INTRODUCTION

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DYSFUNCTION OF THE SINOATRIAL NODE is well recognized in humans, particularly in ischemic heart disease and the elderly, and can cause a variety of arrhythmias (1). Reduced oxidative phosphorylation is a prominent feature of ischemic heart disease and can be simulated by cyanide (CN), which inhibits cytochrome oxidase, preventing mitochondrial ATP production. Early electrophysiological studies showed that either anoxia or CN slowed cardiac pacemaker activity and also caused reductions of both upstroke velocity and overshoot of the pacemaker action potential. It was assumed that all these changes were caused by inhibition of the Ca²⁺ current (19).

It is well established that intracellular Ca²⁺ concentration ([Ca²⁺]_i) plays a important role in ischemic failure of ventricular muscle (22, 32). Different models of ischemia and metabolic inhibition produce variable changes in the systolic increase in [Ca²⁺]_i (the Ca²⁺ transient), but most studies of metabolic inhibition show reductions in Ca²⁺ transients (2, 6). This reduction in the Ca²⁺ transient is often attributed to a coexisting reduction in the Ca²⁺ current (6, 21). However, a more recent study suggested that decreased Ca²⁺ current was not the cause of impaired Ca²⁺ release during metabolic inhibition and proposed instead that metabolic changes inhibited the Ca²⁺ release channels in the sarcoplasmic reticulum (SR) (27).

Despite many studies on the effect of metabolic inhibition on [Ca²⁺]_i and its relation to cardiac arrhythmias (20, 23), little is known about the intracellular signaling pathway(s) involved. A recent study provided evidence that cytochrome P-450, a superfamily of heme-containing monooxygenases, was involved in the modulation of Ca²⁺ channels, [Ca²⁺]_i, and cell contraction in cardiac myocytes (34). The intracellular cAMP concentration ([cAMP]_i) was reduced after ventricular myocytes were treated with a cytochrome P-450 inhibitor or CN, suggesting that a decrease of cAMP-dependent phosphorylation of L-type Ca²⁺ channels might contribute to contractile failure in the heart (34). Because the activity of cytochrome P-450 is reduced by anoxia or CN (18), it was suggested that inhibition of cytochrome P-450 might be an important cellular pathway during cardiac ischemia (34).

Although it is well established that ischemia or metabolic inhibition changes Ca²⁺ handling in ventricular myocytes, there are no equivalent studies in pacemaker cells. This may reflect the fact that the role of the SR in pacemaker cells was not recognized until very recently (for reviews, see Refs. 16 and 33). Recent studies on [Ca²⁺]_i in both amphibian (13) and mammalian pacemaker cells (3, 11, 12, 35) have suggested a novel mechanism for cardiac pacemaking. A key observation is that the heart rate is correlated with the magnitude of the Ca²⁺ transient (13, 14), and it is proposed that Ca²⁺ release from the SR activates the Na⁺/Ca²⁺ exchanger, causing an inward pacemaker current (3, 12, 13). Because the magnitude of the Na⁺/Ca²⁺ exchanger current is dependent on the amplitude of the Ca²⁺ transient, modulation of the Ca²⁺ transient influences the heart rate.

To investigate whether [Ca²⁺]_i also is important in pacemaker activity under ischemic conditions, we studied the effect of CN on Ca²⁺ handling in toad pacemaker cells. The possible involvement of cytochrome P-450 and cAMP was also addressed.

	METHODS

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Preparation of pacemaker cells. These experiments were approved by the Animal Ethical Committee of the University of Sydney and conform to Australian guidelines. Toads (Bufo marinus) were killed by decapitation and pithed. Single pacemaker cells were enzymatically isolated from the sinus venosus as previously described (13, 17). The cells were routinely superfused with the following (standard) solution (in mM): 110 NaCl, 2.5 KCl, 0.5 MgSO₄, 2 CaCl₂, 10 HEPES, and 10 glucose; pH 7.3 equilibrated with air. Drugs were applied from a fine tube positioned within 200 µm of the cell to ensure a rapid onset of action. All experiments were performed at room temperature (22°C), which is close to the physiological temperature for these semitropical amphibia.

