventricular myocyte damage induced by ischemia.

The activation of cardiac sensory neurites associated with vagal or sympathetic afferent axons, which occurs during ischemia-reperfusion (22, 25), can be mimicked by local epicardial application of H₂O₂ (22, 23, 33, 34). Furthermore, these effects are attenuated by administering deferoxamine before the induction of ischemia or epicardial application of H₂O₂ (22, 23, 33, 34). The observation that such cardiac sensory neurites are insensitive to ischemia in the presence of an Fe²⁺ chelator has been interpreted as indicating that ischemia-induced modification of cardiac sensory neurites involves Fe²⁺-induced formation of · OH (22). The same may be true with respect to ischemia-sensitive intrinsic cardiac neurons.

The precise mechanisms that are involved in neuronal responses to H₂O₂ remain to be established. H₂O₂ affects the voltage-gated Na⁺ channels in cardiomyocytes, initially delaying their inactivation to prolong action potential duration, a change that eventually leads to cellular inexcitability (7, 36). The free radical-generating system also modifies Na⁺ channels in myelinated nerve axons, delaying the inactivation of these channels (10). Presumably, such modification of action potential duration would be attenuated in the presence of a K_ATP-channel opener, since such a compound shortens action potential duration. As a matter of fact, cromakalim modified the spontaneous activity generated by intrinsic cardiac neurons on its own (Table 2). Because the activity generated by most identified neurons was suppressed in the presence of cromakalim, it is presumed that this agent acted to increase resting K⁺ efflux of these neurons via the K_ATP channels; such a change would lead to neuronal hyperpolarization and thus possibly to decreased excitability. Because cromakalim influenced intrinsic cardiac neurons without changing the recorded cardiac indexes, it is unlikely that the neuronal responses so induced were secondary to cardiodynamic alterations. The finding that intrinsic cardiac neurons were no longer affected by H₂O₂ when it was administered in the presence of cromakalim (Table 3) supports the contention that K_ATP channels may be involved in H₂O₂-induced effects on the intrinsic cardiac nervous system.

Perspectives. The data obtained in the present series of experiments indicate that locally released H₂O₂ affects a population of intrinsic cardiac neurons in situ by Fe²⁺-dependent generation of · OH radicals. Furthermore, the sensitivity of this population of intrinsic cardiac neurons to local accumulation of H₂O₂ appears to depend in part on functioning K_ATP channels associated with these neurons. These data have implications with respect to modifying the untoward cardiac effects that occur when intrinsic cardiac neurons are affected by myocardial ischemia-reperfusion.