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Am J Physiol Heart Circ Physiol 279: H992-H999, 2000;
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Vol. 279, Issue 3, H992-H999, September 2000

Effect of hydrogen peroxide on the membrane currents of sinoatrial node cells from rabbit heart

Jiqing Guo1, Wayne R. Giles1, and Christopher A. Ward2

1 Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1; and 2 Department of Physiology, Queen's University, Kingston, Ontario, Canada K7L 3N6


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of H2O2 on pacemaker activity and underlying membrane currents were studied in isolated rabbit sinoatrial (SA) node cells using perforated patch current- and voltage-clamp methods. Short-term exposure (<10 min) of the nodal cells to H2O2 (200 µM) resulted in an initial shortening of spontaneous action potential cycle length (from 445 ± 60 to 398 ± 56 ms; P < 0.05) and a prolongation of action potential duration. H2O2 (100 µM) significantly increased peak L-type Ca2+ current (ICa,L) from -384 ± 77 to -439 ± 84 pA (116 ± 2%, n = 6). Additionally, the persistent or non-inactivating component of ICa,L was increased from -52 ± 3 to -88 ± 14 pA (174 ± 19%, n = 6). The hyperpolarization-activated current (If) was decreased from -228 ± 62 to -161 ± 72 pA after exposure to H2O2 (n = 7). There were no changes in the delayed rectifier K+ current (IK) (n = 7). H2O2-induced Ca2+ currents were blocked by 2 µM nicardipine (n = 6), 2 mM Ni2+ (n = 2), and the protein kinase C (PKC) inhibitor bisindolylmaleimide (10-7 M; n = 4) but not by 20 µM tetrodotoxin. These results suggest that H2O2 can increase the spontaneous pacing rate in rabbit SA node cells by enhancing ICa,L and that this effect is mediated by a PKC-dependent pathway.

patch clamp; action potential; potassium ion current; hyperpolarization-activated current; calcium ion current, sinoatrial nodal cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

REACTIVE OXYGEN SPECIES have been shown to be important contributing factors in the generation of rhythm disturbances and cellular damage during reperfusion of previously ischemic myocardium (7, 19). One reactive oxygen species in particular, H2O2, has been suggested to play an important role in these pathological alterations. Evidence favoring this mechanism has been provided by several different means including direct application of exogenous H2O2 to nonischemic myocardial tissues or manipulation of H2O2 formation/degradation pathways during pathological conditions. Indeed, a correlation exists between the tissue content of glutathione peroxidase, which detoxifies H2O2, and the severity of reperfusion injury (17). Direct application of H2O2 has also been shown to initiate proarrhythmic activity (1, 6, 36).

Although some aspects of the underlying mechanisms by which H2O2 generates rhythm disturbances have been studied in some detail, there is little information and no consensus regarding the specific effects of H2O2 on individual transmembrane currents (19). H2O2 has been reported to increase several different currents including the ATP-sensitive K+ current (12) and inward-rectifying K+ current (24); but apparently it can also decrease the delayed rectifier K+ current (3). In addition, in mammalian ventricle, H2O2 appears to have a number of different effects on L-type Ca2+ currents (ICa,L), with increases, decreases, or no change being reported (3, 28, 33, 34). Based on our previous study (36) and other findings, it is likely that the differing effects of the actions of H2O2 on ICa,L in cardiac tissue can be attributed to recording conditions, although variations in animal species and tissue should be considered.

Recently, we confirmed that the effects of H2O2 on rat ventricular action potential waveforms were dependent upon recording conditions, with measurable effects observed only when the myoplasm was not dialyzed (36). Our results showed that in rat ventricular myocytes, H2O2 prolonged action potential duration (APD) by selectively slowing the rate of fast inactivation of the tetrodotoxin (TTX)-sensitive sodium current (INa). These effects of H2O2 appear to be mediated by the intracellular second messenger, protein kinase C (PKC).

