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Departments of 1 Medicine and
2 Surgery, The mechanism of action potential abbreviation
caused by increasing rate in human ventricular myocytes is unknown. The
present study was designed to determine the potential role of
Ca2+ current
(ICa) in the
rate-dependent changes in action potential duration (APD) in human
ventricular cells. Myocytes isolated from the right ventricle of
explanted human hearts were studied at 36°C with whole cell voltage
and current-clamp techniques. APD at 90% repolarization decreased by
36 ± 4% when frequency increased from 0.5 to 2 Hz.
Equimolar substitution of Mg2+ for
Ca2+ significantly decreased
rate-dependent changes in APD (to 6 ± 3%,
P < 0.01). Peak
ICa was decreased
by 34 ± 3% from 0.5 to 2 Hz
(P < 0.01), and
ICa had recovery
time constants of 65 ± 12 and 683 ± 39 ms at
calcium current; ion channels; action potential; electrophysiology; cardiac arrhythmias
IT IS WELL KNOWN that the cardiac action potential
duration (APD) changes with alterations in frequency and APD is
significantly abbreviated at rapid rates (3, 10, 15, 35), but the
underlying mechanisms are unclear. In mammalian cardiac tissue,
rate-dependent abbreviation of APD at rapid rates has been considered
to result from an increase in transmembrane
K+ conductance (22) and/or
an augmentation of
Na+-K+
pump activity (11).
Beeler and Reuter (6) reported that membrane
Ca2+ current
(ICa) is
important for determining the plateau phase of the action potential in
guinea pig ventricular myocardial fibers. Hume and Uehara (19) found
that amplitude and kinetics of
ICa are major determinants of the differences in morphology and duration of the
action potential in guinea pig atrial and ventricular myocytes. We also
found that ICa
plays an important role in determining APD in human atrium (25).
Therefore, ICa
changes may explain rate-dependent changes in cardiac APD. However,
whether ICa
contributes to the rate-dependent abbreviation of APD in human
ventricle is unknown.
The properties of
ICa have been
described in cardiac cells isolated from human atria and human
ventricles (14, 26, 28, 29). However,
ICa was
characterized at room temperature in most of these studies. It has been
demonstrated that changes in temperature profoundly affect the
availability and/or time-dependent properties of
ICa (1).
Therefore, it is important to evaluate the role of
ICa in governing
rate-dependent changes in APD in humans at normal physiological temperature.
The present study was designed to determine the relation between
rate-dependent changes in APD and changes in
ICa in human ventricular myocytes at a physiological temperature (36°C). The action potential-voltage clamp ("action potential clamp")
technique was used to analyze the potential contribution of changes in
ICa to
frequency-dependent changes in APD.
Myocyte isolation.
Right ventricular tissues from explanted hearts were obtained at the
time of heart transplantation from patients. All hearts were initially
placed in cold (4°C) oxygenated Krebs solution and then transferred
to cardioplegic solution for dissection and coronary artery
cannulation. A procedure described previously (24) was used to isolate
ventricular cells. Briefly, a portion of the free wall of the right
ventricle (~2 × 4-5 cm) was removed along with the
coronary artery branch irrigating it, with dissection and arterial
cannulation completed within 20 min of excision of the heart. The free
wall was perfused with oxygenated, nominally Ca2+-free Tyrode solution for
20-30 min, and the solution was then changed to one containing
200-300 U/ml collagenase (CLS II, Worthington Biochemical,
Freehold, NJ) for 60-100 min. Myocytes were isolated from the
digested tissue and placed in a
high-K+ storage solution (24).
