Download Reducing Ryanodine Receptor Open Probability as a Means to

Reducing Ryanodine Receptor Open Probability as a Means
to Abolish Spontaneous Ca2ⴙ Release and Increase Ca2ⴙ
Transient Amplitude in Adult Ventricular Myocytes
L.A. Venetucci, A.W. Trafford, M.E. Dı́az, S.C. O’Neill, D.A. Eisner
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Abstract—The aim of this work was to investigate whether it is possible to remove arrhythmogenic Ca2⫹ release from the
sarcoplasmic reticulum that occurs in calcium overload without compromising normal systolic release. Exposure of rat
ventricular myocytes to isoproterenol (1 ␮mol/L) resulted in an increased amplitude of the systolic Ca2⫹ transient and
the appearance of waves of diastolic Ca2⫹ release. Application of tetracaine (25 to 50 ␮mol/L) decreased the frequency
or abolished the diastolic Ca2⫹ release. This was accompanied by an increase in the amplitude of the systolic Ca2⫹
transient. Cellular Ca2⫹ flux balance was investigated by integrating Ca2⫹ entry (on the L-type Ca2⫹ current) and efflux
(on Na–Ca2⫹ exchange). Isoproterenol increased Ca2⫹ influx but failed to increase Ca2⫹ efflux during systole (because
of the abbreviation of the duration of the Ca2⫹ transient). To match this increased influx the bulk of Ca2⫹ efflux occurred
via Na–Ca2⫹ exchange during a diastolic Ca2⫹ wave. Subsequent application of tetracaine increased systolic Ca2⫹ efflux
and abolished the diastolic efflux. The increase of systolic efflux in tetracaine resulted from both increased amplitude
and duration of the systolic Ca2⫹ transient. In the presence of isoproterenol, those Ca2⫹ transients preceded by diastolic
release were smaller than those where no diastolic release had occurred. When tetracaine was added, the amplitude of
the Ca2⫹ transient was similar to those in isoproterenol with no diastolic release and larger than those preceded by
diastolic release. We conclude that tetracaine increases the amplitude of the systolic Ca2⫹ transient by removing the
inhibitory effect of diastolic Ca2⫹ release. (Circ Res. 2006;98:1299-1305.)
Key Words: spontaneous release 䡲 calcium 䡲 sarcoplasmic reticulum 䡲 arrhythmias
I
n cardiac myocytes, an increase of sarcoplasmic reticulum
(SR) Ca2⫹ load increases both systolic Ca2⫹ release and
force of contraction. This occurs largely through the release
of Ca2⫹ ions from the SR via a release channel known as the
ryanodine receptor or (RyR) (see Bers1 for review). However,
when too much Ca2⫹ enters the SR (Ca2⫹ overload), Ca2⫹ is
also released spontaneously during diastole.2– 4 This occurs as
a result of increased RyR open probability secondary to
increased SR Ca2⫹ content.5 This diastolic Ca2⫹ release
activates the electrogenic sodium/calcium exchanger (NCX)
and produces inward current resulting in delayed afterdepolarizations (DADs).6 – 8 DADs play a role in the genesis of
arrhythmias in many clinical settings such as heart failure,9
digitalis toxicity, catecholaminergic ventricular tachycardia,
and reperfusion arrhythmias.10 In addition, this tendency to
diastolic Ca2⫹ release limits the use of positive inotropes that
work by increasing SR Ca2⫹ content.
The aim of the present work was to develop a strategy to
abolish the potentially arrhythmogenic diastolic Ca2⫹ release
from the SR. The challenge was to do this without interfering
with the normal systolic Ca2⫹ release. Because both forms of
release occur through the RyR, this is not simple. Recent
work has demonstrated that JTV519 (an agent that reduces
RyR open probability by increasing binding of the accessory
protein FKBP12.6 to the RyR) prevents the development of
ventricular arrhythmias following ␤-adrenergic stimulation in
transgenic mice that have a reduced expression of
FKBP12.6.11 (This agent also protected against heart failure
in a canine model12). However, neither of these studies
investigated changes of cellular Ca2⫹ handling. Furthermore,
the fact that an agent affecting FKBP12.6 binding to the RyR
protects against arrhythmias induced by decreased FKBP12.6
does not provide information about whether maneuvers that
affect RyR opening in other ways will also be antiarrhythmic.