To date, however, there have been few studies aimed at determining the effects of H2O2 on cells derived from the primary pacemaker tissue, the sinoatrial node (SAN). In the present study, we examined the effects of H2O2 on the spontaneous action potential generation and underlying ionic currents in isolated SAN cells. In the pacemaker myocytes from the adult rabbit heart, H2O2 causes a biphasic effect on the spontaneous pacemaker rate: an initial increase is followed by a decrease of the spontaneous cycle length with prolonged exposure. Our results show that the increase in pacemaker rate is associated with an increase in ICa,L. There is a slight decrease the hyperpolarization-activated current (If) but no measurable effect on the delayed rectifier K+ current (IK).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation. Rabbit sinoatrial cells were isolated using a method described previously by Tanaka et al. (32) in 1996 with the following modifications. New Zealand White rabbits (1.5-2.0 kg) were anesthetized with pentobarbital sodium (40 mg/kg). The chest was quickly opened, and the aorta was cannulated to allow perfusion of the coronary vessels with a standard Tyrode solution at 37°C. Subsequently, the heart was excised and mounted on a Langendorff-type apparatus. After the blood was cleared from the coronary vessels, the perfusate was switched to nominally Ca2+-free Tyrode solution for 3 min; that was followed by perfusion with the same solution to which 0.025 mg/ml collagenase (Yakult, Tokyo, Japan) and 2.5 mg/ml protease (Sigma, St. Louis, MO) were added. After the SAN region was perfused for 15 min in the presence of these enzymes, the SAN region, bordered by the crista terminalis, atrial septum, and cranial and caudal vena cava, was excised and cut into small strips (0.5-1 mm wide). These strips were further digested for 15-20 min in Ca2+-free Tyrode solution containing 1 mg/ml collagenase (type I, Sigma) and 0.1 mg/ml elastase (Boehringer, Mannheim, Germany). Finally, the tissue strips were put into a high-K+ solution (KB) (14), and individual myocytes were obtained by gentle trituration. The isolated cells were stored at 4°C until used in experiments.

Isolated SAN cells had a fusiform shape and a smooth surface with no discernible cross-striations. Cell size was in the range of 5-10 µm width and 50-100 µm length. The capacitance of an isolated myocyte averaged 38.3 ± 9.2 pF (n = 24).

Solution and drugs. The Tyrode solution contained (in mM) 145 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 1.0 Na2HPO4, 5.0 HEPES, and 5.5 glucose (pH 7.4, adjusted with NaOH). The KB solution contained (in mM) 90 potassium glutamate, 10 potassium oxalate, 25 KCl, 10 KH2PO4, 20 taurine, 0.5 EGTA, 5 HEPES, 1.0 MgCl2, and 10 glucose (pH 7.3, adjusted with KOH). For action potential and whole cell current recordings, the pipette solution contained (in mM) 110 potassium aspartate, 20 KCl, 5.0 MgCl2, 1 CaCl2, 5 disodium ATP, 10 HEPES, and 10 EGTA (pH 7.2, adjusted with KOH). In some experiments, Cs+ was substituted for K+ on an equimolar basis.

H2O2 stock solution (0.5 M) was prepared each day by diluting 30% H2O2 (Fisher Scientific) with distilled water and protected from light to minimize photodegradation. H2O2 was added to the superfusion solution to a final concentration of either 100 or 200 µM and applied to the cells at a rate of 2-3 ml/min. Both concentrations of H2O2 resulted in similar changes of membrane potential and whole cell currents, indicating that there is little concentration dependence over this range. Bisindolylmaleimide (BIS) was obtained from Calbiochem (San Diego, CA). All the other chemicals were obtained from Sigma.

Recording methods. The amphotericin B (0.2 mg/ml) perforated patch-clamp technique, as described previously by Ward and Giles (36) in 1997, was used in both action potential and whole cell membrane current recordings. Total "pipette" resistance was less than 20 MOmega for action potential recordings and was less than 10 MOmega for voltage-clamp measurements. Aspartate-containing pipette solutions resulted in a liquid junction potential of -10 mV, which was corrected. All the experiments were carried out at 34 ± 0.5°C. Data acquisition and analysis was performed using the customized software Cellsoft (D. Bergman, University of Calgary).

