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1 Department of Physiology, University of Leeds, Leeds LS2 9JT, United Kingdom; 2 Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-01, Japan; and 3 Department of Physiology, Iran University of Medical Sciences, Tehran, Iran
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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4-Aminopyridine (4-AP)-sensitive transient outward current (Ito) has been observed in the sinoatrial node, but its role is unknown. The effect of block of Ito by 5 mM 4-AP on small ball-like tissue preparations (diameter ~0.3-0.4 mm) from different regions of the rabbit sinoatrial node has been investigated. 4-AP elevated the plateau, prolonged the action potential, and decreased the maximum diastolic potential. Effects were greater in tissue from the periphery of the node than from the center. In peripheral tissue, 4-AP abolished the action potential notch, if present. 4-AP slowed pacemaker activity of peripheral tissue but accelerated that of central tissue. Differences in the response to 4-AP were also observed between tissue from more superior and inferior regions of the node. In the intact sinoatrial node, 4-AP resulted in a shift of the leading pacemaker site consistent with the regional differences in the response to 4-AP. It is concluded that 4-AP-sensitive outward current plays a major role in action potential repolarization and pacemaker activity in the sinoatrial node and that its role varies regionally.
heart; cardiac; pacemaking; transient outward potassium current
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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IT IS WELL KNOWN THAT transient outward current (Ito) is present in atrial and ventricular muscle and the atrioventricular node (17, 30, 32). Less is known of Ito within the sinoatrial node. Giles and van Ginneken (17) characterized Ito present in the atrial cells of the crista terminalis, which borders the sinoatrial node. Within the sinoatrial node, several authors have commented on the presence of Ito, although they did not characterize it (8, 21, 33). Recently, we (4) confirmed the existence of Ito in the sinoatrial node; 4-aminopyridine (4-AP) is a well-known blocker of Ito, and we showed the existence of substantial 4-AP-sensitive current in sinoatrial node cells of the rabbit. The 4-AP-sensitive current (Ito) was made up of two components, a transient outward component showing time-dependent inactivation and a sustained outward component. The physiological role of Ito in the sinoatrial node is not known.
Functionally, structurally, and electrophysiologically the sinoatrial node is not a homogeneous tissue. Normally, the center of the sinoatrial node, 1-2 mm distant from the crista terminalis, is the leading pacemaker site, i.e., the action potential is normally first initiated here (1, 26). From the center, the action potential is conducted in an oblique cranial direction through the transitional and peripheral regions of the sinoatrial node to the surrounding atrial muscle of the crista terminalis. Although the normal function of the transitional and peripheral regions of the sinoatrial node is to conduct the action potential from the center to the atrial muscle, in response to a wide variety of interventions the leading pacemaker site shifts to the transitional or peripheral region and, therefore, these regions can have a pacemaker role (37, 40). In the center, the cells are smaller than those in the periphery and contain fewer and less well-organized myofilaments (1, 36, 38). Furthermore, in the center, the action potential upstroke is slower, the action potential overshoot is less, the action potential is longer, the maximum diastolic potential is less negative, and intrinsic pacemaker activity is slower than in the periphery (1, 26, 28). The differences in electrical activity are known to be the result of genuine regional differences in membrane properties (rather than electrotonic interactions) because the differences are also seen in both small ball-like tissue preparations isolated from different regions of the sinoatrial node (26, 28) and in single cells (19).
We distinguish between single sinoatrial node cells on the basis of cell capacitance, a measure of cell size (we assume that the small cells are from the center, whereas the large cells are from the periphery; see above) (19). Small cells have a low density of the hyperpolarization-activated current (If) and lack the tetrodotoxin (TTX)-sensitive Na+ current (INa), whereas in large cells the density of both currents is high (19). The possibility that this could explain the faster intrinsic pacemaker activity of the periphery was confirmed by blocking the currents in small ball-like tissue preparations from different regions of the sinoatrial node (28, 35). Although the density of the transient component of Ito may not be correlated with cell capacitance, the density of the sustained component is correlated and is greater in larger cells with a higher capacitance (4). This suggests that the role of Ito will be greater in the periphery of the sinoatrial node. In the present study, we have investigated this possibility by blocking the current with 4-AP in small ball-like tissue preparations from different regions of the rabbit sinoatrial node.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Experiments were carried out on the intact sinoatrial node and small ball-like preparations of sinoatrial node tissue.