Previous work has shown that the local anesthetic tetracaine,
by reducing RyR Po, decreases the frequency of spontaneous
Ca2⫹ release in unstimulated rat ventricular myocytes13 and
has no effect on the systolic Ca2⫹ transient of stimulated
myocytes where spontaneous Ca2⫹ release is absent.14 Those
studies did not, however, investigate the effects of tetracaine
on systolic and diastolic release in stimulated cells also
exhibiting spontaneous Ca2⫹ release as would be observed in
Original received February 17, 2006; revision received March 29, 2006; accepted March 30, 2006.
From the Unit of Cardiac Physiology, University of Manchester, United Kingdom. Present address for M.E.D.: Veterinary Biomedical Sciences, Royal
(Dick) School of Veterinary Studies, The University of Edinburgh, United Kingdom.
Correspondence to D.A. Eisner, Unit of Cardiac Physiology, 3.18 Core Technology Facility, 46 Grafton St, Manchester M13 9PT, United Kingdom.
E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000222000.35500.65
1299
1300
Circulation Research
May 26, 2006
vivo. We have, therefore, investigated whether reduction of
RyR Po is a useful antiarrhythmic strategy in stimulated cells.
The results demonstrate that tetracaine abolishes spontaneous
Ca2⫹ release in the steady state but increases the systolic Ca2⫹
transient. The increased systolic Ca2⫹ transient allows the cell
to balance Ca2⫹ fluxes without generating arrhythmogenic
spontaneous releases of Ca2⫹. We suggest that RyR open
probability reduction could be used as a novel therapeutic
strategy against arrhythmias triggered by DADs.
Materials and Methods
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Experiments were performed on single ventricular myocytes isolated
from Wistar rats. Myocytes were isolated by collagenase/protease
digestion as described previously.15 Voltage clamp was imposed
using the perforated patch technique with amphotericin-B. Electrodes (1.5 to 3 M⍀ resistance) were back filled with solution
containing (in mmol/L) CsCH3O3S 115, CsCl 20, NaCl 12, HEPES
10, Cs2EGTA 0.1, and MgCl2 5, titrated to pH 7.2 with CSOH. The
concentration of amphotericin-B was 240 ␮g/mL. Final access
resistance was typically ⬇20 M⍀ and was overcome using the
switch-clamp facility of a Axoclamp-2A amplifier (Axon Instruments, Union City, Calif). Cells were stimulated with 100-ms pulses
(from ⫺40 to 0 mV) at 0.5 Hz. The superfusing solution contained
(in mmol/L) NaCl 135, glucose 11, CaCl2 1 to 2, HEPES 10, MgCl2
1, KCl 4, 4-aminopyridine 5, BaCl2 0.1; titrated to pH 7.4 with 2
mol/L NaOH. Probenecid (2 mmol/L) was added to decrease the loss
of fluorescent indicators from the cells. Cells were loaded with the
acetoxymethyl ester of the low affinity (Kd⫽9.5 ␮mol/L) indicator
Fluo-4FF (Molecular Probes) to provide a wide range of sensitivity
to changes of [Ca2⫹]. In preliminary experiments using Fluo-3, we
found that the level of fluorescence reached at the peak of the Ca2⫹
transient was saturating. All experiments were performed at room
temperature (24°C). Isoproterenol (1 ␮mol/L; Sigma) was used to
produce Ca2⫹ overload and generate diastolic Ca2⫹ release. The Ca2⫹
influx through the Ca2⫹ current was calculated by integrating the
Ca2⫹ current. The Ca2⫹ efflux associated with the Ca2⫹ transient was
obtained by integration of the NCX current immediately after
repolarization (tail current). The efflux mediated by Ca2⫹ waves was
quantified by integrating the inward current associated with them.
For all of these calculations, the current value corresponding to the
minimum [Ca2⫹]i reached after systole was used as the baseline
current level.
Where applicable, the data are reported as the mean⫾SEM of n
experiments. Significance was tested using either t test or 1-way
ANOVA.
Figure 1. Effects of tetracaine on Ca2⫹ waves. In both A and B,
the traces show [Ca2⫹]i in response to voltage clamp depolarizations applied from a holding potential of ⫺40 mV to 0 mV at 0.5
Hz. From left to right, the traces were obtained in the following
conditions: control; isoproterenol (1 ␮mol/L); isoproterenol plus
the indicated tetracaine concentration. The tetracaine concentration was 25 ␮mol/L in A and 50 ␮mol/L in B.
isoproterenol⫹tetracaine (P⫽0.002). The higher concentration of tetracaine (50 ␮mol/L) abolished diastolic Ca2⫹
release in all 11 cells studied (Figure 1B). The removal of this
diastolic Ca2⫹ release is accompanied by removal of the
arrhythmogenic transient inward current (see Figure 2).