Statistical data are given as mean ± SE. Statistical significance was taken to be P < 0.05 as evaluated by Student's paired t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of H2O2 on spontaneous pacemaker activity and action potentials in SAN cells. Rabbit SAN cell pacemaker activity and action potentials were recorded continuously. These spontaneous action potential waveforms, recorded in control conditions (Fig. 1; Table 1), were very similar to those reported in previous studies (13). In each experiment, data was acquired only after the access resistance had stabilized and the cells generated stable rhythmic action potentials for at least 5 min.


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  		Fig. 1.   Spontaneous action potentials from a rabbit sinoatrial node (SAN) cell recorded under control conditions and following exposure to 200 µM H2O2 for 5 (A) or 18 min (B). The initial rapid depolarization phase of the first action potentials in each recording condition has been aligned to clearly illustrate the H2O2-induced changes in action potential waveform and pacemaker rates. The dotted line indicates 0 mV level. The bath temperature was 34 ± 0.5°C in this and all other experiments.


                              
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  		Table 1.   Early (at 10 min) changes in action potentials parameters of rabbit SA node cells induced by of 200 µM H2O2

Addition of H2O2 to the superfusion solution resulted in time-dependent, biphasic alterations of spontaneous firing rate and action potential waveforms. Both 100 and 200 µM H2O2 gradually increased the spontaneous firing rate of SAN cells (over a period of 5 to 10 min) with no apparent differences between these H2O2 concentrations. A representative effect of 200 µM H2O2 on spontaneous pacemaker activity and action potential is illustrated in Fig. 1. In all cells examined, H2O2 resulted in a shift of the action potential threshold to more negative membrane potentials (Fig. 1A) and a simultaneous prolongation of APD. The overshoot of the action potentials was also increased in five of seven cells examined. No measurable change was found in maximum diastolic potentials (MDP) or in the initial one-third of diastolic depolarization of pacemaker potential. The effects of 200 µM H2O2 on these action potential parameters are summarized in Table 1.

Prolonged superfusion of SAN cells with H2O2-containing solution (for more than 20-30 min) resulted in a progressive decrease of firing rate in SAN cells. This was accompanied by a depolarizing shift of the MDP, a decrease of the action potential amplitude, and a marked change of the action potential waveform (Fig. 1B). To minimize this variability and address the underlying ionic mechanisms for the remainder of these experiments, we focused on the early effects of H2O2, i.e., those due to H2O2 exposures of less than 10 min.

Effect of H2O2 on the whole cell membrane currents. To examine the ionic mechanism(s) responsible for the positive chronotropic effect of H2O2, transmembrane ion currents were recorded using the same conditions as were used for action potential measurements. In each experiment the membrane potential was held at -60 mV to mimic the normal MDP and avoid activation of If. ICa,L was recorded by applying a voltage step to 0 mV for 500 ms immediately following a 100-ms conditioning step to -35 mV to inactivate Na+ (INa) and T-type Ca2+ (ICa,T) currents. The membrane potential was then repolarized to -40 mV for 1 s to record the tail current due to the IK. This clamp protocol was repeated every 20 s during the experiment. After exposure to H2O2, the peak inward current at the onset of the test (P2) depolarization increased gradually (over 5 to 10 min; Fig. 2, A and C), as predicted from the increased firing rate of the spontaneous action potentials (Fig. 1A). In five cells, 200 µM H2O2 increased the peak inward current at 0 mV from -232 ± 91 to -344 ± 107 pA (P < 0.05). In each experiment a persistent inward current was also observed (Fig. 2, A and B). The H2O2-induced increases in both peak and persistent current components were strongly but not completely inhibited by 2 µM nicardipine (n = 6; see also Fig. 3). Subsequent experiments with 2 mM Ni+ (n = 2; data not shown), which does completely block ICa,L, resulted in findings similar to those with nicardipine. Together, these findings suggest that ICa,L is responsible for the increase in inward current. Currents were fit to a double exponential equation to quantify the effects of H2O2 on current inactivation. The fast time constant was accelerated from 13.0 ± 1.6 to 9.9 ± 1.2 ms (n = 6), whereas the slow time constant was significantly slowed from 81.6 ± 33.0 to 157.1 ± 69.9 ms (n = 6) following exposure to H2O2, respectively.