Intact sinoatrial node. New Zealand White rabbits weighing 1.5-2 kg were anesthetized with intravenous pentobarbital sodium (30-40 mg/kg). The chest was opened, and the heart was rapidly excised into modified Krebs-Ringer solution at 32°C. The right atrium was separated from the rest of the heart and opened by a longitudinal incision in the free wall to expose the endocardial surface. The right atrium was then trimmed to leave a preparation ~15 × 15 mm, which included the whole sinoatrial node and some of the surrounding atrial muscle. A typical preparation is illustrated in Fig. 1A. The preparation (endocardial surface up) was fixed in a tissue bath.
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Small ball-like preparations of sinoatrial node tissue. After the sinoatrial node had been isolated as described above, four strands of tissue (0.3-0.4 mm in width and 3-4 mm in length) were cut from the sinoatrial node in a direction perpendicular to the crista terminalis. A typical position of the strands in the intact sinoatrial node is shown in Fig. 1A; the cut end of the crista terminalis is marked CT. The crista terminalis runs from top to bottom in the diagram, and its position is marked by the right branch of the sinoatrial ring bundle (RSARB), a thin flap of tissue that also marks the border between the atrial muscle (left of the RSARB in Fig. 1A) and the sinoatrial node (right of the RSARB in Fig. 1A). The strands were cut around the expected leading pacemaker site and were numbered 1-4. It is known that the peripheral part of the sinoatrial node overlaps the atrial muscle of the crista terminalis, and a razor blade was used to remove this muscle from the strands as well as the lipid tissue on the epicardial surface of the remainder of the sinoatrial node. After they had been trimmed, the strands were ~0.3 mm in width, ~0.2 mm in depth, and ~3 mm in length. The strands were tied into a series of small balls (typically 5) with diameters of ~0.3-0.4 mm. The ball closest to the crista terminalis included the RSARB on its surface and was named A. The remaining balls were named B-E. The nomenclature used in relation to the balls is shown in Fig. 1B. Strand 1 was from the more superior (or cranial) part of the sinoatrial node, whereas strand 4 was from the more inferior (or caudal) part. Balls A and B, being closest to the atrial muscle of the crista terminalis, were from the periphery of the sinoatrial node; balls D and E, being distant from the crista terminalis, were from the center; and ball C was typically from a transitional region between the periphery and center. In the intact sinoatrial node, the leading pacemaker site would typically be one of the four balls represented by shading in Fig. 1B (middle-center). Figure 1C shows a drawing of a strand tied into a series of balls. The dissection procedure took several hours to complete because after each step the tissue was allowed sufficient time to recover and resume spontaneous activity. Once the dissection procedure was complete, a strand of balls (endocardial surface up) was fixed in the tissue bath.