It is also clear from Figure 1 that the abolition of diastolic
Ca2⫹ release is accompanied by an increase of the amplitude
of the systolic Ca2⫹ transient. In 11 cells, tetracaine
(50 ␮mol/L) increased the amplitude of the Ca2⫹ transient by
16% from 1.36⫾0.29 to 1.59⫾0.26 ␮mol/L (P⫽0.007).
It is known that tetracaine can also inhibit the ICa,L.14,18 This
raises the question of whether the effects of tetracaine on Ca2⫹
Results
Effects of Tetracaine on Diastolic Ca2ⴙ Release
Spontaneous Ca2⫹ release was induced using 1 ␮mol/L
isoproterenol in both 1 and 2 mmol/L [Ca2⫹]o. As expected,16,17 isoproterenol increased the amplitude of both the
systolic Ca2⫹ transient and the L-type Ca2⫹ current (ICa,L) in all
of the cells studied. Isoproterenol also produced diastolic
Ca2⫹ release, and this occurred in 11 of 49 of cells studied in
1 mmol/L [Ca2⫹]o and 28 of 38 cells studied in 2 mmol/L
[Ca2⫹]o. When a steady state had been achieved (2 to 3
minutes), tetracaine was applied to reduce the RyR Po. Two
concentrations of tetracaine were used: 25 and 50 ␮mol/L. At
both concentrations, tetracaine had dramatic effects on diastolic Ca2⫹ release: 25 ␮mol/L tetracaine abolished diastolic
Ca2⫹ release in 50% (3 of 6) of the cells studied and reduced
its frequency in the remainder (Figure 1A). In these latter 3
cells, the mean frequency of Ca2⫹ waves was 1.0 per Ca2⫹
transient in isoproterenol and 0.46⫾0.02 per Ca2⫹ transient in
Figure 2. Effects of tetracaine on sarcolemmal Ca2⫹ fluxes and
Ca2⫹ balance. Traces show (from top to bottom): [Ca2⫹]i; membrane current (Im) (note that the current on repolarization is also
shown amplified ⫻25); calculated Ca2⫹ flux (upward represents
net Ca2⫹ uptake into cell). The various components identified
are: influx (I); Ca2⫹ efflux associated with the systolic Ca2⫹ transient (SE); and Ca2⫹ efflux mediated by diastolic Ca2⫹ release
(DE). These were calculated by integration of the membrane
currents. Systolic efflux was assumed to end when the level of
[Ca2⫹]i had returned to within 5% of the lowest level reached in
diastole and the calculation of diastolic efflux then began.
Shown are: control (a); isoproterenol (1 ␮mol/L) (b); and isoproterenol⫹
tetracaine (50 ␮mol/L) (c). The box in b highlights the diastolic
Ca2⫹ release and accompanying transient inward current.
Venetucci et al
Ryanodine Receptor as an Antiarrhythmic Strategy
handling are attributable to a pure inhibition of RyR or to a
combined effect of RyR and ICa,L inhibition. We found that, at
a concentration of 50 ␮mol/L, tetracaine had no effect
(P⫽0.24) on the peak amplitude of ICa,L (1.28⫾⫺0.12 nA in
isoproterenol and 1.32⫾0.13 nA in isoproterenol⫹tetracaine).
At a higher concentration of tetracaine (100 ␮mol/L), there was
a 9.1⫾1.2% decrease in the amplitude of the current, and,
therefore, all subsequent analysis is based on the results obtained
using 50 ␮mol/L tetracaine in cells that exhibited spontaneous
release in isoproterenol.
Effects of Tetracaine on Sarcolemmal Ca2ⴙ
Flux Balance
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Previous work has shown that the Ca2⫹ released during
diastolic waves activates Na–Ca exchange and that the Ca2⫹
efflux resulting from this is important for cellular Ca2⫹
balance.19 The question then arises as to how cellular Ca2⫹
flux balance is maintained when the efflux produced by
diastolic Ca2⫹ release is removed. The upper traces of Figure
2 show the effects of isoproterenol (b) and tetracaine (c) on
[Ca2⫹]i and membrane current. Tetracaine removes the diastolic Ca2⫹ release and transient inward current. The lower
panel shows the calculated Ca2⫹ fluxes across the surface
membrane obtained by integrating Ca2⫹ entry on ICa,L (I) and
efflux by NCX on depolarization (SE) and during the diastolic Ca2⫹ wave (DE). Isoproterenol increases Ca2⫹ entering via
ICa,L (I) from 3.96⫾0.65 to 11.97⫾1.45 ␮mol/L (P⫽0.001).