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  		Fig. 2.   Changes of L-type Ca2+ current (ICa,L), delayed rectifier K+ current (IK), and hyperpolarization-activated current (If) after exposure to H2O2. A: current changes in response to a depolarization in a SAN cell before exposure to H2O2 (trace a), following early exposure to H2O2 (trace b), and after long-term exposure to H2O2 (trace c) are superimposed. Both ICa,L and IK were activated by a 500-ms depolarizing step to 0 mV from a holding potential of -60 mV. Na+ current (INa) was inactivated by a 100-ms conditioning pulse to -35 mV immediately preceding the test pulse. The membrane potential was then repolarized to -40 mV to record tail currents due to deactivation of IK. B: superimposed current traces of If were activated by a hyperpolarization test pulse to -100 mV. Prepulse was the same as that in A. C: representative plots of the time course for the changes in ICa,L, If, and IK following exposure to H2O2. ICa,L was measured as the peak inward current during the depolarization to 0 mV. IK was measured as the initial amplitude of the outward tail current at -40 mV. If was measured as the maximum inward current during the hyperpolarization to -100 mV. The current records in A and B were obtained at the times indicated by corresponding letters.



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  		Fig. 3.   The effects of 100 µM H2O2 on ICa,L, IK, and If obtained with ruptured patch recordings. All protocols were identical to those described in the legend of Fig. 2.

In SAN cells, activation of IK initiates and controls action potential repolarization, and its deactivation is an important contributor to the rate of pacemaker depolarization (13). Possible effects of H2O2 on IK were evaluated by monitoring changes of the tail current of IK at -40 mV. The IK tail current (which is a gradual decay of outward current after repolarization from 0 to -40 mV) showed no significant change in either amplitude or decay rate during exposure to H2O2 (Fig. 2C). Peak tail currents, determined prior to and at 5 min after exposure to H2O2 were 39 ± 8 and 44 ± 10 pA, respectively (P > 0.05).

If, in many pacemaker cells, contributes to the MDP and modulates the last one-third of diastolic depolarization (5). In principle, an increase in If could augment the spontaneous firing rate of SAN cells. To examine this possibility, If was recorded by hyperpolarizing the membrane potential to -100 mV for 0.5 s. A slow but progressive decrease of If was observed following exposure to H2O2 (Fig. 2, B and C). It should be noted that this gradual decrease occurred in the same cells and over an identical time period for which the increasing ICa,L was observed (Fig. 2C), indicating that this decrease is not likely due to a general rundown of membrane currents. In seven cells observed, If at -100 mV was decreased from -228 ± 62 pA in control conditions to -161 ± 72 pA following 5-min exposure to H2O2.

Effect of intracellular dialysis. Previously, we and others have demonstrated that, in ventricular myocytes, the effects of H2O2 are mediated by second messengers and therefore are strongly dependent upon the recording conditions utilized (36). To evaluate this phenomenon in SAN cells, we repeated some experimental protocols using standard ruptured patch recording techniques (Fig. 3). Under these experimental conditions, H2O2 caused no changes in spontaneous rate or action potential waveform. As well, H2O2 had no effect on ICa,L under these conditions other than a gradual decline in both ICa,L and If (n = 5), consistent with current rundown associated with this recording method.

H2O2 increases ICa,L. To further examine whether changes in ICa,L may be responsible for the positive chronotropic effect of H2O2, more detailed voltage-clamp experiments were performed. ICa,L was isolated using Cs+-rich pipette solutions, and 5 mM Cs+ was added to the superfusate. These experimental conditions block both K+ currents. However, complete block of outward K+ currents with Cs+ is very difficult to achieve under perforated patch conditions. From a holding potential of -80 mV, ICa,L was activated by depolarizing steps to selected test membrane potentials, following a conditioning pulse to -35 mV for 100 ms to inactivate INa and ICa,T. Figure 4A shows a typical record of ICa,L at -10 mV. H2O2 (100 µM) induced an increase in inward current similar to that observed when using K+-rich pipette solutions. Current-voltage (I-V) relationships measured using the peak inward current or the current at the end of the step showed that H2O2 increased both parameters over a broad voltage range (Fig. 4B), with no significant shift of the I-V relationship. All of this inward current was completely blocked by 2 µM nicardipine, suggesting that the H2O2-induced current can be attributed to ICa,L. In six cells, ICa,L measured as the nicardipine-sensitive current at -10 mV was increased from -384 ± 77 to -439 ± 84 pA (116 ± 2%, P < 0.05) at the peak and from -52 ± 3 to -88 ± 14 pA (174 ± 19%, P < 0.05) near the end of 500-ms depolarizing pulses.