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
80 mV. Action potential clamp demonstrated a decreasing
contribution of
ICa during the
action potential as rate increased. The rate-dependent slow component
of the delayed rectifier K+
current (IKs)
was not observed in four cells with an increase in frequency from 0.5 to 3.3 Hz, perhaps because the
IKs is so small
that the increase at a high rate could not be seen. These results
suggest that reduction of Ca2+
influx during the action potential accounts for most of the
rate-dependent abbreviation of human ventricular APD.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Solutions and chemicals. Tyrode solution contained (mM) 136 NaCl, 5.4 KCl, 1.0 MgCl2, 2.0 CaCl2, 0.33 NaH2PO4, 10.0 glucose, and 10 HEPES; pH was adjusted to 7.4 with NaOH. For cell dissociation, Ca2+ was omitted. A choline solution containing (mM) 136 choline chloride, 5.4 CsCl, 1.0 MgCl2, 2.0 CaCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES was used when ICa was measured directly; pH was adjusted to 7.4 with CsOH. The pipette solution for ICa recording (whole cell voltage-clamp mode) contained (mM) 20 CsCl, 110 cesium aspartate, 1.0 MgCl2, 10 HEPES, 10 EGTA, 0.1 GTP, and 5 Mg2ATP; pH was adjusted to 7.2 with CsOH. The pipette solution for action potential recording (current-clamp mode) contained (mM) 20 KCl, 110 potassium aspartate, 1.0 MgCl2, 10 HEPES, 0.05 EGTA [5 for the slow component of the delayed rectifier K+ current (IKs) recording], 0.1 GTP, 5 Mg2ATP, and 5 sodium phosphocreatine; pH was adjusted to 7.2 with KOH.
Electrophysiology and data analysis.
The tight-seal whole cell patch-clamp technique was used. Borosilicate
glass electrodes (1.0 mm OD) were pulled with a Brown-Flaming puller
(model P-87); tip resistances were 2-3 M
when filled with pipette solution. Data were acquired with the use of an Axopatch 200A
and/or 1-D amplifier (Axon Instruments, Foster City, CA). Command pulses were generated by a 12-bit digital-to-analog converter controlled by pCLAMP software (Axon Instruments). Recordings were low-pass filtered at 2 kHz and stored on the hard disk of an
IBM-compatible computer. Tip potentials were compensated before the
pipette touched the cell. After a gigaseal (>10 G) was obtained,
the cell membrane was ruptured by gentle suction to establish the whole
cell configuration.
60 mV divided by the voltage drop). Membrane capacitance was 179 ± 10 pF (n = 25). To
control for differences in cell size, all mean data are expressed as
current densities (i.e., normalized to capacitance). Before
compensation, the capacitive time constant and
Rs averaged
1,314.2 ± 113.9 µs and 8.6 ± 0.5 M, and after compensation
corresponding values were 381.3 ± 26.2 µs and 2.0 ± 0.3 M.
ICa rarely
exceeded 2 nA, and the mean maximum voltage drop across the
Rs, therefore,
did not exceed 4 mV. Cells with significant leak current were rejected.
The liquid junction potentials between the external and pipette
solutions were 10-11 mV and were not corrected.
The average length of single human ventricular myocytes was 148.3 ± 8.8 µm (n = 15, range 112-185
µm), and the diameter was 18.5 ± 1.2 µm (range 12-25
µm); the estimated cell surface area was therefore 9.2 ± 0.5 × 10
5
cm2 on the basis of assumed right
cylinder geometry. The input resistance (Rin) was
determined by using four consecutive 5-mV hyperpolarizing steps from a
holding potential of 60 mV, with the resulting change in current
used to calculate
Rin (16). Mean
Rin as estimated in 11 cells was 56.7 ± 5.4 M. The resting space constant
(sc) was estimated as follows:
sc = 
, where r is the cell radius,
Rm is the
specific membrane resistance, and
Ri is
intracellular resistivity.
Rm was estimated
from the product of
Rin and surface
area, providing a mean value of 5.1 ± 0.2 K · cm2, and
Ri was assumed to
be 100-200 · cm (18, 32, 40). The mean
values of sc were 1.54 ± 0.22 and
1.09 ± 0.16 mm, which are more than seven times the single cell
length. These could be underestimated, since the surface area estimated
on the basis of a specific capacitance of 1 µF/cm2 is 17.9 × 105
cm2, twice as large as the value
above, which indicates that membrane infolding results in a true
surface area larger than that of a right cylinder (40).