In two successful experiments, strands were cut parallel to the crista terminalis and tied into a series of eight balls. The approximate position of such a strand is shown (dotted lines) in Fig. 1A. The strands were from the transitional-central region of the sinoatrial node and extended from the superior part of the sinoatrial node to the inferior part. The tissue bath was superfused with modified Krebs-Ringer solution at 32°C. Solution flowed under the action of gravity at a rate of 20-25 ml/min through a heat exchanger into the chamber. The bath temperature was monitored using a miniature thermistor to ensure that the temperature remained at 32°C. Experiments were carried out at 32°C, because our experience is that all electrophysiological properties are stable for much longer periods (>8 h) at 32°C than at 37°C. In some experiments, activation maps of the intact sinoatrial node were made by recording extracellular potentials from 90-100 sites with a pair of modified bipolar electrodes (see Fig. 10). The electrodes were positioned using a calibrated XYZ micromanipulator with 0.1-mm precision. Another pair of modified bipolar electrodes was used to record the extracellular potential from the atrial muscle as a reference signal. Each pair of modified bipolar electrodes consisted of two 100-µm stainless steel wires (one wire 1 mm shorter than the other) insulated to the tip and taped together. High-gain (50-88 dB) amplification and filtering (0.5-30 Hz band-pass filter used) of the signals from the modified bipolar electrodes resulted in a sharp negative deflection at the instant of activation of the recording site (confirmed by action potential recording by conventional glass microelectrodes). The time interval between the time of activation at the recording site and the time of activation at the reference site on the atrial muscle was measured (average time interval over 10 beats measured). The site showing the earliest activation (at which this interval was longest) was taken to be the leading pacemaker site. The time of activation of other sites with respect to the time of initiation of the action potential at the leading pacemaker site was shown as a series of isochrones at 5- to 10-ms intervals. The activation pattern was stable in all experiments reported. During the mapping procedure cycle length was measured 10 times at 5-min intervals (from beginning to end of mapping procedure), and the mean cycle length was then calculated from the 10 values. Intracellular action potentials were recorded from small balls of sinoatrial node tissue using conventional glass microelectrodes (resistance, 30-40 M
; filling solution, 3 M KCl). Action
potential duration at 
30 mV and spontaneous cycle length (time
interval between successive spontaneous action potentials) were
measured using an electronic device (24). Action potentials, action
potential duration, and spontaneous cycle length were recorded using a
thermal array recorder (RTA-1200, Nihon Kohden), tape (sampling rate, 5 kHz; digital magnetic tape recorder, PC-108M, Sony), and Axotape software (Axon Instruments, Burlingame, CA) for later analysis. The
modified Krebs-Ringer solution contained (in mM) 120 NaCl, 4 KCl, 1.3 MgSO4, 1.2 NaH2PO4,
1.2 CaCl2, 25.2 NaHCO3, and 4 glucose. The
solution was equilibrated with 95%
O2-5%
CO2 to give a pH of 7.4. A stock
solution of 0.5 M 4-AP was prepared in distilled water (pH titrated to
7.4 using HCl). This was added to modified Krebs-Ringer solution to
give the required concentration of 4-AP.
Data are presented as means ± SE for the indicated number of
preparations. Student's t-test
(paired or unpaired as appropriate) or a one-way analysis of variance
was used to test differences for normally distributed data. For data
not normally distributed, an equivalent nonparametric test was used
(Mann-Whitney rank sum test, Wilcoxon signed-rank test, Kruskal-Wallis
ANOVA on ranks). SigmaStat (Jandel Scientific Software) or Microsoft
Excel was used. A difference was considered significant if
P < 0.05. Linear regressions were
carried out using SigmaStat or Fig.P (Fig.P Software).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effect of 4-AP on small ball-like tissue preparations from different regions of sinoatrial node: Regional differences from periphery to center. Five millimolar 4-AP was principally used in this study (see DISCUSSION for justification of concentration used). In experiments on small ball-like tissue preparations, when a microelectrode impalement was steady, action potentials were recorded under control conditions and then 4-AP was applied for 2 min, after which 4-AP was washed off and the tissue was allowed to recover. All effects of 4-AP shown were recorded once the preparation had reached a steady state and were reversible on washoff of 4-AP.
Action potentials recorded from tissue taken from the periphery (ball A) and center (ball D) of the sinoatrial node are shown in Fig. 2. Under control conditions, typical differences in electrical activity between the periphery and center of the sinoatrial node can be seen: in the peripheral ball, the action potential upstroke was faster, the action potential overshoot was greater, the maximum diastolic potential was more negative, and pacemaking was faster. The effects of 5 mM 4-AP are also shown in Fig. 2. 4-AP increased both the overshoot and the duration of the action potential. The increase in duration was greater in ball A from the periphery than in ball D from the center. In the peripheral ball, 4-AP decreased the maximum diastolic potential (i.e., made it more positive; Fig. 2A), but in the central ball it increased the maximum diastolic potential (Fig. 2B). Although a decrease in the maximum diastolic potential was observed in all peripheral balls (A and B) studied (n = 19), an increase in the maximum diastolic potential was seen in 6 of 13 central balls (D and E). A decrease in the maximum diastolic potential was observed in the remaining seven central balls. Finally, 4-AP altered the cycle length, increasing it in the peripheral ball (Fig. 2A) but decreasing it in the central ball (Fig. 2B).
