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Krannert Institute of Cardiology, Indianapolis, Indiana 46202
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Sudden
increases in heart rate cause accumulation of K+ in the
extracellular space. However, the exact relationship between rate and
extracellular K+ concentration
([K+]o) in vivo is unknown. We measured
[K+]o in right atria of anesthetized dogs by
using K+-sensitive electrodes. Peak increase in
[K+]o ranged from 0.18 ± 0.04 mM
[means ± SE; cycle length (CL) = 350 ms] to 0.80 ± 0.09 mM (CL = 250 ms) above baseline (3.50 ± 0.08 mM at
CL = 380 ms; n = 5). During rapid pacing-induced
atrial fibrillation, peak increase in [K+]o
averaged 0.80 ± 0.07 mM (n = 5). Whole cell
current-clamp measurements in single right atrial myocytes
(n = 5) showed that raising
[K+]o from 3 to 5 mM in 1-mM steps
progressively depolarized resting membrane potential and reduced both
phase 0 action potential amplitude and maximal upstroke
velocity. Multisite epicardial mapping (n = 4)
demonstrated that sudden rate increases changed longitudinal conduction
velocity (CVL) by 
3.6 ± 1.8% to 5.9 ± 1.2% over a CL range of 330 to 250 ms. Our observations suggest that
rate-related [K+]o accumulation in vivo is of
sufficient magnitude to modulate those cellular electrophysiological
properties that determine atrial CVL.
atrium; fibrillation; tachycardia; potassium
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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A SUDDEN INCREASE IN
RATE causes transient net loss of K+ from isolated
preparations of cardiac tissue (17), from whole heart preparations (5), and from hearts in vivo
(23). Net K+ loss indicates efflux of
K+ in excess of K+ influx. If there is a
restriction to diffusion away from the cell membrane, a net
K+ efflux would be expected to increase extracellular
K+ concentration ([K+]o) and
thereby generate a concentration gradient between the extracellular
space and the perfusion fluid. Previously, Kunze (16) used
K+-sensitive electrodes to study isolated superfused rabbit
atria and showed that transient increases in
[K+]o of 0.4-1.8 mM occurred as the rate
was raised to frequencies between 60 and 300 min
1.
Because fluctuations in [K+]o activity within
this range have been shown to influence cardiac electrophysiological
properties by altering K+ equilibrium potential and
K+ conductance (1, 3, 4, 11, 13-15, 28),
rate-dependent changes in K+ distribution must be
considered when studying the pattern of electrical activation and
inactivation in the atrium. Because the magnitude and time course of
K+ accumulation critically depend on the diffusion distance
between the cells and the capillary blood (or in the case of isolated superfused preparations the bathing solution), the shorter distances between the cells and the capillaries in the intact heart, as opposed
to the superfused tissue, would be expected to reduce the extent of
[K+]o accumulation. Absence of an intact
vascular supply in isolated superfused cardiac muscle, therefore,
raises the interesting and important question of whether the previous
in vitro observations can be applied to in vivo function. Despite their
importance to electrophysiological events in the atria, rate-related
changes in [K+]o in the mammalian atrium in
vivo have not been described. The response of
[K+]o to acute atrial fibrillation (AF) is
also not known. The present study was, therefore, undertaken
1) to measure atrial tachycardia- and AF-induced increases
in [K+]o in right atrial myocardium of
anesthetized open-chest dogs, 2) to determine the effects of
these changes in [K+]o on
electrophysiological properties of single atrial myocytes in vitro, and
3) to correlate the magnitude and time course of rate-dependent changes in [K+]o with those in
atrial conduction velocity.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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[K+]o Measurement with K+-Sensitive Electrodes
Mongrel dogs (n = 6) were anesthetized with thiopental sodium (25 mg/kg iv). Anesthesia was maintained with pentobarbital sodium (5 mg · kg1 · h1 iv). After
intubation with a cuffed endotracheal tube, positive pressure
ventilation was begun. Care was taken to adjust respiratory rate and
tidal volume to obtain normal arterial blood gases and pH. Values for
pH, PO2, PCO2, and
HCO
K+-sensitive miniature electrodes were made according to the methods of Johnson et al. (11). The electrodes were fabricated by coating the tips of chloridized silver wires (outer diameter, 0.007 in.) with a cellulose acetate-titanium dioxide sponge. A polyvinyl chloride-valinomycin-based coating provided the K+-selective membrane. The sponge was applied to achieve a tip length of 0.5-1.0 mm, and the ion-selective membrane coat, extended beyond the sponge by ~0.5 mm. Sponged silver wires without the ion-selective membrane served as reference electrodes. The electrodes were calibrated in vitro at 37°C with 3 and 10 mM KCl-NaCl solutions (total ionic strength 150 mM). Electrodes were used if they exhibited a 56-61 mV shift per 10-fold change in [K+] (11).