The subsequent application of tetracaine had no significant
effect on influx 10.18⫾1.36 ␮mol/L (P⫽0.397). In control,
the systolic efflux (SE) is 4.83⫾0.42 ␮mol/L, and this is
decreased to 3.08⫾0.46 ␮mol/L (P⫽0.086) in isoproterenol,
where the bulk of the Ca2⫹ efflux (7.82⫾0.76 ␮mol/L) now
occurs in diastole (DE). In tetracaine, this situation is reversed and the bulk of the Ca2⫹ efflux is associated with the
systolic Ca2⫹ transient (SE) (6.92⫾1.20 ␮mol/L) and diastolic Ca2⫹ efflux (DE) is reduced to 0.91⫾0.35 ␮mol/L.
If NCX mediated Ca2⫹ efflux occurs during the depolarizing pulse, the above analysis would lead to an overestimate of
Ca2⫹ entry by ICa,L (attributable to inward NCX current) and
underestimate of the amount of Ca2⫹ efflux associated with
the systolic Ca2⫹ transient (as the integral begins only after
the pulse ends). This is more problematic in the presence of
isoproterenol, where the Ca2⫹ transient is larger and decays
more quickly than under control conditions, ie, more of the
transient takes place during the pulse. To address these issues
and obtain a more precise measure of Ca2⫹ influx and efflux
during the pulse, we have calculated the Ca2⫹ efflux on NCX
during the depolarizing pulse.20 In any given cell, we measure
the relationship between changes of [Ca2⫹]i and NCX current
on repolarization to ⫺40 mV. In 7 cells, we measured the
relationship between [Ca2⫹]i and NCX current in response to
caffeine and found that at 0 mV, the slope was 58% of that at
⫺40 mV. From this ratio and the observed relationship at
⫺40 mV, we calculated the slope of the relationship at 0 mV
and used this to estimate the NCX current during the pulse.
This estimation of NCX current was then used to correct both
ICa,L-mediated systolic Ca2⫹ influx and NCX mediated Ca2⫹
efflux.
1301
Calculation of Ca2ⴙ Fluxes After Correcting for Ca2ⴙ Efflux on
NCX During the Depolarizing Pulse
Isoproterenol⫹
Tetracaine
(␮mol/L)
Control
(␮mol/L)
Isoproterenol
(␮mol/L)
Ca2⫹ influx via ICa,L
3.72⫾0.65
11.15⫾1.34
9.07⫾1.26
Systolic efflux
5.60⫾0.60
5.54⫾0.73
10.27⫾1.33
Diastolic efflux
Ca2⫹ balance
0.14⫾0.09
7.28⫾0.75
0.79⫾0.39
⫺2.03⫾0.71
⫺2.21⫾0.89
⫺1.99⫾0.71
As described in the text, the Ca2⫹ efflux on NCX during the depolarizing pulse
was calculated from the measured 关Ca2⫹兴i and the relationship between 关Ca2⫹兴i
and NCX current. The calculated NCX current was then subtracted from the
measured current to give the corrected ICa,L and hence the corrected Ca influx.
From top to bottom, the rows show: Ca2⫹ influx on the L-type current
calculated as above; calculated efflux during systole (as above); measured Ca2⫹
efflux during diastole; net Ca2⫹ balance (negative values indicates a net
calculated loss).
The results obtained from this analysis are summarized in
the Table. Isoproterenol greatly increases Ca2⫹ influx via ICa,L
from 3.72⫾0.64 ␮mol/L in control to 11.15⫾1.34 ␮mol/L in
isoproterenol (P⬍0.001); the application of tetracaine results
in a reduction in Ca2⫹ influx to 9.07⫾1.26 (P⫽0.027),
representing an 18.7% reduction. The Ca2⫹ transient–mediated Ca2⫹ efflux (systolic efflux) does not change after
application of isoproterenol (5.61⫾0.60 ␮mol/L in control
and 5.54⫾0.73 ␮mol/L in isoproterenol), despite the increase
in transient amplitude. This is attributable to the fact that the
increase in transient amplitude is offset by the acceleration of
the decay of the Ca2⫹ transient decreasing the time available
for NCX to remove Ca2⫹ from the cell (see below). The
addition of tetracaine causes a substantial increase in Ca2⫹
efflux associated with the systolic transient
(10.27⫾1.33 ␮mol/L; P⬍0.001) and obviates the need for
diastolic Ca2⫹ release to mediate Ca2⫹ loss from the cell.