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action potential duration). For
example, in ball A from the periphery,
4-AP increased the cycle length by 91 ± 8 ms
(n = 9 preparations), but the
diastolic interval increased by only 28 ± 5 ms
(n = 9). Therefore, ~70% of the
increase in cycle length in response to 4-AP in ball
A is attributed to the increase in action potential
duration. In contrast, in ball E from
the center, 4-AP still prolonged the action potential (although by a
small amount) and both the cycle length and diastolic interval were
shortened (by 205 ± 23 and 243 ± 22 ms, respectively;
n = 5 preparations). In this case, the
change in cycle length obviously cannot be explained by the change in
action potential duration and is more likely to be a direct effect of
4-AP as considered in DISCUSSION.
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8 M TTX was applied to
inhibit nerve fibers in four balls of tissue, two peripheral balls
(A) and two central balls
(D). The effects of 5 mM
4-AP were similar in the absence and presence of TTX; for example,
under control conditions 4-AP increased action potential duration by 56 ± 10%, whereas in the presence of TTX it increased it by 59 ± 16% (paired t-test,
P = 0.68). It is concluded that the
effects of 4-AP are not indirect.
Effect of 4-AP on small ball-like tissue preparations from different regions of sinoatrial node: Regional differences in superior-inferior direction. The action potential in the sinoatrial node not only varies from the periphery to the center, it also varies from the superior to the inferior region, although less is known about the variation in this direction. The superior sinoatrial node-inferior sinoatrial node differences are important because pacemaker shift almost invariably involves a shift in the superior or inferior direction (37). In two successful experiments, a strand from the transitional-central region of the sinoatrial node was made running parallel to the crista terminalis (i.e., running from the superior part to the inferior part of the sinoatrial node; Fig. 1). The strand was tied into a series of eight small ball-like tissue preparations, each ~0.3-0.4 mm in diameter. Figure 8 is taken from one of these experiments and shows that the response to 4-AP varied in the superior and inferior regions. Figure 8A shows the effect of 4-AP on the action potential from a ball from the superior region (1st ball, i.e., most superior, in the strand), whereas Fig. 8B shows the effect on the action potential from a ball from the inferior region (7th ball in the strand). In both balls the action potential was prolonged, but the prolongation was greatest in the ball from the more inferior region. A similar result was obtained from the second experiment. To quantify this regional difference, strands 1-4 cut perpendicular to the crista terminalis as shown in Fig. 1 were used. Mean data for action potential duration are shown in Fig. 9, A and B (data for balls C and D are shown combined). Under control conditions there was a small (but not significant) increase in action potential duration from strand 1 from the more superior region to strand 4 from the more inferior region. In all balls, 4-AP caused a significant increase (P < 0.005) in action potential duration (Fig. 9A), but the percent increase was significantly greater (ANOVA on ranks, P = 0.002) in the balls from the more inferior region (Fig. 9B). In the presence of 4-AP there was a significant gradient (ANOVA, P < 0.001) in action potential duration from the more superior to the more inferior region, unlike under control conditions.
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Effect of 4-AP on intact sinoatrial node. In the intact sinoatrial node, the changes in the intrinsic pacemaker activity of different regions caused by 4-AP could result in pacemaker shift. The leading pacemaker site is the site showing the fastest pacemaker activity. Although inspection of Fig. 6A suggests that the periphery will be the leading pacemaker under control conditions, this is not the case, because in the intact sinoatrial node the periphery is suppressed by the atrial muscle and the leading pacemaker site is in the transitional or central region (25). In the presence of 4-AP the intrinsic pacemaker activity of the peripheral and transitional regions is suppressed, whereas that in the center is accelerated (Fig. 6), and, therefore, the leading pacemaker site is expected to shift further toward the center (i.e., away from the atrial muscle of the crista terminalis). Furthermore, because 4-AP accelerates the intrinsic pacemaker activity of the more superior region but suppresses the intrinsic pacemaker activity of the more inferior region (Fig. 9), 4-AP is expected to shift the leading pacemaker site toward the superior region of the sinoatrial node. These predictions were tested in a series of eight experiments on the intact sinoatrial node.