Wires from the reference and K+ electrodes were threaded in a retrograde fashion into the tip of a 21-gauge needle. After the electrode was inserted in a thicker portion of the right atrial myocardium (either crista terminalis or pectinate muscle), the needle was withdrawn, leaving the electrodes in place. The entire K+-selective region was inside the atrial muscle. The distance between the K+-sensitive electrode and reference electrode was ~1 mm. The integrity of the K+ electrodes in vivo was assessed by responses to intravenous injections of concentrated KCl (0.12 mmol/kg) in 3 ml of saline (11). The response pattern was stable over the duration of our measurements (usually <60 min). At the end of the study, the right atrium was excised to confirm the position of the K+ electrode. A total of six K+ electrodes (1 electrode per dog) were successfully placed into the atrial myocardium. At autopsy, four electrodes were found in the crista terminalis, and two electrodes were found in the pectinate muscle.
Lead II of the electrocardiogram, the bipolar left ventricular
electrogram, arterial blood pressure, and the K+ signal
were displayed on a monitor (Bard Electrophysiology, Marcom, Minneapolis, MN) and recorded on videotape. Voltage signals from the
K+ electrodes were differentially amplified with filter
settings of direct current to 1 Hz (custom-made amplifier, Krannert
Institute of Cardiology). All signals were stored on a personal
computer and displayed on-line on a computer monitor. The calibration
curve obtained in vitro for the K+ electrode was also
stored on computer. Results are presented as millimolar
[K+]o, with the activity coefficient for
K+ equal to 0.746 (8). The millivolt changes
(
mV) recorded from the K+ electrodes, in vivo, were
directly converted to [K+]o according to the
equation (31) mV = slope · log
([K+]o/[K+]o,serum),
where slope indicates the voltage difference for a 10-fold change in
[K+] at 37°C, and
[K+]o,serum is the
[K+]o in the arterial serum. Because
K+ activities in the arterial serum and extracellular space
are not significantly different in the steady state (8),
changes in [K+]o were referenced to the serum value.
The reference [K+]o was measured from an arterial blood sample withdrawn during constant baseline pacing. In preliminary experiments, we had found that arterial [K+]o remained unchanged during rapid pacing and AF, and therefore, only one sample was drawn at the beginning of each protocol.
Experimental Protocols for Measurement of [K+]o
To determine the effect of changes in pacing CL on [K+]o accumulation in right atrial myocardium, the right atrium was stimulated at CLs of 350, 330, 300, 280, and 250 ms for 2 min from a baseline CL of 380 ms. Five minutes at the baseline CL separated each 2-min pacing period. The sequence of CLs was randomly selected. At each stimulation rate, we measured the maximal change in [K+]o to obtain the[K+]o-rate relationship.
AF was induced by rapid right atrial pacing at a CL of 100 ms for 5 min
by using 2-ms pulses with current amplitudes of four times the
diastolic threshold for atrial capture. This maneuver reliably
initiated and maintained AF, which usually converted to sinus rhythm
<30 s after cessation of rapid pacing. Atrial pacing at a CL of 380 ms
was resumed immediately on conversion to sinus rhythm.
[K+]o was continuously monitored before,
during, and after AF. We determined the magnitude of the maximal
[K+]o during AF, as well as the time
course of accumulation and resolution of changes in
[K+]o.