Despite the above corrections for Ca2⫹ efflux occurring
during depolarization, net Ca2⫹ flux balance is not attained
(see Discussion) and equals ⫺2.03⫾0.71 ␮mol/L in control,
␮ mol/L
in
isoproterenol,
and
⫺2.21⫾0.89
⫺1.99⫾0.71 ␮mol/L in isoproterenol and tetracaine (not
different from each other; P⫽0.633).
Mechanisms Responsible for the Increase of
Systolic Ca2ⴙ Efflux After Tetracaine Application
The above analysis demonstrates that cells can balance Ca2⫹
fluxes without recourse to waves of Ca2⫹ release caused by an
increase of the amount of Ca2⫹ efflux that occurs during the
systolic Ca2⫹ transient. This increased Ca2⫹ efflux could be
mediated by 3 mechanisms: (1) increased amplitude of Ca2⫹
transient that causes greater activation of NCX; (2) slower
decay of the Ca2⫹ transient that gives more time for NCX to
pump Ca2⫹ of the cell; or (3) change in NCX function such
that there is more Ca2⫹ efflux for the same level of [Ca2⫹]i.
We have already shown that tetracaine increases Ca2⫹ transient amplitude. To determine whether the 2 remaining
mechanisms contribute to the increase in the Ca2⫹ efflux
associated with the Ca2⫹ transient, we studied both the rate of
decay of the Ca2⫹ transient and the function of NCX. We
measured the rate constant of decay in isoproterenol and
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May 26, 2006
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Figure 3. Effects of tetracaine on transient decay and NCX function. A, Systolic Ca2⫹ transients in response to depolarizing
pulses from ⫺40 to 0 mV at 0.5 Hz. Traces show average systolic Ca2⫹ transient traces (a); normalized data (b); tail currents
associated with Ca2⫹ transients (c); and rate constants of decay
of the systolic Ca2⫹ transient quantified by fitting an exponential
equation to the decay phase of the transient (d). B, The membrane current during decay of the Ca2⫹ transient plotted vs
[Ca2⫹]i, in isoproterenol (gray points) and in
isoproterenol⫹tetracaine 50 ␮mol/L (black points). The linear
regression equation was applied to the relationship, and the
regression coefficients in the 2 conditions were compared. The
histogram on the right shows results from nine cells. *P⬍0.05
tetracaine in 9 cells (Figure 3A). The rate constant of decay
was decreased from 20.2⫾1.1 sec⫺1 in isoproterenol to
15.5⫾1.3 sec⫺1 in 50 ␮mol/L tetracaine (P⫽0.001). Therefore, both the slowing of decay of the transient and the
increase in its amplitude contribute to the increased Ca2⫹
efflux. Finally, the function of NCX was assessed by plotting
the NCX tail current during decay of the Ca2⫹ transient as a
function of [Ca2⫹]i. The slope of this relationship gives a
measure of the degree of activation of NCX. In all 9 cells
(Figure 3B), 50 ␮mol/L tetracaine had no significant effect
and the mean regression coefficient was ⫺0.127⫾0.015
nA/␮mol per liter in isoproterenol and ⫺0.141⫾0.017 nA/
␮mol per liter in isoproterenol⫹50 ␮mol/L tetracaine
(P⫽0.111). On the basis of this finding, we can conclude that
tetracaine does not change NCX function.
What Causes the Increase of Ca2ⴙ Transient
Amplitude After Application of Tetracaine?
The finding that an agent such as tetracaine that decreases
RyR opening increases the amplitude of the systolic Ca2⫹
transient is surprising. Previous work found that, in the steady
state, tetracaine did not have any effect on either the Ca2⫹
transient amplitude or contraction.14 The main difference
between our experiments and this previous work is that our
cells show diastolic Ca2⫹ release. Previous work has also
Figure 4. Diastolic Ca2⫹ release reduces the amplitude of the
following Ca2⫹ transient. A, Record of [Ca2⫹]i in isoproterenol
(left) and isoproterenol⫹tetracaine (right). B, Specimen transients: following a wave (a); no previous wave (b); and in tetracaine (c). C, Mean Ca2⫹ transient amplitude data from 5 cells.