Activation maps were constructed as described in METHODS under control conditions and after the application of 5 mM 4-AP for at least 40 min (to allow the preparation to reach a steady state). Although the small ball-like tissue preparations were exposed to 4-AP for a shorter time, our experience is that the intact sinoatrial node preparations always take longer to reach steady state after application of a drug than the small ball-like tissue preparations, presumably because of the more extensive tissue mass (see also Refs. 28, 35). A result is shown in Fig. 10. The isochrones show the extent of propagation of the action potential in a given time (in ms) after the action potential was first initiated at the leading pacemaker site (0-ms isochrone); the set of isochrones shows the sequence of activation. Under control conditions, spontaneous excitation first occurred at a site 0.7 mm from the RSARB (Fig. 10A); this is typical (1). After the application of 4-AP, there was no significant change in the cycle length (in 8 preparations, cycle length was 565 ± 30 ms under control conditions and 580 ± 29 ms in presence of 4-AP). After the application of 4-AP, the leading pacemaker site shifted in the superior direction and away from the crista terminalis (Fig. 10B). This is consistent with our predictions. In seven preparations, the leading pacemaker site shifted by 1.8 ± 0.3 mm. The shift was 1.5 ± 0.3 mm along the RSARB in the superior direction and 0.5 ± 0.3 mm away from the crista terminalis. In one other preparation, there was no pacemaker shift in the presence of 4-AP.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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This study shows for the first time that 4-AP-sensitive current plays an important role in the electrical activity of the sinoatrial node and that this role varies from the periphery to the center and from the superior part of the sinoatrial node to the inferior part.
Viability of preparations used. The majority of the experiments in this study were carried out on strands of tissue divided into small balls of tissue (~0.3-0.4 mm in diameter) by ligatures. Several lines of evidence suggest that although the balls of tissue were small they were unlikely to be damaged significantly by the dissection procedure: 1) all balls studied showed stable spontaneous activity; 2) the regional differences in electrical activity of the small balls have been consistently observed in various studies in our laboratories in Japan and England (see, e.g., Refs. 28, 35) since we first reported them in 1985 (26); and 3) the electrical activity of the small balls is similar to the electrical activity recorded from similar sites in the intact sinoatrial node (26), and the electrical activity of the small balls prepared using ligatures is similar to that of small balls prepared by cutting (39).
Nature of 4-AP-sensitive current. Ito is blocked by 4-AP with EC50 values of 0.2-0.5 mM (6, 41). Kv4.2 and Kv4.3 channels are probably responsible for Ito, although it has been suggested that Kv1.4 is responsible (6, 10, 15). Ultrarapid delayed rectifier K+ currents (IK,ur) are also blocked by 4-AP, with EC50 values ranging from 5 µM to 1 mM (44). Kv1.5 and possibly Kv3.1 channels may be responsible for such currents (13, 14, 44). In sinoatrial node cells, 4-AP blocks a transient outward current (8, 33) and, in addition, a sustained outward current (4, 21). Here it is assumed that the sustained current is a noninactivating component of Ito. However, it is possible that it is a separate current (possibly an IK,ur). We have recently cloned a Kv4.2 channel from a rabbit sinoatrial node cDNA library, and the channel when expressed in Xenopus oocytes is similar (but not identical) to Ito in the rabbit sinoatrial node (E. Conley, J. Hancox, and M. R. Boyett, unpublished observations). In addition, using immunocytochemistry we have shown the presence of Kv1.5 in the guinea pig sinoatrial node (11). The concentration of 4-AP used in the present study is sufficient to block any of the currents above. The dose dependence of the effect of 4-AP on action potential duration (Fig. 7B) is comparable to that of Ito.