Isolation of atrial myocytes. The heart was rapidly removed after an intravenous bolus injection of 10,000 international units of heparin and immersed in oxygenated (100% O2) normal Tyrode solution at room temperature. The right coronary artery was cannulated, and perfusion with Tyrode solution was started at a constant rate of 12 ml/min. The right atrium was then dissected free and any leaks from right coronary artery branches were stopped by ligation with 3-0 silk suture to ensure adequate perfusion of right atrial tissue. As soon as the coronary effluent was clear of blood, perfusion was switched to nominally Ca2+-free Tyrode solution for 20 min, followed by 60-80 min of perfusion with the same solution containing collagenase type II (100 U/ml; Worthington Biochemical, Lakewood, NJ) and 0.1% BSA (Sigma, St. Louis, MO). A small piece of tissue was taken from the crista terminalis and minced, and single cells were obtained by trituration with a wide-bore Pasteur pipette. Cells were kept in storage solution at room temperature until use. A small aliquot of the cell suspension was placed in a 0.5-ml heated perfusion chamber (Harvard Apparatus, Holliston, MA) on the stage of an inverted microscope. Cells were allowed to adhere to the bottom of the chamber, and then they were superfused at 2 ml/min with Tyrode solution described below. Experiments were performed at 36°C. Only quiescent, rod-shaped myocytes with clear cross striations were used.
Tyrode solution for cell isolation contained (in mM) 136 NaCl, 2 CaCl2, 5.4 KCl, 0.8 MgCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES (pH 7.4 adjusted with NaOH). The same solution was used for action potential studies except for the [K+], which was modified as indicated in the text. The storage solution contained (in mM) 136 NaCl, 5 KCl, 1 MgCl2, 0.33 NaH2PO4, 10 glucose, 10 HEPES, 1 CaCl2, and 0.2% BSA. The pipette solution for action potential recording contained (in mM) 110 potassium aspartate, 20 KCl, 1 MgCl2, 5 ATP-Mg, 0.1 GTP, 10 HEPES, 5 Na2-phosphocreatine, and 0.05 EGTA (pH 7.2 adjusted with 5 N KOH). The pipette solution for measurement of steady-state whole cell currents contained (in mM) 86 potassium aspartate, 20 KCl, 1 MgCl2, 5 ATP-Mg, 0.1 GTP, 10 HEPES, 5 Na2-phosphocreatine, and 10 EGTA (pH 7.2 adjusted with 5 N KOH). The final [K+] after pH adjustment was 142.2 mM in both pipette solutions. The K+ equilibrium potentials (EK) as determined by the Nernst equation {EK =61.5 log
([K+]i/[K+]o)}
were 103, 95, and 89 mV at 3, 4, and 5 mM
[K+]o, respectively, assuming that
intracellular [K+] ([K+]i)
remains constant.
The whole cell patch-clamp technique (6, 24) was used to
record action potentials in the current-clamp mode, and membrane currents were recorded in the voltage-clamp mode. Electrodes made of
borosilicate glass were filled with the pipette solution and connected
to a patch-clamp amplifier (Axopatch 200A; Axon Instruments, Union
City, CA). Action potentials were evoked by applying 2-ms depolarizing
current pulses. Stimulus strength was adjusted to produce a latency of
2-2.5 ms. Stimulus strength was generally at 1.1- to 1.5-times
diastolic threshold. Atrial myocyte membrane potential was recorded in
the normal current (INormal) mode of the
Axopatch 200A patch-clamp amplifier. Action potential measurements were
begun 5 min after cell membrane rupture. Voltage-command pulses were
generated by a 12-bit digital-to-analog converter controlled by pClamp
6 software (Axon Instruments). Recordings of whole cell currents and
action potentials were low-pass filtered at 300 Hz and 10 kHz and
sampled at 1 and 20 kHz, respectively, and then stored on the hard disk
of a personal computer.
Junction potentials between the bath and pipette solution averaged
approximately 5 mV and were corrected for action potentials only.
Seal resistance was >5 G
. The series resistance was electronically compensated. Membrane capacitance (Cm) was
calculated as published previously (26).
Activation mapping. Epicardial mapping of right atrial activation was performed as described previously (27). Maps were recorded before and 10, 40, 70, and 100 s into each 2-min period of constant atrial pacing at CLs of 330, 300, 280, and 250 ms. Four maps were recorded at 30-s intervals starting 10 s after resumption of baseline CL (380 ms). There was a 5-min pacing period between each episode of rate increase. The sequence of pacing CLs was random. Electrical stimuli of 2-ms duration and twice diastolic threshold were delivered through Teflon-coated stainless steel electrodes inserted into the right atrial appendage by using a programmable stimulator and a constant current isolator (Krannert Engineering, Indianapolis, IN). Activation maps for each time point were generated off-line after the experiment. Longitudinal conduction velocity (CVL) was measured by analyzing activation times at a series of electrode sites in the direction of the longitudinal propagation as determined from activation maps (18). An activation time was defined to be the time of the greatest negative downslope of an electrogram. The distance of each electrode site from the first of the series of electrodes was plotted as a function of activation time, and CVL was measured by using linear regression analysis. The number of electrode sites for each measurement always exceeded three and the correlation coefficient was always >0.99. Results from three consecutive beats were averaged for each CVL determination.