Cells were current clamped to record action potentials and/or
voltage clamped with rectangular steps or individual action potential
waveforms at 0.5 Hz with pClamp6. In the action potential clamp
experiments, ICa
was identified by subtracting currents before and after the
substitution of external Ca2+ with
equimolar Mg2+, as described by
Bouchard et al. (9). Similar results were obtained with use of
Cd2+ (200 µM) to
suppress ICa, but
all the results were with Mg2+
substitution, because Cd2+ can
also affect Na+ current
(INa).
Nonlinear curve-fitting techniques (Clampfit in pClamp6 or Sigmaplot,
Jandel Scientific, San Rafael, CA) based on the Marquardt procedure
were used to fit equations to experimental data. Paired and nonpaired
Student's t-tests were used as
appropriate to evaluate the statistical significance of differences
between two group means, and ANOVA was used for multiple groups.
P < 0.05 was considered to indicate
significance. Group data are expressed as means ± SE.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Relation between changes in APD and ICa.
To study the relation between rate-dependent abbreviation of APD and
ICa, we recorded
action potentials in current-clamp mode at 0.5, 1, and 2 Hz before and
after external Ca2+ substitution
with equimolar external Mg2+. The
resting membrane potentials were between 69 and 73 mV without any artificial current injection. Figure
1A shows
action potentials recorded from a representative human ventricular
myocyte at frequencies of 0.5, 1, and 2 Hz in the presence of 2 mM
external Ca2+ (control). APD
decreased as stimulus rates increased. The average changes in APD at 50 and 90% repolarization (APD50 and
APD90) are shown in Fig.
1B.
APD50 and
APD90 decreased from 325 ± 23 and 439 ± 37 ms, respectively, at a frequency of 0.5 Hz to 181 ± 12 and 281 ± 21 ms, respectively, at 2 Hz
(P < 0.01, n = 18). The mean rate-dependent
reductions were 44 ± 4% for
APD50 and 36 ± 4% for
APD90. Replacement of external
Ca2+ by 2 mM external
Mg2+ greatly attenuated APD
adaptation to rate. Figure 1C shows
APD recordings from the same cell and at the same frequencies as in Fig. 1A but in the presence of 2 mM
external Mg2+ to replace external
Ca2+. APD was substantially
reduced and showed little alteration with changes in rate. As indicated
in Fig. 1C, inset, substitution of
external Ca2+ with external
Mg2+ totally suppressed
ICa on
depolarization from 40 to 0 mV in the same cell. In eight cells,
replacement of external Ca2+
virtually abolished rate-dependent changes in APD, as shown by the mean
data in Fig. 1D. The reduction in
APD50 and
APD90 caused by increasing rate
from 0.5 to 2 Hz averaged 5 ± 2 and 6 ± 3%, respectively,
which is substantially less (P < 0.01) than under control conditions over the same range of frequencies.
On restoration of external Ca2+
concentration to 2 mM, rate-dependent changes in
APD50 and
APD90 were restored to 39 ± 5 and 33 ± 5% as rate increased from 0.5 to 2 Hz
(n = 4).
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Action potential clamp and ICa. To obtain more information about how ICa contributes to rate-dependent abbreviation of APD, we used the action potential waveform to clamp individual cells and to quantify ICa during the action potential at different frequencies. The action potential was acquired from each cell at 0.5 Hz and used as a voltage-clamp waveform in the same cell. External Ca2+-dependent current (ICa) was measured by digital subtraction of the currents before and after substitution of external Ca2+ with equimolar external Mg2+, as described by Bouchard et al. (9). Figure 2 shows representative recordings from a ventricular myocyte. Figure 2A demonstrates the action potential waveform, and Fig. 2B shows currents elicited by the action potential waveform in the presence (control) and absence of 2 mM external Ca2+ (Mg2+). Figure 2C shows the ICa obtained by subtracting currents of Fig. 2B in the absence of external Ca2+ (Mg2+) from those in its presence (control). ICa increased rapidly to a peak and then decreased rapidly to a plateau, which terminated with repolarization.
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Frequency-dependent reduction of ICa.