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  		Fig. 4.   Effect of H2O2 on ICa,L using experimental conditions that block potassium currents. A: representative current traces of ICa,L at -10 mV prior to exposure (circles), following exposure to 100 µM H2O2 (squares), and after addition of 2 µM nicardipine (+) are superimposed. The holding potential was -80 mV, and the pipette solution KCl was substituted with cesium aspartate (110 mM). B: current-voltage (I-V) relationship of the peak (solid symbols) and noninactivating component of ICa,L (open symbols) illustrated in A. C: H2O2-induced current from A shown as a difference current. D: I-V relationship of peak () and noninactivating components (open circle ) of the H2O2-induced current (n = 6).

To gain more insight into the effect of H2O2 on ICa,L, the current traces recorded before and after H2O2 exposure were subtracted. The resulting difference in current defines the H2O2-induced effect (Fig. 4C). Although there was some variation in rate and extent of the decay of the H2O2-induced current component among cells, a consistent finding was that this difference current was much slower compared with the ICa,L recorded under control conditions. These results demonstrate that H2O2 can augment the peak current and may slow the inactivation rate of ICa,L as well (see DISCUSSION).

Effect of TTX on H2O2-induced inward current(s). It has been reported previously that H2O2 slows the rate of INa inactivation in rat ventricular myocytes (2, 36). We therefore examined the possible involvement of the TTX-sensitive INa in H2O2-induced current in SAN cells. This was done by applying the selective inhibitor TTX. In the presence of 20 µM TTX, H2O2 still increased the inward current activated by a depolarizing step to 0 mV (Fig. 5). ICa,L was increased from -333 ± 96 to -385 ± 107 pA (117 ± 2%) at the peak and from -38 ± 4 to -72 ± 14 pA (174 ± 19%) near the end of 500-ms depolarizing pulses (n = 4). This finding confirms that TTX had no significant effect on the H2O2-induced current and that the initial effects (1-5 min) of H2O2 in SAN cells is mediated primarily by ICa,L.


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  		Fig. 5.   H2O2-induced current in the presence of 20 µM tetrodotoxin (TTX). A: current traces of ICa,L at -10 mV prior to and following exposure to 100 µM H2O2 are superimposed. The experimental protocol was identical to the one described in the legend of Fig. 4. B: I-V relationships of peak ICa,L under control () and following exposure to H2O2 (open circle ) were obtained from the experiment results in A.

Involvement of PKC in the effect of H2O2 on ICa,L. Previously, it has been demonstrated that inhibition of PKC attenuates the effects of H2O2 on action potential waveform in rat ventricular myocytes (36). Since the negative findings using ruptured patch recordings in this study (Fig. 3) suggest that second messengers are also involved in H2O2-induced changes in ICa,L, we examined the potential role of PKC as a modulator of the effects of H2O2 on SAN cells. In these experiments, SAN cells were incubated with BIS, a potent PKC inhibitor, for at least 3 h to ensure complete PKC inhibition. Under these conditions, the ability of 100 µM H2O2 to increase ICa,L was almost completely abolished (Fig. 6A). In each of four cells examined, the peak and the persistent ICa,L currents in the presence of H2O2 were compared with the control levels (102.8 ± 0.5% and 102.6 ± 3.4% of the control, respectively). Neither value was significantly different from those without BIS incubation (Fig. 6B). Thus PKC inhibition strongly decreases the effect of H2O2 on ICa,L in SAN cells.