Data Analysis
Data are presented as means ± SE. Responses of [K+]o accumulation to changes in stimulation rate, serial [K+]o measurements, [K+]o-induced changes in single-cell action potential parameters, and steady-state, current-voltage relationships were analyzed by repeated-measures ANOVA. Multiple comparisons were made by using the Newman-Keuls multiple comparison test. Differences were considered significant at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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[K+]o Accumulation in Response to Sudden Rate Changes
A representative response of [K+]o to atrial pacing at progressively shorter CLs from a baseline CL of 380 ms is illustrated in Fig. 1. A stepwise decrease in stimulation CL increased [K+]o to a maximum, which then stayed elevated throughout the remainder of each 2-min stimulation period. As the initial CL was resumed, [K+]o levels declined to their respective baseline values. Time course and magnitude of changes in [K+]o were similar between crista terminalis and pectinate muscle. Fig. 2A summarizes the CL dependence of[K+]o, in
right atrial myocardium. Maximal [K+]o
monotonically increased with decreasing CL. Because
[K+]o in the arterial serum averaged
3.50 ± 0.08 mM, [K+]o increased to
maximum mean values ranging from ~3.7 mM at a CL of 350 ms to ~4.3
mM at a CL of 250 ms. Mean arterial blood pressure at the time of
maximal increase in [K+]o was unchanged at
CLs 
280 ms compared with baseline, but was slightly reduced to
104 ± 7 mmHg at the shortest pacing CL (Fig. 2B;
P < 0.001).
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[K+]o Accumulation in Response to AF
Figure 3 shows representative recordings of [K+]o (top trace), mean arterial blood pressure (middle), and RR intervals immediately before and during rapid atrial pacing-induced AF, and over a 3-min period after resumption of right atrial pacing at the baseline CL of 380 ms. [K+]o rapidly rose to its peak, and then declined slowly until it reached a stable value during the last minute of maintained AF. [K+]o levels rapidly returned to control values after conversion to sinus rhythm and resumption of baseline pacing rate. Note that the effects of electrically maintained and spontaneous AF on [K+]o were similar. Mean arterial blood pressure immediately fell with the onset of AF and tended to return to control values as AF continued. RR intervals were significantly decreased and remained unchanged as long as AF persisted. Figure 4 summarizes[K+]o as a function of the duration of AF.
When AF persisted for 5 min, [K+]o initially
rose to a maximum, averaging 0.8 ± 0.07 mM above the
prefibrillation level (range, 0.58-1.01 mM), and stabilized at
~0.6 mM during the last 2-3 min of continuous AF. The peak [K+]o during AF averaged 0.8 ± 0.07 mM. This value was not significantly different from that during pacing
at a CL of 250 ms (0.8 ± 0.09 mM; P > 0.05).
Mean arterial blood pressure decreased from 121 ± 5 mmHg
(control) to 92 ± 4 mmHg during the initial phase of AF and
tended to return to control values.
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In two experiments, we simultaneously recorded
[K+]o in the right atrium and left
ventricular midmyocardium. A representative example is shown in Fig.
5. Both the magnitude and time course of
accumulation during incremental atrial pacing were similar in atrial
and ventricular myocardium.
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Effect of Changes in [K+]o on Electrophysiological Properties of Single Atrial Myocytes
We then determined the response of single cell action potential properties to elevations in [K+]o within the range encountered during incremental atrial pacing and AF in vivo. Typical records of transmembrane action potentials from an atrial myocyte measured in response to an increase in [K+]o from 3 to 5 mM in 1 mM steps are shown in Fig. 6A. [K+]o-dependent changes in action potential parameters at a stimulation frequency of 1 Hz are summarized in Table 1. Stepwise increase of [K+]o significantly depolarized take-off potential and reduced both phase 0 action potential amplitude and maximal upstroke velocity in a concentration-dependent manner. Because phase 0 amplitude changes resulting from depolarization may significantly exaggerate the timing changes of action potential duration at 50 and 90% repolarization, we quantified the time course of repolarization by measuring the intervals from the peak of phase 0 amplitude to repolarization potentials of30 mV [action potential duration (APD30mV)] and 60
mV (APD60mV), respectively. Raising [K+]o had no significant effect on
APD30mV or APD60mV (P > 0.05). In two cells, we were able to record transmembrane action
potentials in response to an increase of
[K+]o from 3 to 4 mM during continuous
stimulation at 3.33 Hz (Fig. 6, B and C). The
increase in stimulation frequency appeared to reduce the magnitude of
[K+]o-dependent changes in action potential
properties compared with those measured at 1 Hz. For example, in the
two cells shown, a 1 mM increase of [K+]o
from a baseline value of 3 mM during continuous stimulation at 1 Hz
depolarized the take-off potential by 4.0 and 5.8 mV, respectively,
whereas the same change in [K+]o at a
stimulation frequency of 3.33 Hz reduced the magnitude of these changes
by approximately half.