To study why Ca2+ influx decreased
during the action potential when the rate increased, conventional step
voltage-clamp protocols were used to determine use and frequency
dependence of ICa
under conditions designed to suppress other currents
(K+ replacement by
Cs+ in the pipette and
extracellular Na+ replacement by
choline). Figure 4 shows the use and
frequency dependence of
ICa under these
conditions. ICa
was recorded with trains of 15 pulses (300 ms from 80 to +10 mV)
at 0.2, 0.5, 1, and 2 Hz (60 s between trains). Figure
4A displays representative current
traces from the 1st and 15th pulses at 2 Hz.
ICa was clearly smaller during the 15th than during the 1st pulse, and time-dependent inactivation of
ICa appeared
faster at the 15th than that at the 1st pulse. Figure
4B shows changes in
ICa during each
beat expressed as a function of the first pulse at each frequency.
Statistically significant use dependence was noted at frequencies
>0.2 Hz (P < 0.05, 0.01, and 0.01 for 0.5, 1, and 2 Hz, respectively). The use-dependent reductions in
ICa resulted in
frequency dependence of the current, as shown in Fig.
4C, which illustrates the relation between pulse frequency and steady-state peak
ICa, with
currents of each frequency normalized to values at 0.2 Hz.
ICa was reduced significantly at each frequency, with a reduction of 36 ± 2% at 2 Hz.
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Time-dependent reactivation of ICa.
The time dependence of
ICa reactivation
was studied with the paired-pulse protocol illustrated in Fig.
5. Identical 300-ms pulses (P1 and P2) from
a holding potential of 80 to +10 mV were delivered every 10 s,
with varying P1-P2 intervals. The current during P2 relative to the
current during P1 was determined as a function of the P1-P2
reactivation interval (Fig. 5A). The
curves in Fig. 5B show nonlinear curve
fits to mean data at holding potentials of 80 mV
(n = 11) and 60 mV
(n = 7).
ICa recovery
reached 97% and was well fit by a biexponential function with time
constants of 65 ± 12 and 638 ± 39 ms for a holding potential of
80 mV and 164 ± 15 and 697 ± 45 ms for a holding
potential of 60 mV.
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Current-voltage relation of ICa.
Two types of ICa
["T type"
(ICa,T) and
"L type"
(ICa,L)]
have been found in cardiac cells from a variety of species (4, 5, 30,
38). To determine whether
ICa,T is present
in human ventricular cells and contributes to rate-dependent changes in APD, current-voltage (I-V) relations
of ICa were
determined using 300-ms depolarizing steps every 10 s from holding
potentials of 80 and 40 mV. The magnitude of
ICa was measured
as the difference between the peak inward current and the steady-state
current at the end of the depolarizing step. For analyses of average
currents, ICa was
normalized to cell capacitance to control for variations in cell size.
80 mV in the absence and presence of the L-type
Ca2+ channel blocker nifedipine
(10 µM). Nifedipine abolished the membrane current, which suggests
that the current elicited by the voltage protocol shown is
ICa,L. Figure
6B shows mean
ICa density-voltage relations.
ICa density was
significantly less at 40 mV (n = 10) than at 80 mV (n = 12)
over a broad range of test potentials. Maximum current density was
obtained at the same voltage (+10 mV) at each holding potential. No
evidence for ICa,T, in terms
of a discrete component that activates and inactivates at more negative
voltages (4, 5, 30, 31), was observed in any cell.
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Voltage-dependent activation and inactivation of
ICa.
The voltage dependence of
ICa activation
can be determined from the I-V
relation of ICa
on the basis of the following formulation: dt = It/[Gx(Vt Vr)],
where dt is the
activation variable and It is
ICa at a test
potential Vt,
Gx is the maximum
conductance, and
Vr is the
reversal potential, estimated from a linear fit of the terminal portion
of the ascending limb of the I-V
relation of ICa.
We used this equation to calculate the
ICa activation variable from the I-V relation at a
holding potential of 80 mV (Fig.
6B). Values of
dt were
normalized to the maximum value in each cell to obtain the activation
variable d.