*P⬍0.01.
shown that the occurrence of diastolic Ca2⫹ waves depresses
the subsequent systolic Ca2⫹ release.21–24 Together with our
data, these observations are consistent with the hypothesis
that Ca2⫹ waves decrease the following Ca2⫹ transient and
that tetracaine increases the transient amplitude by removing
Ca2⫹ waves. This hypothesis predicts that tetracaine will have
no effect on the amplitude of transients that are not preceded
by a Ca2⫹ wave. Figure 4A and 4B show that, in isoproterenol, waves are not seen in every diastolic period. Ca2⫹
transients preceded by a wave (a) tend to be smaller than
those where the previous diastolic period did not have a wave
(b). Figure 4C shows mean data from 5 cells. The mean
amplitude of the systolic Ca2⫹ transients preceded by a wave
was 1.08⫾0.06 ␮mol/L, and this was less than the value
when no diastolic wave occurred (1.44⫾0.11 ␮mol/L,
P⫽0.001). Importantly, the amplitude of the Ca2⫹ transient in
isoproterenol and tetracaine (1.47⫾0.13 ␮mol/L) was not
different from that obtained in isoproterenol alone without a
preceding diastolic Ca2⫹ release (P⫽0.882).
These results demonstrate that Ca2⫹ waves depress the Ca2⫹
transient that follows them and that the increase in transient
amplitude produced by tetracaine is caused by the removal of
this inhibitory effect. If this explanation is correct then one
would predict that tetracaine should not increase the amplitude of the Ca2⫹ transient in cells where the application of
isoproterenol did not produce Ca2⫹ waves. That this is the
case is shown by the experiment of Figure 5A, where
tetracaine (50 ␮mol/L) decreases the Ca2⫹ transient amplitude. The mean data from 14 cells demonstrate that
Venetucci et al
Ryanodine Receptor as an Antiarrhythmic Strategy
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Figure 5. Effects of tetracaine on cells that were not Ca2⫹ overloaded in the presence of isoproterenol. A, Record of cytosolic
Ca2⫹ in isoproterenol (left) and isoproterenol⫹tetracaine
(50 ␮mol/L) (right). Tetracaine produces a small reduction in
transient amplitude. B, Mean data from 14 cells. *P⬍0.05.
50 ␮mol/L tetracaine produced a significant (P⫽0.006)
reduction in transient amplitude from 1.26⫾0.03 ␮mol/L to
1.16⫾0.02 ␮mol/L.
Discussion
Inhibition of RyR achieved using JTV519 has been shown to
prevent the onset of ventricular arrhythmias after
␤-adrenergic stimulation.10 The 2 aims of the present work
were (1) to establish whether, at a cellular level, selective
RyR inhibition (achieved by using tetracaine) could be used
to remove the unwanted diastolic Ca2⫹ release from the SR
and the associated arrhythmogenic inward current and (2) to
study the effects of RyR inhibition on the systolic Ca2⫹
transient under Ca2⫹-overloaded conditions and determine the
effects of RyR inhibition on cardiac contractility. The experiments show that tetracaine abolished diastolic waves of Ca2⫹
release yet increased the systolic Ca2⫹ transient amplitude.
The finding that an agent that decreases RyR opening
increases the systolic Ca2⫹ transient is unexpected and warrants discussion.
Previous work has investigated the effects of tetracaine
under 2 conditions. (1) When applied to Ca2⫹-overloaded
cells clamped at a constant membrane potential, tetracaine
decreased the frequency of spontaneous Ca2⫹ waves and
increased the Ca2⫹ efflux on each wave.13 This is explained
by tetracaine increasing the threshold SR Ca2⫹ content at
which a wave occurs. As a result, there is an increase in the
amount of Ca2⫹ released and the wave is larger. This,
however, results in increased Ca2⫹ efflux and, therefore, at a
constant influx of Ca2⫹ into the cell, it takes longer for the SR
to reaccumulate this calcium and the frequency of Ca2⫹
release is decreased. (2) When applied to nonoverloaded cells
stimulated to produce Ca2⫹ transients, tetracaine produces a
transient decrease in the amplitude of the Ca2⫹ transient and
contraction but, after a few beats, contraction returns to levels
similar to control.14 The transient nature of the response is
explained by the fact that the initial decrease in systolic Ca2⫹
1303
on application of tetracaine decreases the Ca2⫹ efflux from the
cell thereby increasing SR Ca2⫹ and hence allowing the
systolic Ca2⫹ transient to recover. In the steady state, the Ca2⫹
efflux from the cell must balance the Ca2⫹ entry that is largely
via the ICa,L. If this influx is constant, then the efflux must be
constant and therefore the amplitude of the Ca2⫹ transient in
tetracaine must be the same as in control.25 (See below for
consideration of the consequences of the slight increased
duration of the Ca2⫹ transient in tetracaine.)