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Typical records of steady-state current density-voltage relationships
of atrial myocytes measured in response to a voltage ramp protocol
(during which the test potential was changed from 100 to 50 mV over
6.3 s) are shown in Fig.
7A. There was a marked inward
rectification at test potentials negative to the zero current potential. Raising [K+]o progressively
increased the inward component of steady-state current over a certain
range of negative test potentials. For example, the mean amplitudes of
the inward current densities measured at 100 mV were 1.0 ± 0.2, 1.4 ± 0.3, and 1.9 ± 0.3 A/F in 3, 4, and
5 mM [K+]o, respectively (Fig.
7B; P = 0.005). There was also a small, yet significant, increase in
the outward steady-state current density measured at 50 mV in 5 mM
[K+]o (0.24 ± 0.06 A/F) compared with
those measured in 3 and 4 mM [K+]o at the
same potential (0.20 ± 0.05 and 0.19 ± 0.07 A/F,
respectively; P = 0.005). In addition, raising
[K+]o from 3 to 5 mM caused an 8.6 ± 0.8 mV (P < 0.001) shift of the reversal potential in
the positive direction. The magnitude of the shift was not
significantly different from that of the resting membrane potential on
increases of [K+]o (12.0 ± 1.0 mV;
P > 0.05). We also estimated the changes in steady-state membrane input resistance, Rm, of
single atrial myocytes caused by increasing
[K+]o. The Rm was
calculated from the slope conductance of the steady-state current-voltage relationship close to the point of zero current potential. The Rm of atrial myocytes was
528 ± 104 M in 3 mM [K+]o and was
not significantly changed by raising [K+]o to
4 [508 ± 85 M and 5 mM (498 ± 99 M), respectively
(P > 0.05)]. The chord conductance between 80 and
100 mV significantly increased with each increment in
[K+]o, averaging 0.44 ± 0.08 s in
3 mM, 0.56 ± 0.13 s in 4 mM, and 0.69 ± 0.15 s in
5 mM [K+]o (P < 0.001).
Chord conductance increased with [K+]o in a
linear fashion (r2 = 0.99;
P = 0.04).
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Rate-Dependent Changes in CVL in the Right Atrium In Vivo
CVL was measured by using multisite epicardial activation mapping (27). Figure 8 shows examples of activation maps, electrograms, and regressions of distance on activation time used to determine CVL at various basic CLs. Atrial activation pattern and electrogram morphologies remained unchanged with an increase in stimulation rate. Figure 9 shows the time course of changes in mean CVL obtained in four dogs during and after 2-min periods of constant atrial pacing at progressively shorter CLs. On a sudden increase in stimulation rate, CVL decreased gradually and reached a new steady state between 40 and 100 s after the onset of rate change. At each CL, changes in CVL occurring between 40 and 100 s after the sudden rate increase were significant compared with control at a CL of 380 ms (Fig. 9A; P = 0.021). Changes in CVL occurring 100 s after rate increase averaged between3.6 ± 1.8% and 5.9 ± 1.2% over a CL range of 330 to 250 ms (Fig.
9B; P < 0.05 vs. control at CL of 380 ms). Changes in CVL were of similar magnitude at the various CLs
tested (P > 0.05). CVL remained
significantly depressed at the first measurement after resumption of
the initial pacing CL and returned to its respective control values,
thereafter (Fig. 9A).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Major Observations
Results show that [K+]o in right atrial myocardium rises in response to a sudden increase in rate and then plateaus as stimulation is maintained. [K+]o declines to the control level within 60 to 120 s on resumption of baseline pacing CL. Mean peak[K+]o
increases monotonically as pacing CL decreases, ranging from 0.18 ± 0.04 mM at a CL of 350 ms to 0.8 ± 0.09 mM at a CL of 250 ms.