4-AP can affect other currents. In skeletal muscle, 4-AP blocks the ATP-sensitive K+ current (IK,ATP), with an EC50 value of 3.3 mM at 0 mV (7). However, IK,ATP is not expected to be present in the sinoatrial node under normal conditions. In sheep cardiac Purkinje fibers, 4-AP blocks the background inward rectifying K+ current (IK,1) (42), but this current is absent from the sinoatrial node (20). Finally, another K+ current, the muscarinic K+ current (IK,ACh), is reported to be activated by 4-AP (34). In our experiments on rabbit sinoatrial node cells, 4-AP only blocked a transient outward current and a sustained outward current during depolarizing pulses (4); it had no effect on the holding current at60 mV (see also Ref. 21).
This suggests that in our experiments
IK,ATP,
IK,1, and
IK,ACh (if
present) were not being affected, because at 60 mV (with a
normal extracellular K+
concentration),
IK,ATP,
IK,1, and
IK,ACh are
expected to be large and a change in one of the currents should have
resulted in a discernible change in whole cell current. If 4-AP does
generally block
IK,1, it is
curious that a depolarization of the resting membrane is not a
characteristic feature reported (see, e.g., Ref. 9). Furthermore, in
our experiments there was no 4-AP-sensitive tail current after
depolarizing pulses (4); this confirms that 4-AP did not block the
rapid or slow delayed rectifying
K+ currents
(IK,r and
IK,s,
respectively). Finally, 4-AP has been shown to affect the
hyperpolarization-activated current
If (42); it
shifts the If
activation curve in the depolarizing direction, although it also partly
blocks the current. This effect of
If is expected to
hasten pacemaker activity and perhaps to cause a decrease in the
maximum diastolic potential but to have no effect on action potential
duration.
Role of 4-AP-sensitive current in sinoatrial node. In atrial, Purkinje, and ventricular tissue, block of 4-AP-sensitive current can greatly slow the initial rapid phase of repolarization (phase 1) after the action potential peak, abolish the action potential notch, elevate the action potential plateau, and prolong the action potential (3, 9, 17). Block of Ito by 4-AP can explain all of the actions of 4-AP. However, block of IK,ur alone is known to produce a prolongation of the action potential, and, therefore, block of IK,ur could contribute to the prolongation of the action potential in the presence of 4-AP (see, e.g., Ref. 44). In the present study, 4-AP produced all of the above effects in the sinoatrial node: it slowed phase 1 repolarization and abolished the action potential notch (if present), elevated the plateau, and prolonged the action potential (Figs. 2-5). Like previous authors, we suggest that these 4-AP-dependent changes are the result of the block of Ito (and possibly IK,ur); they cannot be the result of a change in IK,ATP, IK,1, IK,ACh, or If for the reasons discussed above. The results obtained suggest that the 4-AP-sensitive current plays a more important role in the periphery of the sinoatrial node, because the notch and its abolition by 4-AP were only observed in the periphery (Fig. 3), there was only a significant increase in the action potential overshoot by 4-AP in the periphery (Fig. 4A), and the increase in action potential duration was significantly greater in the periphery (Fig. 5B). There are several reasons why the effects of 4-AP were greater in the periphery. First, the density of Ito may be greater in the periphery. We have previously shown (4) that the density of the sustained component of Ito is greater in larger sinoatrial node cells presumably from the periphery of the sinoatrial node. Second, because the diastolic potentials are more negative in the periphery of the sinoatrial node compared with those in the center, the voltage-dependent inactivation of Ito during diastole is expected to be less in the periphery (and, consequently, greater Ito will be activated during the action potential in the periphery). There is another reason why the action potential notch may only be evident in the periphery. In the center, Ito may activate during the slow upstroke of the action potential and, therefore, activation of the current will not lead to a notch, whereas in the periphery activation of Ito will follow the rapid upstroke and may lead to a notch.