100 and +60 mV, and then
ICa was recorded
during a 300-ms test pulse to +10 mV. The inactivation variable
(f) was determined as
ICa at a given
prepulse potential divided by the maximum
ICa in the
absence of a prepulse.
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4.8 ± 0.9 mV (n = 10), and the slope factor was 6.2 ± 1.1 mV. Inactivation reached a
maximum at +10 mV and was incomplete. At more positive voltages, the
extent of inactivation decreased. The inactivation curve was
nonmonotonic or "U shaped," compatible with an important
Ca2+-dependent component to
inactivation previously seen in human atrial cells (12, 25), and could
be fit by a Boltzmann relation with the following equation (33, 34):
f = 1/{1 + exp[(V V0.5)/K]} + R, where
V0.5 is the
estimated half-maximum inactivation voltage,
V is prepulse potential,
K is the slope factor, and
R characterizes the degree of
incomplete inactivation (R = 0.18/{1 + exp[(43 V)/K]}),
providing the curve shown in Fig. 7B.
V0.5 averaged
28.5 ± 2.8 mV (n = 7), and
K averaged 7.8 ± 1.4 mV.
Rate-dependent IKs.
Because a rate-dependent increase in
IKs is believed
to contribute to the shortening of APD at high rates in guinea pig
ventricular myocytes (20), we determined a possible effect of changing
depolarization rates on
IKs in human
ventricular cells.
IKs was examined
by a 2-s ramp protocol from 50 to +40 mV after a train (0.5 or
3.33 Hz) of 30 step voltage pulses (500-ms; Fig.
8A). The
experiment was performed with an external solution that was free of
Na+ and
K+ in the presence of 5 µM
E-4031 [to block the rapid component of the delayed rectifier
K+ current
(IKr)], 5 mM 4-aminopyridine [to block transient outward K+ current
(Ito1)],
and 200 µM Cd2+ (to block
ICa). Figure
8B shows the ramp-activated
IKs with small tail current superimposed at 0.5 and 3.33 Hz in a representative human
ventricular cell. Similar results were observed in a total of four
cells. No significant difference in
IKs or tail
current was observed, perhaps because
IKs is so small
that the rate-dependent change in human ventricular cells could not be
seen.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We have demonstrated that rate-dependent abbreviation of APD in human ventricular myocytes is closely related to changes in Ca2+ influx during the action potential caused by frequency-dependent reduction of ICa due, at least in part, to incomplete time-dependent recovery at higher frequencies.
Comparison with previously published studies of mechanisms for rate-dependent changes of APD in cardiac cells. Rate-dependent abbreviation in cardiac APD has been considered to be related to an increase in transmembrane K+ conductance (22) and/or an increase in Na+-K+ pump activity (11) on the basis of the observation that extracellular K+ was elevated with a parallel increase in frequency (21, 22). Transient outward K+ current (Ito) has been described in human ventricular cells (8, 23, 41); however, its property of rate-dependent reduction does not account for the increase in extracellular K+ or for rate-dependent reduction in APD. Inward rectifier background current (IK1) has been reported to show a rate-dependent decrease in guinea pig ventricular cells that is dependent on the presence of ICa (13), but a decrease of the outward current would not account for rate-dependent abbreviation of APD.
IKs has been reported to show a rate-dependent increase in guinea pig ventricular cells and to contribute to APD abbreviation in that species (20). IKr and IKs have been described in human ventricular cells (24); however, IKs is much smaller in human than in guinea pig ventricular cells. With the use of a ramp protocol, a clear rate-dependent increase in IKs was not observed in human ventricular cells with an increase in frequency from 0.5 to 3.3 Hz (Fig. 8), perhaps because the IKs is so small that the increase at a high rate could not be seen. The small IKs in human ventricular cells we reported previously (24) and recorded in this study may be due to an artifact of the cell isolation procedure and/or the reduced IKs expression in myocytes from failing hearts (but not in normal myocytes). The present study reveals a close relation between the rate-dependent abbreviation of APD and reduction of Ca2+ influx during the action potential. Earlier studies demonstrate that ICa is very important for determining the plateau phase of cardiac action potential (6), and ICa blockade dramatically shortens cardiac APD (19), whereas ICa increases induced by
-adrenergic stimulation (25, 39) and
Ca2+ channel agonists (39) prolong
APD in myocardium, supporting the findings of the present study.