In the present experiments (performed on cells that were
both stimulated and Ca2⫹ overloaded), tetracaine produced a
maintained increase of the amplitude of the systolic Ca2⫹
transient. In isoproterenol, much of the Ca2⫹ efflux occurs
during the diastolic Ca2⫹ wave. The application of tetracaine
abolishes these waves and, therefore, the Ca2⫹ efflux associated with them. However, in tetracaine, Ca2⫹ efflux is now
fully associated with the systolic Ca2⫹ transient, with no
requirement for a diastolic Ca2⫹ wave to maintain Ca2⫹
balance. This is achieved by a combined effect on Ca2⫹ influx
via ICa,L and Ca2⫹ efflux. When allowance is made for the
contaminating effects of NCX current during the depolarizing
pulse (Table), the Ca2⫹ influx via ICa,L is decreased by 18.7%
(P⫽0.027). This decrease is solely attributable to a higher
rate of decay of ICa,L because the peak amplitude of the current
is not significantly different. This faster inactivation of ICa,L is
probably caused by the bigger Ca2⫹ transient rather than a
direct effect of tetracaine on ICa,L inactivation because when
tetracaine is applied to non–Ca2⫹-overloaded cells, there is no
effect on Ca2⫹ influx via ICa,L (7.84⫾0.49 ␮mol/L in isoproterenol and 8.24⫾0.45 ␮mol/L in isoproterenol⫹tetracaine;
P⫽0.305).
The increased Ca2⫹ efflux associated with the Ca2⫹ transient produced by tetracaine is caused by the combined
effects of increased amplitude and duration of the Ca2⫹
transient stimulating Ca2⫹ efflux on NCX. As shown in
Figure 3, the properties of NCX, itself, are unaffected by
tetracaine. The mechanism of the increased amplitude of the
Ca2⫹ transient is dealt with below. The decrease of the rate of
decay of the Ca2⫹ transient is surprising, as one might expect
an increase in the Ca2⫹ transient to activate SERCA via
CaMKII dependent mechanisms (for review, see Maier and
Bers26). There are 3 possible explanations. (1) As shown,
previously, Ca2⫹ release in tetracaine may be less uniform
thereby slowing the Ca2⫹ transient.27 (2) Changes in RyR
gating produced by raising luminal Ca2⫹ in tetracaine may
prolong the release phase of systole.28 (3) The increased SR
Ca2⫹ content produced by tetracaine may decrease SERCA
activity by increasing the gradient against which Ca2⫹ is
pumped.29
The calculation of Ca2⫹ balance shows that the Ca2⫹
balance is always negative and there is an unidentified Ca2⫹
influx that could be attributable to (1) a background Ca2⫹
influx during diastole or (2) Ca2⫹ entry on reverse NCX
during systole. In the former case, the required background
influx would be approximately 1 ␮mol 䡠 L⫺1 per second, in
approximate agreement with previous estimates of Ca2⫹
influx in rat ventricular myocytes.30 The latter possibility is
suggested by recent work indicating that during adrenergic
stimulation, the calcium current can activate Ca2⫹ entry on
1304
Circulation Research
May 26, 2006
NCX.31 However, on the basis of this explanation, one would
expect the unresolved influx to be greater in isoprenaline than
control and this is not the case.
Tetracaine Increases the Ca2ⴙ Transient
Amplitude by Removing Ca2ⴙ Waves
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A novel finding is that in addition to abolishing Ca2⫹ waves,
tetracaine increases the amplitude of the systolic Ca2⫹ transient. That these phenomena are linked causally is suggested
by the observation of Figure 4 that Ca2⫹ transients preceded
by Ca2⫹ waves are smaller than those not preceded by waves.