The results obtained also suggest that 4-AP-sensitive current plays a greater role in the inferior part of the sinoatrial node, because the prolongation of the action potential caused by 4-AP was greater in the inferior part compared with that in the superior part (Fig. 9). If the peripheral-central and superior-inferior differences are caused by differences in the expression of an ion channel, then the regional differences in ion channel expression are complex. In the sinoatrial node, in addition to the classical effects of 4-AP, 4-AP also affected the maximum diastolic potential and pacemaking (Figs. 2, 4B, and 6). Generally, 4-AP decreased the maximum diastolic potential (Figs. 2 and 4B). The decrease suggests that 4-AP-sensitive current contributes to the maximum diastolic potential. In sinoatrial node cells, the activation threshold of Ito is about70 mV and, therefore, it is possible that there will be
activation of 4-AP-sensitive current during diastole (4). Figure
4B shows that the decrease in the
maximum diastolic potential was greater in the periphery of the
sinoatrial node; this is consistent with the other evidence summarized
above showing that the role of 4-AP-sensitive current is greater in the
periphery. However, another explanation of the depolarization is that
it is the result of the 4-AP-induced depolarizing shift in the
If activation
curve (42).
In some central balls, 4-AP increased the maximum diastolic potential
(Fig. 2B). This is perhaps an
indirect consequence of the block of the 4-AP-sensitive current; the
elevation and prolongation of the plateau caused by 4-AP is expected to
enhance the activation of delayed rectifying
K+ currents
(IK,r and
IK,s), which in
turn will increase the maximum diastolic potential. The increase in
maximum diastolic potential was only observed in central balls, in
which the increase in action potential duration was small; perhaps if
the role of the 4-AP-sensitive current is slight (manifested as a small
increase in action potential duration), the direct effect of the
decrease in 4-AP-sensitive current on the maximum diastolic potential
is slight and the indirect effect of the increase in the delayed
rectifying K+ currents dominates.
In the central balls, 4-AP accelerated pacemaker activity (Figs. 2 and
6), and Figs. 2B and
8A show that this was the result of an
acceleration of the pacemaker potential. This could be a direct effect
of the block of 4-AP-sensitive current flowing during diastole, the
evidence for which is considered above. If correct, this shows that
4-AP-sensitive current helps control the pacemaker potential and
pacemaker activity. Alternatively, the acceleration in rate could again
be the result of the 4-AP-induced depolarizing shift in the
If activation
curve (42). In some central balls, 4-AP resulted in an increase of the
maximum diastolic potential, whereas in the majority it resulted in a
decrease; the change in cycle length in the central balls was not
correlated with the change in maximum diastolic potential.
If the role of 4-AP-sensitive current is greater in the periphery, the
acceleration of pacemaker activity by 4-AP may be expected to be
greater in the periphery, whereas the reverse was true. In peripheral
balls, 4-AP prolonged the cycle length rather than shortening it (Figs.
2A and 6). The prolongation of the
cycle length is likely to be another indirect effect of 4-AP; as shown in Effect of 4-AP on small ball-like tissue
preparations from different regions of sinoatrial node: Regional
differences from periphery to center (see Fig.
7A and related text), much of this was
the result of the marked increase in action potential duration in the
peripheral balls.
Physiological importance of 4-AP-sensitive current and its region dependence. From the center of the sinoatrial node through the periphery of the node to the atrial muscle there is a gradient in action potential duration, with the action potential in the center being the longest and the action potential in the atrial muscle being the shortest. This gradient is observed in the intact sinoatrial node (35) and also in the small balls of sinoatrial node tissue (Fig. 5A; see also Ref. 28). We suggest that this is a protective mechanism. Because the action potentials more distant on the conduction path are shorter than those earlier in the conduction path, repolarization occurs in the same direction as depolarization and occurs last in the center of the sinoatrial node. This will prevent reexcitation arrhythmias by preventing reexcitation of the sinoatrial node by the atrial muscle. A similar gradient in action potential duration prevents reexcitation at other points in the excitation pathway: of the atrial muscle of the crista terminalis by the atrial muscle of the atrial appendage (43), of the Purkinje fibers by the ventricular muscle, and of the ventricular subendocardium by the ventricular subepicardium. Figure 5A shows that the gradient in action potential duration from the center to the periphery was abolished by 4-AP, and therefore, the 4-AP-sensitive current must be responsible for it. It is interesting that Ito is responsible for other region-dependent differences in the action potential (9, 30, 43) and contributes to remodeling of electrical activity in development (22), hypertrophy (see, e.g., Ref. 31), heart failure (23), and the phenomenon of cardiac memory (16).