Comparison with previously published studies of ICa in human cardiac cells. Several groups have described the properties of ICa in human cardiac cells (14, 26, 28, 29). Our study differs from these in that we determined Ca2+ influx during the action potential and recorded ICa at body temperature (all previous studies in human ventricular myocytes have been performed at room temperature), characterized recovery and frequency-dependent properties in detail (not reported in previous work), and evaluated the role of ICa in human ventricular action potential behavior (not previously performed). Previous studies have not examined the presence and amplitude of ICa,T in the human ventricle. In the present study we did not find evidence for ICa,T in human ventricular myocytes.
We found that ICa recovery from steady-state inactivation is biexponential, with time constants of 65 and 683 ms at80 mV and 164 and 697 ms at
60 mV, which indicates that slight membrane depolarization may
slow ICa
recovery. We also found that
ICa shows significant steady-state inactivation at 1 Hz. These findings imply
that ICa is
partially inactivated at normal heart rates (70 beats/min) and that
physiological increases in heart rate are likely to cause further
ICa inactivation.
The time dependence of
ICa reactivation
in ventricular cells is different from our previous findings in human
atrial myocytes, in which
ICa reactivation
is monoexponential at 80 mV, with a time constant of 56 ms.
Correspondingly, the frequency-dependent decrease in
ICa at 80
mV was more important in ventricular cells (35% reduction at 2 Hz)
than in human atrial cells (10% reduction at 2 Hz) (25). Therefore,
changes in ICa
would contribute more to rate-dependent abbreviation of APD in
ventricular cells than in atrial cells in humans. We were unable to
find reports of
ICa frequency
dependence in human ventricular myocytes with which to compare our findings.
Significance of our observations. A better understanding of the ionic mechanisms governing human ventricular repolarization is important for designing improved antiarrhythmic strategies. The rate-dependent properties of ventricular APD and refractoriness are known to be an important determinant of the occurrence of reentrant cardiac arrhythmias (36). Attuel et al. (2) showed that refractoriness abbreviation with increased rates characterizes patients with vulnerability to atrial reentrant arrhythmias. The present study suggests that frequency-dependent ICa reduction contributes significantly to frequency-dependent ventricular APD abbreviation in humans. There is considerable interest in developing novel antiarrhythmic drugs that act selectively during tachycardia, and a better understanding of the mechanisms of physiological APD adaptation to tachycardia in humans, as developed in the present study, is important for this effort.
The properties of ICa have been well described in animals with the use of conventional rectangular depolarizing clamp steps. Little direct information is available in the literature regarding the ICa contribution to rate-dependent changes in APD. Bouchard et al. (9) employed the action potential clamp technique to determine the effects of APD change on excitation-contraction coupling in rat ventricular myocytes. They evaluated ICa during an action potential by subtracting membrane currents before and after the addition of 100 µM Cd2+ or after the substitution of external Ca2+ with external Mg2+. They quantified
ICa
in rat ventricular myocytes during action potentials and showed that
ICa was
inactivated before the action potential terminated in rat ventricular
cells (9). In the present study we applied the action potential clamp to examine how much
ICa was present
during the plateau of the action potential, by subtracting membrane
currents before and after the substitution of external
Ca2+ with external
Mg2+ in human ventricular
myocytes, and found considerable inward ICa during the
action potential plateau. Tachycardia reduced plateau ICa, accounting
for action potential abbreviation. Reduced APD during tachycardia
shortens the refractory period and the wavelength and may promote the
occurrence of reentrant arrhythmias.