Fluorescence measurements. After isolation, cells were incubated with 5 µM indo 1-AM for 10-15 min (13). Loaded cells were illuminated at 360 ± 5 nm with an ultraviolet light source whose intensity was reduced 30-fold with a neutral-density filter. The emitted fluorescence was guided to two photomultiplier tubes via either a 400 ± 5-nm or 510 ± 5-nm interference filter. The light signal at each wavelength was filtered at 10 Hz, and the background was subtracted. The analog signals were digitized, and the ratio of fluorescence signals at 400 to 510 nm (R) was calculated and converted to [Ca²⁺]_i using the following equation (8)
An in vivo calibration method was used and gave the following values: R_min = 0.11, R_max = 1.94,  = 2.84, and K_d = 606 nM (13), where R_min is the ratio at zero [Ca²⁺]_i, R_max is the ratio at saturating [Ca²⁺]_i, and  is the ratio of fluorescence at 510 nm at zero and saturating [Ca²⁺]_i.

Patch-clamp procedure. The perforated-patch technique was used to record either spontaneous action potentials or Ca²⁺ currents. The pipette solution contained (in mM) 100 KCl, 10 KH₂PO₄, 10 TES, 5 NaH₂PO₄, and 2 MgSO₄, pH 7.3. Nystatin (240 µg/ml) or amphotericin B (200 µg/ml) was added to the pipette solution. An Axopatch 200A (Axon Instruments; Foster City, CA) was used in current-clamp mode to record membrane potentials or voltage-clamp mode to record Ca²⁺ current. The series resistance of the perforated patches was <20 M; cell capacitances were 30-50 pF. Series resistance and capacitance compensation were made once cell access had been acheived. Tetrodotoxin (TTX; 10⁷ M) was used in some experiments to block Na⁺ currents (17). Membrane currents and fluorescence signals were sampled at 2 kHz; membrane potentials were sampled at 0.5 kHz.

Statistics. All statistical data are presented as means ± SE, with the number of cells studied given as n. Student's paired t-test was used except where noted, with P < 0.05 accepted as significant.

	RESULTS

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Effect of CN on spontaneous Ca²+ transients in pacemaker cells. Figure 1A shows [Ca²⁺]_i signals recorded from a single pacemaker cell loaded with indo 1. After application of 2 mM CN, the amplitude of the Ca²⁺ transients was reduced in association with the reduced firing rate (Fig. 1B). In these indo-loaded cells, the control firing rate was 27 ± 2 beats/min (n = 9), and in the presence of CN it fell to 12 ± 4 beats/min (P < 0.002, n = 9); in some cases, the cells became quiescent (see, e.g., Figs. 4B and 6B). The changes of [Ca²⁺]_i usually occurred within 3-5 min after application of CN. The amplitude of the Ca²⁺ transients decreased from 338 ± 30 to 189 ± 37 nM (n = 9, P < 0.005), i.e., to 56% of control. Although diastolic [Ca²⁺]_i declined (Fig. 1B), when all experiments were considered there was no significant change (P  0.2, n = 9). The time course of the decay of the Ca²⁺ transients was also significantly slowed by CN, as shown in Fig. 1, C and D. The average time constant of the exponential decay increased from 0.33 ± 0.03 to 0.46 ± 0.04 s (n = 9, P < 0.02). These data show that CN reduced the amplitude of the Ca²⁺ transients and simultaneously reduced the spontaneous firing rate. The present experiments were restricted to short exposures to CN, and the Ca²⁺ transients and firing rate were fully reversible when CN was washed out (see, e.g., Fig. 6D).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   The effect of CN on spontaneous Ca2+ transients. A: spontaneous Ca2+ transients recorded from a toad pacemaker cell. B: amplitude and frequency of Ca2+ transients were decreased in the presence of 2 mM CN. C: representative control Ca2+ transient (dotted line) with exponential fit to the declining phase (continuous line), with a time (t) constant of 0.4 s. D: as in C, but with the Ca2+ transient in the presence of CN. Note the slower decline of the Ca2+ transient. [Ca2+]i, intracellular Ca2+ concentration.

Effect of CN and cytochrome P-450 inhibitor on Ca²+ current and [Ca²+]_i. Reduced Ca²⁺ transients could result from a reduction of the Ca²⁺ current, as has been reported previously for ventricular myocytes during metabolic inhibition (6, 21). A recent study suggested that CN inhibits cytochrome P-450, causing reduced [cAMP]_i levels and reduced phosphorylation of the L-type Ca²⁺ channels (34). We tested whether similar mechanisms occur in pacemaker cells. To explore these issues, we simultaneously recorded Ca²⁺ currents and Ca²⁺ transients evoked by depolarizing voltage pulses from a holding potential of 60 mV (Fig. 2A). TTX was added to the bath solution to block residual Na⁺ currents.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   The effect of CN and clotrimazol on the Ca2+ current and Ca2+ transient evoked by depolarization. The Ca2+ current and [Ca2+]i signal were simultaneously recorded from a single pacemaker cell. The cell was voltage clamped at a holding potential of 60 mV, and a depolarizing step to 0 mV was imposed on the cell. Na+ current was blocked by 107 M tetrodotoxin. A: control. B: in the presence of 2 mM CN. C: in the presence of the cytochrome P-450 inhibitor clotrimazole. Note that the Ca2+ current shows a greater reduction in clotrimazole than CN, whereas the reduction of Ca2+ transients is similar.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Current-voltage relationships in the presence of CN and clotrimazole. A: peak Ca2+ current-voltage relationships obtained from voltage-clamp experiments similar to Fig. 3 (n = 9). There was a 23% deduction of peak current in the presence of NaCN, but this was not significantly different compared with the control. B: effect of 5 µM clotrimazol on Ca2+ current-voltage relationship compared with control. Clotrimazole significantly reduced the Ca2+ currents at all voltages.