Figure 6A shows that 4-AP reduced the regional difference in pacemaker activity (difference in cycle length in balls A and E is smaller in presence of 4-AP). Regardless of the reasons for the changes in cycle length in the presence of 4-AP (see Role of 4-AP-sensitive current in sinoatrial node for a discussion of possibilities), the result shows that 4-AP-sensitive current, as well as If and INa (see Regional differences in role of membrane currents in sinoatrial node), must be responsible for the regional differences in pacemaker activity. The regional differences in pacemaking are expected to be important physiologically, because they will make the sinoatrial node more robust. An intervention that may adversely affect one region may be better tolerated by another. Ito is modified by a variety of different interventions, e.g.,
-adrenergic agonists
(5). Because our data show that 4-AP-sensitive current is able to
alter, directly or indirectly, pacemaker activity (Fig. 6), it is
possible that these interventions may be able to alter pacemaker
activity via an effect on
Ito.
The maximum diastolic potential is greater in the periphery of the
sinoatrial node, and Fig. 4B shows
that 4-AP-sensitive current is involved in the difference, because the
difference was smaller in the presence of 4-AP.
Regional differences in role of membrane currents in sinoatrial node. The greater importance of 4-AP-sensitive current in the periphery compared with the center of the sinoatrial node is only one of a number of regional differences in membrane currents to emerge. Above it is argued that pacemaking is faster in the periphery than in the center partly because the action potential is shorter in the periphery (because of 4-AP-sensitive current). However, there are at least two other reasons for the faster pacemaking in the periphery. 1) If plays a greater role in pacemaking in the periphery, possibly as a result of a greater density of If (29, 35). 2) Although the Ca2+ current (ICa) is involved in pacemaking in the center, the TTX-sensitive INa is involved in the periphery, possibly because INa is absent in the center but is large in the periphery (19). (ICa and INa are involved in pacemaking by triggering the action potential, and pacemaking is faster when INa is responsible because the threshold for INa is more negative than that of ICa.) Because INa is present in the periphery but not the center, the action potential upstroke is faster in the periphery than in the center. Guo et al. (18) reported that the sustained inward current (Ist) is present in central sinoatrial node cells but is absent from peripheral cells; the significance of this is not known. Finally, partial block of the rapid delayed rectifying K+ current (IK,r) by E-4031 has greater effects on the center of the sinoatrial node (I. Kodama, M. R. Boyett, M. R. Nikmaram, H. Honjo, and R. Suzuki, unpublished observations); this suggests that the density of IK,r may be less in the center (and, therefore, the center is more sensitive to partial block of the current), and, if correct, this difference in IK,r would also contribute to the regional difference in action potential duration and maximum diastolic potential. All of the published information concerning regional differences in membrane currents within the sinoatrial node is concerned with differences between the periphery and center. Evidence for a greater role of 4-AP-sensitive current in the inferior part of the sinoatrial node from the present study is the only information about differences in the superior-inferior direction, although we have previously shown (27) that the response to vagal stimulation not only varies in the peripheral-central direction but also varies in the superior-inferior direction.
Although reported space constants of the rabbit sinoatrial node are variable (12), Bonke (2) reported a mean value of 465 µm. This is small relative to the size of the sinoatrial node of the rabbit (see, e.g., Fig. 1). Therefore, the regional differences seen in small balls of tissue are also expected to be seen in the intact sinoatrial node, and this is indeed the case (see, e.g., Ref. 1).| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by the British Heart Foundation, the British Council, the Japanese Ministry of Education, Science, Sports and Culture, and the Japan Society for the Promotion of Science.
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FOOTNOTES |
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Address for reprint requests: M. R. Boyett, Dept. of Physiology, Univ. of Leeds, Leeds LS2 9JT, UK.
Received 24 June 1997; accepted in final form 29 May 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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) in action potential overshoot (OS;
A) or MDP
(B) in peripheral and central balls
on application of 4-AP. n, No. of
preparations.
, Data from
balls A and
B; 
, data from
balls D and
E. Means ± SE are shown
(n = 4 or 5 preparations). Data are
fitted with typical dose-response curves to guide the eye.
and arrow, leading pacemaker site under control conditions;
, position of reference electrode (see
METHODS).