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  		Fig. 6.   Effect of the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIS, 100 nM) on the H2O2-induced increase in ICa,L. A: representative ICa,L traces in control (con) and following exposure to H2O2 are superimposed. Currents were elicited from a holding potential of -60 mV by a 500-ms depolarizing step to -10 mV. A 100-ms conditioning pulse to -35 mV was used to inactivate INa. B: relative changes of the peak and the noninactivating components of ICa,L with and without preincubation in BIS. Values are means ± SE for n = 6 myocytes in the control group and n = 4 myocytes pretreated with BIS. *Significant difference (P < 0.05) from control (no-BIS) conditions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous reports have shown that t-butyl hydroperoxide, a reactive oxygen agent, produced an initial increase followed by a decrease in spontaneous pacemaker rate in a multicellular SAN preparation (27, 28). These findings were interpreted in terms of a corresponding biphasic change in ICa,L, If, and IK (27). Our results from single cell recordings showed a similar biphasic rate change induced by H2O2. We found that the initial positive chronotropic effect of H2O2 is primary due to an increase in the amplitude and/or slowing of the inactivation rate of ICa,L. A slow decrease in If and no change in IK were observed in same period after exposure to H2O2. Our study focused on the initial positive chronotropic effect of H2O2 on SAN cells, since these effects are likely to be produced by a changes in specific ionic currents as opposed to being due to general rundown of the cell.

ICa,L is one of the transmembrane ionic currents that generates pacemaker activity in adult rabbit SAN cells (13). Inhibition of ICa,L can result in decrease of heart rate, as a result of a slowing of the pacemaker depolarization and a shift of the threshold of the action potential to more positive potentials (23). In contrast, enhancing ICa,L results in an increased heart rate and a shift of the firing threshold to more negative potentials (32). The functional role of ICa,L on the pacemaker action potential is illustrated in the mathematical model of SAN by Demir et al. (4) in 1994. Our results show that the initial effects of H2O2 on the SAN action potential include 1) a negative shift of the firing threshold and 2) prolongation of APD. These findings strongly suggest that H2O2 causes an increased inward current in the voltage range of the pacemaker depolarization and the action potential. Voltage-clamp experiments demonstrated that H2O2 enhanced the amplitude of ICa,L (Fig. 4). This current change was blocked by the Ca2+ channel antagonists nicardipine and Ni2+ but was not changed by TTX. These findings suggest that this current is ICa,L rather than INa or ICa,T, which are present in SAN cells and activated at similar membrane potentials.

The analysis of ICa,L following H2O2 treatment consistently showed significantly slowed inactivation compared with control records. A similar change in ICa,L has been reported recently by Thomas et al. (34) in 1998 in guinea pig ventricular myocytes. These results suggest that H2O2 can slow the inactivation of this Ca2+ current and/or shift the voltage dependence of its inactivation to more negative potentials. Further study is needed to identify the mechanisms of H2O2 on ICa,L inactivation, as this decrease in inactivation appears to be the major mechanism by which H2O2 enhances both peak and persistent ICa,L. Consistent with our experimental data, mathematical modeling of the SAN pacemaker action potential suggests that an incomplete inactivating component of ICa,L could alter the plateau phase and the APD (4). Although ICa,L inactivation kinetics alterations were not reported following oxidant exposure in multicellular SAN preparations (27, 28), this may be due to altered intercellular coupling between the individual SAN cells, which can change the spatial uniformity under voltage-clamp conditions, making it difficult to record small current changes. The interaction between heterogeneous cells in SAN tissue (18) in the presence of reactive oxygen species may also contribute to the reported differences in results obtained using single myocytes vs. those from multicellular preparations.

Prolongation of APD following H2O2 exposure has been reported previously in other cardiac myocytes (3, 24, 36). When coupled with depolarization of the resting membrane potential, this is thought to be one cause of reperfusion arrhythmias (7, 19). Other ionic current changes, e.g., a slowing of the rate of fast inactivation of INa, a decrease in IK, and/or an altered inward-rectifying K+ current (IK1), have also been suggested to be responsible for the observed changes in APD in the heart (3, 24, 36). Pacemaker cells from the SAN express very little IK1 and those from the central region of the SAN have no detectable INa (13, 18) (see also Fig. 4, at conditional pulse at -35 mV in nicardipine). Our experiments demonstrated that the H2O2-induced current was not sensitive to TTX pretreatment. Thus the mechanism of prolongation of APD in SAN cells is different from that previously identified in ventricular myocytes (36).