Effect of cAMP on the change of action potential and Ca²+ current caused by metabolic inhibition. If the effect of CN was due to inhibition of cytochrome P-450 through modulation of [cAMP]_i, as proposed above, one would expect that the effect of CN could be reversed by elevating [cAMP]_i. To test this idea, we recorded normal pacemaker action potentials (Fig. 4A). After pacemaker action potentials had been abolished by CN (Fig. 4B), 1 mM dibutyryl cAMP was added to the CN-containing solution. Over the first 6 min of application of dibutyryl cAMP, the pacemaker potentials gradually recovered (Fig. 4, C-E). However, after a longer period (8 min; Fig. 4F), the spontaneous firing again failed. Similar results were seen in three other cells. We found that adrenaline or isoprenaline also produced similar effects as dibutyryl cAMP (data not shown). Because dibutyryl cAMP temporarily reversed the effect of CN on the action potential, it was possible that the effect of CN was due to reduced [cAMP]_i through the inhibitory effects of CN on cytochrome P-450.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   cAMP reverses the effect of CN on spontaneous firing of a single pacemaker cell. Spontaneous action potentials were recorded from a single pacemaker cell. A: control conditions. B: after 4 min of NaCN; note that regular firing has ceased. C: after 2 min of dibutyryl cAMP; note the start of recovery. D: after 4 min of dibutyryl cAMP; partial recovery. E: after 6 min of dibutyryl cAMP; complete recovery. F: after 8 min of dibutyryl cAMP; note that spontaneous firing is failing again despite the continuing presence of dibutyryl cAMP.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effect of dibutyryl cAMP on Ca2+ current in the presence of CN or clotrimazole. Ca2+ currents were recorded at 0 mV from a holding potential of 60 mV. A: Ca2+ current in control conditions, after the addition of CN, and after the further addition of 1 mM dibutyryl cAMP in the continuing presence of CN. B: identical format to A but with clotrimazole instead of CN.

SR Ca²+ store content of pacemaker cells in metabolic inhibition. There are variable reports on how SR Ca²⁺ content changes during metabolic inhibition. Different studies have reported that SR Ca²⁺ content decreased (30), did not change (31), or increased (6, 27). To study the effect of metabolic inhibition on SR Ca²⁺ content in pacemaker cells, we measured the SR Ca²⁺ content by rapid caffeine application (4) and measured the amplitude of the peak increase in [Ca²⁺]_i above the resting level.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effect of CN and dibutyryl cAMP on sarcoplasmic reticulum (SR) Ca2+ content of toad pacemaker cells. Spontaneous Ca2+ transients were recorded from a single toad pacemaker cell. A: initially, the cells shows spontaneous Ca2+ transients; rapid application of 10 mM caffeine then produced a large Ca2+ signal representing SR Ca2+ content. B: after a 4-min exposure to CN. Note that this cell became quiescent in CN. The SR Ca2+ signal produced by caffeine was also greatly inhibited. C: 2 min after the addition of dibutyryl cAMP in the continuing presence of CN. Note the recovery of spontaneous Ca2+ transients, whereas SR Ca2+ content showed no recovery. D: 6 min after washout of CN and dibutyryl cAMP. Both Ca2+ transients and Ca2+ stores have returned to normal.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effect of clotrimazole and dibutyryl cAMP on SR Ca2+ content of toad pacemaker cells. Spontaneous Ca2+ transients were recorded from a single toad pacemaker cell. A: control spontaneous Ca2+ transients followed by a rapid appication of 10 mM caffeine to determine SR Ca2+ content. B: same as in A, but in the presence of 5 µM clotrimazole. Note the reduced amplitude and firing rate of spontaneous Ca2+ transients; however, the SR Ca2+ content is little affected. C: addition of dibutyryl cAMP produced recovery of spontaneous Ca2+ transients, but SR Ca2+ content showed little change.