During 5-min episodes of acute AF, [K+]o
gradually increases, peaks within 90 to 120 s after its onset (0.8 ± 0.07 mM), and then slowly declines to a slightly smaller plateau value (0.54 ± 0.06 mM) as AF persists.
[K+]o completely resolves on spontaneous
conversion to sinus rhythm.
Changes of [K+]o within the range encountered during sudden rate changes in vivo are of sufficient magnitude to significantly depolarize take-off potential and reduce both phase 0 amplitude and maximal upstroke velocity of single right atrial myocytes in a concentration-dependent manner. Abrupt increases in rate exert a negative dromotropic effect. However, we were unable to detect a relationship between the magnitude of CVL changes and CL.
Previous Studies
Previously, Kunze (16) showed that stimulation of the quiescent superfused rabbit atrium at incremental rates produced progressive increases in [K+]o, ranging from 0.4 mM at 60 min1 to 1.8 mM at 300 min1.
Henning et al. (7) found rate-dependent
[K+]o increases as high as 1.0 mM on
stimulation of the quiescent canine coronary sinus to a rate of 
120
min1 in the continued presence of adrenaline. In the
latter study, [K+]o accumulation was
accompanied by depolarization of the maximal diastolic potential,
varying from 0.5 to 8 mV. In the present study, increases in atrial
rate between 13 and 82 beats/min above baseline similarly resulted in
progressive increases in the magnitude of
[K+]o, although the change in
[K+]o per single heartbeat (~0.010
mmol · l1 · min1) was
larger than in superfused rabbit atria (~0.006
mmol · l1 · min1)
(16) and in the isolated canine coronary sinus (~0.008
mmol · l1 · min1)
(7). Assuming that the increase in
[K+]o as rate is increased reflects increased
K+ efflux during the action potential, our results suggest
that an increase in atrial rate causes a progressively increasing
mismatch of cellular K+ efflux and K+ influx.
Recently, Stambler et al. (25) recorded monophasic action
potentials from fibrillating human atria and showed that action potentials during AF occur at rates exceeding 400 beats/min. Linear extrapolation of the [K+]o-rate
relationship (Fig. 2A) would predict a magnitude of peak [K+]o accumulation of at least 4 mM above
baseline during the initial phase of acute AF. We found, however, that
the change in [K+]o in the acutely
fibrillating atrium was not significantly different from that during
stimulation at 240 beats/min. Although we did not measure action
potential frequency at or near the K+ electrodes, it is
possible that the actual rate of atrial electrical activation in the
vicinity of the electrodes was closer to 240 than 400 beats/min.
Alternatively, the [K+]o-rate relationship
could be nonlinear at rates exceeding 240 beats/min.
The increase in steady-state [K+]o in the present study was sustained or declined only slightly during continued stimulation and AF, respectively, and [K+]o values did not fall below previous baseline activity before returning to their original baseline level. This is in contrast to previous in vitro results on mammalian cardiac tissue (7, 12, 13, 16). The decline during prolonged stimulation as well as the [K+]o depletion that followed cessation of stimulation have previously been ascribed to Na-K-ATPase-mediated active processes redistributing K+ from the extracellular space (7, 12, 15). The reasons for the discrepancy in the time course of rate-related changes in [K+]o between our measurements and those obtained in isolated mammalian cardiac muscle are unclear, but include differences in the experimental conditions [e.g., presence of adrenaline (7)], regulation of Na-K-ATPase by the intracellular levels of Na+ and ATP (21, 33). Also, the shape of the [K+]o curve may critically depend on the length of the stimulation period, as previously demonstrated (7, 16). Had we extended the duration of continued pacing beyond 2 or 5 min, respectively, [K+]o levels might have declined to prestimulation values.
Changes in Action Potential and Membrane Current
The magnitude of the [K+]o-induced shift in take-off potential (12 mV) of single atrial myocytes closely approximates that predicted by changes in the K+ equilibrium potential, assuming [K+]i remains unchanged. Provided that the resting membrane potential of atrial myocytes in vivo exhibits a similar sensitivity to changes in the transmembrane [K+]o gradient, elevations of [K+]o within the range encountered during sudden rate changes (i.e., 0.2 to 0.8 mM; see Fig. 2A) will cause myocyte depolarizations ranging from 1.4 to 5.5 mV, assuming baseline [K+]o equals 3.5 mM. In the isolated superfused canine coronary sinus, successive increases of [K+]o from 2.7 to 4 mM and from 4 to 6 mM caused depolarization of the diastolic membrane potential from80 to
74 and 71 mV, respectively.