Potential limitations. We studied right ventricular cells from patients with severe left ventricular failure, and the patients were receiving medications including captopril, digoxin, dobutamine, and furosemide. It is likely that medications were washed out in the cell isolation process, but we cannot fully exclude an effect of medication on our results. Although the cells used in this study were from hearts that were assessed by the pathologist to have relatively mild or no changes in the right ventricular myocardium, we cannot exclude the possibility that our results were affected by the presence of heart disease. The small IKs we recorded may be related to the reduced expression during heart disease.
To study the effect of ICa on rate-dependent changes in APD, ICa must be blocked; however, pure Ca2+ channel blockers are not available. The inorganic Ca2+ channel blocker Cd2+ can affect INa, whereas the 1,4-dihydropyridine Ca2+ antagonists may also block Ito (17). We therefore replaced external Ca2+ with equimolar Mg2+ (9). The elimination of ICa may limit Ca2+-induced Ca2+ release from sarcoplasmic reticulum and, therefore, inhibit Na+/Ca2+ exchanger current during the action potential (7). This may also induce action potential shortening, which may cause an overestimation of the direct role of ICa in action potential abbreviation. On the other hand, possible Ca2+-activated currents, such as Ca2+-activated Cl and
K+ currents, are also reduced by
the removal of external Ca2+, but
the effect would not account for rate-dependent shortening of APD,
since Ca2+-activated
Cl and
K+ currents are repolarizing
currents, which will tend to shorten APD.
In the present study,
ICa and
ICa kinetics were
studied using Na+-free superfusate
to prevent contamination by
INa. In the
absence of extracellular Na+,
Ca2+ extrusion via the
Na+/Ca2+
exchanger is inhibited and cellular integrity is threatened by progressive Ca2+ overload. Cells
were therefore dialyzed with 10 mM EGTA via the pipette solution.
However, this may result in strong intracellular Ca2+ buffering, limit
ICa inactivation
because of sarcoplasmic reticulum Ca2+ release, and slow the rapid
phase of inactivation (37). Therefore, we may have underestimated the
rate of this process and the extent of physiological
frequency-dependent
ICa inactivation.
ICa rundown may
be problematic when patch-clamp techniques are used. In our experience,
cell quality is a major factor determining rundown, and we did not
observe important rundown over the course of our experiments. We
recorded current amplitude before and after each protocol. If maximum
current amplitude decreased by >5% over the course of an
experimental protocol, the data were discarded.
At room temperature, upregulation of
ICa has been
reported at high frequency in atrial myocytes from a subgroup of
patients (32). We did not find such an increase, perhaps because of
differences in experimental protocols and conditions, cell types,
and/or patient population.
In conclusion, we have demonstrated that rate-dependent abbreviation of
human ventricular APD is largely attributable to rate-dependent changes
in ICa.
Time-dependent recovery of
ICa and its
consequent frequency dependence are likely to be significant
contributors to the physiological control of human ventricular APD and
to be important in controlling the occurrence of ventricular reentrant arrhythmias such as ventricular tachycardia and/or fibrillation.
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ACKNOWLEDGEMENTS |
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The authors thank Haiying Sun for development of the action potential-clamp technique with pClamp6 and for data analysis and technical assistance and Caroll Boyer for secretarial help.
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FOOTNOTES |
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This work was supported by the Heart and Stroke Foundation of Quebec, the Fonds de la Recherche en Santé du Québec, the Medical Research Council of Canada, and the Fonds de Recherche de l'Institut de Cardiologie de Montréal. G.-R. Li was a research scholar of the Fonds de la Recherche en Santé du Québec. R. F. Bosch holds a fellowship of the Deutscheforschungsgemeinschaft.
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. §1734 solely to indicate this fact.
Address for reprint requests: G.-R. Li, Dept. of Physiology, Medical College of Virginia, Virginia Commonwealth University, 1101 East Marshall St., PO Box 980551, Richmond, VA 23298-0551.
Received 13 February 1998; accepted in final form 18 September 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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1.
Allen, T. J. A.
Temperature dependence of macroscopic L-type calcium channel currents in single guinea pig ventricular myocytes.
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Attuel, P.,
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Failure in the rate adaptation of the atrial refractory period: its relationship to vulnerability.
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Attwell, D.,
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