In multicellular SAN preparations, biphasic changes in If and IK have been reported after exposure to t-butyl hydroperoxide (27). However, we found no enhancement of these currents by H2O2. This difference may be explained, in part, by differences in study techniques and/or reactive oxygen agents we used in this study. If (measured at -100 mV) was consistently decreased after exposure to H2O2. It is difficult to ascertain whether this decrease is attributed to a rundown of the current during the experiment. Nevertheless, we can conclude that H2O2-induced alterations in this current are not responsible for the observed effects on action potential waveform: a decrease of If would have been expected to decrease the automaticity (5, 35). However, it should be noted that when electrically coupled to other SAN cells, as in multicellular preparations, it is possible that the small effects that we report on If could be an important mediator for the effects of H2O2.

In previous reports examining the effects of reactive oxygen species in guinea pig ventricular and frog atrial cells, the IK was decreased (3, 33). Deactivation of IK is an important contributor to the pacemaker depolarization in SAN cells. A decrease of IK would result in a prolongation of the APD. Therefore, it was important to examine the effects of H2O2 on this current. We failed to find any changes in IK tail current following exposure to H2O2. This lack of effect might be explained by a difference in channel subtypes in this cell. In rabbit SAN cells, IK has been reported to be mainly due to the rapid delayed rectifier current (21, 25). In contrast, in guinea pig ventricular and frog atrial myocytes, in which the previous studies were done, the slow delayed rectifier current predominates (10, 26).

The possible involvement of second messenger systems in the observed effects of H2O2 was examined in the final part of this study. Previous studies have shown that H2O2 exposure leads to alterations in intracellular calcium homeostasis (11, 31, 37). This could alter the activity of ionic currents regulated by intracellular calcium. PKC has also been identified as a mediator for the effects of H2O2 (34, 36, 37). Our results suggest that a second messenger mediates the effects of H2O2 on SAN cells as well. Thus, under experimental conditions in which the myoplasm was dialyzed with the pipette solution, the initial positive chronotropic effects of H2O2 were blunted or abolished. PKC is implicated as this soluble mediator, since pretreatment of cells with the PKC inhibitor BIS significantly attenuated the effects of H2O2. This is somewhat similar to our previous findings (36, 37). However, the direct effects of PKC activation on ICa,L are some what controversial. ICa,L has been reported to be increase (and then decrease) (8, 20, 30), decrease (29, 31), or remain unchanged (16). These discrepancies might be explained by variations of endogenous PKC levels and different PKC isoforms being expressed in different cell types (9, 15, 16, 22). In addition to PKC, it has also been shown that glutathione is a necessary cofactor for H2O2-induced effects (31). However, when glutathione was present H2O2 decreased ICa,L (31), whereas we report an increased current in the present study.

In summary, our results demonstrate a significant positive chronotropic effect followed by a negative chronotropic effect of H2O2 in cells isolated from the rabbit SAN. As well, H2O2 prolonged the duration of the SAN action potentials. Voltage-clamp measurements established that H2O2 induced a nicardipine- and Ni2+-sensitive and TTX-insensitive inward current, ICa,L. Pretreating the cells with an inhibitor of PKC prevented this increase of ICa,L. These findings suggest that reactive oxygen species generated during reperfusion of previously ischemic myocardium can alter heart rate by enhancing pacemaker activity of SAN cells.


    ACKNOWLEDGEMENTS

This research was supported by operating grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Alberta (to W. R. Giles) and by Heart and Stroke Foundation of Ontario Grant NA4173 (to C. A. Ward). W. R. Giles holds a Medical Scientist Award from the Alberta Heritage Foundation for Medical Research (AHFMR). J. Guo is an AHFMR Fellow. C. A. Ward is a Research Scholar of the Heart and Stroke Foundation of Canada.


    FOOTNOTES

Address for reprint requests and other correspondence: C. A. Ward, Dept. of Physiology, Queen's Univ., Kingston, Ontario, Canada K7L 3N6 (E-mail: wardc{at}post.queensu.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 December 1999; accepted in final form 23 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 279(3):H992-H999
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