[K+]o-induced depolarization was associated
with a 9% decrease in phase 0 action potential amplitude
and a 34% reduction of maximal rate of rise over time
(dV/dtmax), during phase
0. Similarly steep voltage dependences of steady-state phase
0 dV/dtmax and phase
0 amplitude had previously been found in ordinary fibers of
isolated rabbit atria (29). In the latter study,
K+-induced membrane depolarizations from approximately 85
to approximately 75 mV resulted in ~30 and ~20% decreases in
phase 0 dV/dtmax and
phase 0 amplitude, respectively. Because the inward
Na+ current (INa) is the major
conductance during the action potential upstroke, the decrease in the
phase 0 action potential amplitude and upstroke velocity
must predominantly reflect a decrease in the magnitude of inward
INa, most likely due to an increase in steady-state inactivation (32) along with a reduction of
the electrical driving force for inward INa.
Decreases in upstroke amplitude and velocity may result from a decrease in myocyte excitability (32), which in turn is determined by the input resistance, Rm, and the difference between take-off and threshold potential. Elevation of [K+]o did not significantly influence steady-state Rm of single atrial myocytes. This is in accordance with two previous experimental studies, which showed that increases in [K+]o from 4.8 to 8.0 mM and from 5 to 24 mM, respectively, failed to significantly alter steady-state Rm of single guinea pig and rabbit atrial myocytes (20, 32). Furthermore, our results are consistent with recent mathematical simulations demonstrating that the effect of raising [K+]o on Rm of isolated atrial myocytes is likely to be small over the range of fluctuations in [K+]o that can be expected to occur under physiological conditions (22). It is, therefore, unlikely that raising [K+]o affected excitability by means of altering Rm. However, the number of myocytes examined in our study may have been too small to detect minor effects of changes in [K+]o on atrial Rm, should they exist. We also cannot exclude the possibility that increases of [K+]o modulated excitability by means of altering the difference between take-off potential and excitation threshold (4). Nevertheless, the reduction in dV/dtmax is consistent with reduced availability of inward INa along with a diminished electrical driving force for passive Na+ influx during the action potential upstroke resulting from depolarization of the resting membrane potential.
Magistretti et al. (19) previously reported discrepancies between action potentials recorded in the same cell when using a classical microelectrode amplifier with e.g., a bridge circuit headstage or an Axopatch 200A patch-clamp amplifier operating in the INormal current-clamp mode. In the latter study, errors introduced by the patch-clamp amplifier appeared to distort e.g., action potential amplitude and the slope of phase 0 depolarization. However, these amplifier-generated voltage distortions are unlikely to affect our conclusions regarding phase 0 dV/dtmax, because the same amplifier in the same INormal current-clamp mode was used for all single cell studies.
Marked inward rectification of whole cell steady-state currents at test
potentials negative to the zero current potential most likely
corresponds to activation of the inwardly rectifying K+
current (IK1). Slope conductance measured
between test potentials of 80 and 100 mV increased with
[K+]o, in accordance with the previously
demonstrated square root dependence of IK1
conductance on [K+]o (9).
Increases of [K+]o failed to significantly
alter the time course of membrane repolarization. Although the outward
component of the whole cell steady state at a test potential of 50 mV
was significantly larger in 5 mM [K+]o than
in 3 or 4 mM [K+]o, this effect may have been
too small and/or the number of myocytes examined may have been
insufficient to result in detectable changes in repolarization times.
Rate-dependent changes in CVL. A rise in [K+]o, by depolarizing the take-off potential, partially inactivating the inward INa and reducing the electrical driving force for inward INa during the action potential upstroke, slows conduction velocity (2). Given the monotonic increase in peak [K+]o accumulation with an increase in rate and the [K+]o-induced myocyte depolarization and associated reduction of phase 0 dV/dtmax, we expected 1) CVL to slow in a monotonic fashion as pacing CL shortens, and 2) the time course of rate-dependent CVL changes to parallel that of changes in [K+]o.