	DISCUSSION

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Mechanism of decreased Ca²+ transients during early metabolic inhibition. Early studies of the effect of metabolic inhibition on ventricular myocytes have suggested that the decreased Ca²⁺ transient was caused by a reduction of the Ca²⁺ current. Reports of the percent reduction of the Ca²⁺ current produced by metabolic inhibition in ventricular myocytes vary from 17 to 55% (6, 21, 27, 34). We found that the amplitude of the Ca²⁺ current decreased by 23 ± 11% in pacemaker cells, which was small and not statistically significant, making it unlikely that the decrease in the Ca²⁺ transient was entirely due to the inhibition of the Ca²⁺ current.

Possible involvement of cAMP during the metabolic inhibition. In the present study, we found that elevated [cAMP]_i could temporarily reverse the effects of CN on the amplitude of the Ca²⁺ transient and on pacemaker activity. One possible explanation is that CN acts through cytochrome P-450 (34). Cytochrome P-450 forms a superfamily of proteins that metabolize a range of substrates, including steroids, fatty acids, and drugs (for a review, see Ref. 7). Cytochrome P-450 degrades arachidonic acid to epoxyeicosatrienoic acids (28), which stimulate adenylyl cyclase and/or inhibit phosphodiesterases, causing increased cAMP production (29, 34). Because oxygen is required for cytochrome P-450-mediated reactions, it has been suggested that failure of cytochrome P-450 activity could contribute to hypoxic and ischemic damage (34). CN also binds to cytochrome P-450 and inhibits its metabolic activity (18, 25). These results are all consistent with the hypothesis that cytochrome P-450 may play a role in early metabolic inhibition. However, there are several features of our results that argue that the main cellular mechanisms of CN are not exerted through cytochrome P-450: 1) CN had only a small effect on the Ca²⁺ current; and 2) although increasing [cAMP]_i increased the Ca²⁺ current and firing rate, the effect was only transient. Similar observations on the transience of cAMP effects were made in the rabbit sinoatrial node (19). We believe the initial recovery of the firing rate after the addition of cAMP is because the increased Ca²⁺ current increases Ca²⁺ release from the depleted store. With further exposure to CN, we suspect that SR Ca²⁺ stores decline further, causing the subsequent decline in the Ca²⁺ transient and firing rate. Thus, although CN and clotrimazole produce equivalent effects on the Ca²⁺ transient and firing rate, they appear to operate by different mechanisms. Clotrimazole did not change SR Ca²⁺ content but inhibited the Ca²⁺ current, impairing SR Ca²⁺ release and causing reduced Ca²⁺ transients. In contrast, CN had minor effects on the Ca²⁺ current but a major effect on the SR Ca²⁺ content, so that the net effect was a similar reduction in the Ca²⁺ transient. These results suggest that, whereas CN has multiple effects on Ca²⁺ handling, at least in pacemaker cells, the effects of inhibition of cytochrome P-450 are relatively minor.

Possible relationship between reduced Ca²+ transients and slowing of the firing rate. Many pacemaker currents may contribute to the slower firing rate during ischemia or metabolic inhibition, including activation of ATP-sensitive K⁺ channels (10), inhibition of L-type Ca²⁺ current (21), and modification of inward rectifying K⁺ currents (9). Nevertheless, the temporal association of reduced Ca²⁺ transients and slowing of the firing rate raises the possibility that the two are associated. It is known from earlier work that agents such as the SR channel blocker ryanodine and the SR pump inhibitor 2,5-di(tert-butyl)-1,4-benzohydroquinone, which reduce the amplitude of Ca²⁺ transients without affecting the Ca²⁺ current, cause a slowing of the firing rate (13, 14). Thus the reduction of the Ca²⁺ transient would be expected to slow the firing rate independent of any effect on the Ca²⁺ current. We have previously demonstrated that there is an active Na⁺/Ca²⁺ exchanger in pacemaker cells and suggested that [Ca²⁺]_i influences the heart rate through its effect on the amplitude of the Na⁺/Ca²⁺ exchange current (13). The importance of the Na⁺/Ca²⁺ exchanger as a pacemaker current has also been recognized in mammalian pacemaker cells (3, 12, 35). Thus a possibility that arises from the present study is that during metabolic inhibition, the reduced Ca²⁺ transient causes a reduction in Na⁺/Ca²⁺ exchange that, by virtue of its role as a pacemaker current, contributes to the slow and irregular firing rate.