The amplitude of the transients in isoproterenol plus tetracaine are identical to those in isoproterenol alone, which were
not preceded by waves. These data suggest that Ca2⫹ waves
exert an inhibitory effect on the following Ca2⫹ transient and
that, in Ca2⫹-overloaded cells, tetracaine increases the amplitude of the Ca2⫹ transient by removing Ca2⫹ waves and hence
their inhibitory effects. This is supported by the observation
(Figure 5) that tetracaine does not increase the amplitude of
the Ca2⫹ transient in the absence of diastolic Ca2⫹ waves.14
Indeed, there was a small decrease in the amplitude of the
Ca2⫹ transient. This is presumably attributable to the decreased rate of decay of the Ca2⫹ transient (Figure 3)
providing more Ca2⫹ efflux such that the combination of
decreased amplitude and slowed decay produces the same
amount of Ca2⫹ transient associated efflux as in isoproterenol
alone.
It is unclear what the mechanisms responsible for the
inhibitory effects of Ca2⫹ waves are. It could be attributable to
(1) Ca2⫹-dependent inhibition of ICa,L32; (2) Ca2⫹-dependent
adaptation or inactivation of the RyR33,34; or (3) depletion of
SR Ca2⫹ content.35 The results of this report do not allow us
to distinguish among these possibilities.
Are the Effects of Tetracaine Exclusively
Attributable to Actions on RyR?
Tetracaine has actions at sites other than the RyR. (1) It
inhibits the Na current. This is not a problem in the present
experiments because of the holding potential used. (2) It can
also decrease the ICa,L.36 However, on average we found no
effect of 50 ␮mol/L tetracaine on the peak calcium current.
Theoretically a change in the function of NCX could remove
diastolic Ca2⫹ release by increasing Ca2⫹ efflux associated
with the Ca2⫹ transient. The analysis of the relationship
between [Ca2⫹]i and tail current during the decay of the Ca2⫹
transient clearly shows that there is no change in NCX
function. Furthermore, tetracaine has no direct effect on
SERCA.28 We conclude that the effects of tetracaine we
report are exclusively mediated by RyR inhibition.
What Would Be the Effect of Tetracaine on Entire
Heart Contractility?
One important question regarding the use of a RyR inhibitor
as an antiarrhythmic concerns its effects on the contraction of
the whole heart. Many of the available antiarrhythmic agents
have profound negative inotropic effects that make them
unsuitable for use in heart failure, among the most common
causes of arrhythmias. From our experiments, it is very
difficult to reach a conclusion on the overall effect of RyR
inhibition on cardiac contractility; however, it is worth
considering a few points. (1) The observation that tetracaine
(50 ␮mol/L) has mild negative inotropic effects only in
nonoverloaded cells and that, in overloaded cells, tetracaine
produces positive inotropic effects suggests that the overall
effect will depend on the percentage of cells that are Ca2⫹
overloaded and that the higher this percentage is, the more
likely a positive inotropic effect is. (2) At higher concentrations of tetracaine, and therefore higher levels of RyR
inhibition, the negative inotropic effects will become more
pronounced. (3) In multicellular preparations, the overall
negative inotropic action produced by Ca2⫹ waves is much
bigger than would be expected from the simple summation of
the negative inotropic effects in single cells. This is mainly
attributable to the fact that the cells are mechanically connected and weakly activated cells are stretched by the fully
activated ones and dissipate some of the work produced by
the fully activated cells.2 A maneuver that reduces the number
of cells that exhibit waves and are weakly activated will
increase force of contraction. One can hypothesize that at
relatively low concentrations (50 ␮mol/L), tetracaine could
produce a positive inotropic effect (leaving aside the effect
on INa).
The main conclusion of this report is that selective reduction of the open probability of the RyR abolishes diastolic
Ca2⫹ release, while potentiating systolic release. This may
provide the basis of a new antiarrhythmic strategy that could
be valuable in many clinical situations. To pursue this
strategy further, an agent which selectively and reversibly
decreases the open probability of the RyR must be developed.
Acknowledgments
This work was supported by The British Heart Foundation.
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Reducing Ryanodine Receptor Open Probability as a Means to Abolish Spontaneous Ca2+
Release and Increase Ca 2+ Transient Amplitude in Adult Ventricular Myocytes
L.A. Venetucci, A.W. Trafford, M.E. Díaz, S.C. O'Neill and D.A. Eisner
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Circ Res. 2006;98:1299-1305; originally published online April 13, 2006;
doi: 10.1161/01.RES.0000222000.35500.65
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