Variations in stimulation frequency had a negative dromotropic effect. CVL decreases, however, were independent of stimulation frequency. Magnitude and direction of rate-dependent changes in CVL in the present study are in excellent agreement with those previously reported for atrial (30) and ventricular preparations (2). Assuming that changes in dV/dtmax are proportional to changes in the square of conduction velocity (2) as predicted by one-dimensional cable theory, the 25% and 12% decreases in dV/dtmax seen in vitro after 1-mM increases of [K+]o from baseline levels of 3 and 4 mM, respectively, (see Table 1) should be associated with 14 and 6% reductions of atrial CVL. The degree of reduction in CVL encountered in the present study (maximum, 5.9%) is well within these theoretical boundaries, suggesting that rate-dependent [K+]o accumulation in vivo may diminish phase 0 dV/dtmax sufficiently to significantly slow CVL. Lack of a strictly monotonic relationship between peak CVL change and stimulus interval, however, is compatible with the notions that 1) other factors beside [K+]o control the speed of action potential propagation over the range of stimulation frequencies used in our study, and/or 2) the effect of [K+]o accumulation on atrial CVL diminishes when stimulation frequency increases. As Fig. 6, B and C, suggest, [K+]o-induced differences in take-off potential become less at higher stimulation rates, because stimulation occurs before complete repolarization, thereby attenuating voltage-dependent differences in phase 0 Na+ channel conductance, and thus CVL. This mechanism might have resulted in a flattening of the CVL-rate relationship in our study. Alternatively, rate-dependent [K+]o accumulation may act as an innocent bystander, and the shape of the rate-CVL relationship may reflect the rate dependence of other processes. Given the role of the L-type calcium current (ICa,L), as a major determinant of canine atrial action potential duration (34), the amplitude and kinetics of ICa,L will importantly impact on membrane responsiveness during rapid rates, i.e., when phase 0 depolarization occurs before repolarization is complete. The rate-CVL relationship in our study could, therefore, be determined primarily by the properties of ICa,L and, to a lesser degree, by the effect of [K+]o accumulation on membrane responsiveness. When the stimulus interval was suddenly augmented or diminished, the conduction velocity changed gradually rather than abruptly, respectively, to a new steady state. A similar pattern was previously reported for the isolated crista terminalis from rabbit (30). Time required to reach a new steady state was not related to the magnitude of interval change. Rate-dependent redistribution of various ions has been implicated in the development of and recovery from tachycardia-induced CVL depression (10). Similarity in the kinetics of changes in atrial CVL and [K+]o suggests that [K+]o may influence, by means of voltage-dependent mechanisms discussed above, the kinetics of development of and recovery from tachycardia-induced conduction slowing.Study Implications and Limitations
Our measurements of [K+]o activity are confined to portions of the right atrium that are the thickest, i.e., crista terminalis and pectinate muscle. Because atrial tissue structure impacts on changes in [K+]o during rapid pacing and AF, with the relatively thin, free wall being less affected than the thicker portion of the right atrium, both the magnitude and kinetics of rate-dependent [K+]o accumulation are likely to be nonuniform throughout the atrium. Thus the relatively minor changes reported in this study could give rise to spatial heterogeneity of membrane potential and action potential duration, thereby promoting the development and perpetuation of reentrant arrhythmia (i.e., AF).Placing a miniature K+-sensitive electrode in the atria may distort the extracellular space, such that, e.g., diffusion of K+ from a region of the extracellular space to the capillaries becomes impaired. This may affect the magnitude and/or kinetics of K+ accumulation.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the expert technical assistance of Nahid Akhtar. We also thank Dr. L. Gettes and Connie Engle for invaluable help in the fabrication of K+-sensitive electrodes. We also thank Naomi S. Fineberg for statistical analysis.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant P50 HL-52323 (to D. P. Zipes), a Showalter Trust and American Heart Association, Midwest Affiliate Grant (to M. Rubart), and an American Heart Association Award as Student Scholar in Cardiovascular Disease and Stroke (to J. D. Dowell).
Address for reprint requests and other correspondence: M. Rubart, Wells Center for Pediatric Research, Riley Hospital, 702 Barnhill Dr., Indianapolis, IN 46202 (E-mail: mrubartv{at}iupui.edu).
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.
May 2, 2002;10.1152/ajpheart.00721.2001
Received 13 August 2001; accepted in final form 15 April 2002.
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REFERENCES |
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TOP
ABSTRACT
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METHODS
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DISCUSSION
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