This Article Abstract Full Text (PDF) All Versions of this Article: 98/10/1299    most recent 01.RES.0000222000.35500.65v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Venetucci, L.A. Articles by Eisner, D.A. Search for Related Content PubMed PubMed Citation Articles by Venetucci, L.A. Articles by Eisner, D.A. Related Collections Contractile function Arrythmias-basic studies Calcium cycling/excitation-contraction coupling

(Circulation Research. 2006;98:1299.)
© 2006 American Heart Association, Inc.

Cellular Biology

Reducing Ryanodine Receptor Open Probability as a Means to Abolish Spontaneous Ca²⁺ Release and Increase Ca²⁺ Transient Amplitude in Adult Ventricular Myocytes

L.A. Venetucci, A.W. Trafford, M.E. Díaz, S.C. O’Neill, D.A. Eisner

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 eisner{at}man.ac.uk

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 µmol/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 µmol/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.

Key Words: spontaneous release • calcium • sarcoplasmic reticulum • arrhythmias

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In 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 ß-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 P_o, 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 vivo. We have, therefore, investigated whether reduction of RyR P_o 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 µg/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 (K_d=9.5 µmol/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 µmol/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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of Tetracaine on Diastolic Ca²⁺ Release
Spontaneous Ca²⁺ release was induced using 1 µmol/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 (I_Ca,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 P_o. Two concentrations of tetracaine were used: 25 and 50 µmol/L. At both concentrations, tetracaine had dramatic effects on diastolic Ca²⁺ release: 25 µmol/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 isoproterenol+tetracaine (P=0.002). The higher concentration of tetracaine (50 µmol/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).

View larger version (22K): [in this window] [in a new window]   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 µmol/L); isoproterenol plus the indicated tetracaine concentration. The tetracaine concentration was 25 µmol/L in A and 50 µmol/L in B.

View larger version (20K): [in this window] [in a new window]   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 x25); 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 µmol/L) (b); and isoproterenol+ tetracaine (50 µmol/L) (c). The box in b highlights the diastolic Ca2+ release and accompanying transient inward current.

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 µmol/L) increased the amplitude of the Ca²⁺ transient by 16% from 1.36±0.29 to 1.59±0.26 µmol/L (P=0.007).

It is known that tetracaine can also inhibit the I_Ca,L.^14,18 This raises the question of whether the effects of tetracaine on Ca²⁺ handling are attributable to a pure inhibition of RyR or to a combined effect of RyR and I_Ca,L inhibition. We found that, at a concentration of 50 µmol/L, tetracaine had no effect (P=0.24) on the peak amplitude of I_Ca,L (1.28±–0.12 nA in isoproterenol and 1.32±0.13 nA in isoproterenol+tetracaine). At a higher concentration of tetracaine (100 µmol/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 µmol/L tetracaine in cells that exhibited spontaneous release in isoproterenol.

Effects of Tetracaine on Sarcolemmal Ca²⁺ Flux Balance
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 I_Ca,L (I) and efflux by NCX on depolarization (SE) and during the diastolic Ca²⁺ wave (DE). Isoproterenol increases Ca²⁺ entering via I_Ca,L (I) from 3.96±0.65 to 11.97±1.45 µmol/L (P=0.001). The subsequent application of tetracaine had no significant effect on influx 10.18±1.36 µmol/L (P=0.397). In control, the systolic efflux (SE) is 4.83±0.42 µmol/L, and this is decreased to 3.08±0.46 µmol/L (P=0.086) in isoproterenol, where the bulk of the Ca²⁺ efflux (7.82±0.76 µmol/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 µmol/L) and diastolic Ca²⁺ efflux (DE) is reduced to 0.91±0.35 µmol/L.

If NCX mediated Ca²⁺ efflux occurs during the depolarizing pulse, the above analysis would lead to an overestimate of Ca²⁺ entry by I_Ca,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 I_Ca,L-mediated systolic Ca²⁺ influx and NCX mediated Ca²⁺ efflux.

The results obtained from this analysis are summarized in the Table. Isoproterenol greatly increases Ca²⁺ influx via I_Ca,L from 3.72±0.64 µmol/L in control to 11.15±1.34 µmol/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 µmol/L in control and 5.54±0.73 µmol/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 µmol/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 µmol/L in control, –2.21±0.89 µmol/L in isoproterenol, and –1.99±0.71 µmol/L in isoproterenol and tetracaine (not different from each other; P=0.633).

View this table: [in this window] [in a new window]   Table 1. Calculation of Ca2+ Fluxes After Correcting for Ca2+ Efflux on NCX During the Depolarizing Pulse

Mechanisms Responsible for the Increase of Systolic Ca²⁺ 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 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 µmol/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 µmol/L tetracaine had no significant effect and the mean regression coefficient was –0.127±0.015 nA/µmol per liter in isoproterenol and –0.141±0.017 nA/µmol per liter in isoproterenol+50 µmol/L tetracaine (P=0.111). On the basis of this finding, we can conclude that tetracaine does not change NCX function.

View larger version (25K): [in this window] [in a new window]   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 µmol/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

What Causes the Increase of Ca²⁺ 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 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 µmol/L, and this was less than the value when no diastolic wave occurred (1.44±0.11 µmol/L, P=0.001). Importantly, the amplitude of the Ca²⁺ transient in isoproterenol and tetracaine (1.47±0.13 µmol/L) was not different from that obtained in isoproterenol alone without a preceding diastolic Ca²⁺ release (P=0.882).

View larger version (21K): [in this window] [in a new window]   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.

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 µmol/L) decreases the Ca²⁺ transient amplitude. The mean data from 14 cells demonstrate that 50 µmol/L tetracaine produced a significant (P=0.006) reduction in transient amplitude from 1.26±0.03 µmol/L to 1.16±0.02 µmol/L.

View larger version (24K): [in this window] [in a new window]   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 µmol/L) (right). Tetracaine produces a small reduction in transient amplitude. B, Mean data from 14 cells. *P<0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of RyR achieved using JTV519 has been shown to prevent the onset of ventricular arrhythmias after ß-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⁺ 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 I_Ca,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 I_Ca,L and Ca²⁺ efflux. When allowance is made for the contaminating effects of NCX current during the depolarizing pulse (Table), the Ca²⁺ influx via I_Ca,L is decreased by 18.7% (P=0.027). This decrease is solely attributable to a higher rate of decay of I_Ca,L because the peak amplitude of the current is not significantly different. This faster inactivation of I_Ca,L is probably caused by the bigger Ca²⁺ transient rather than a direct effect of tetracaine on I_Ca,L inactivation because when tetracaine is applied to non–Ca²⁺-overloaded cells, there is no effect on Ca²⁺ influx via I_Ca,L (7.84±0.49 µmol/L in isoproterenol and 8.24±0.45 µmol/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 µmol · 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 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 Ca²⁺ Transient Amplitude by Removing Ca²⁺ Waves
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 I_Ca,L³²; (2) Ca²⁺-dependent adaptation or inactivation of the RyR^33,34; 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 I_Ca,L.³⁶ However, on average we found no effect of 50 µmol/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 µmol/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 µmol/L), tetracaine could produce a positive inotropic effect (leaving aside the effect on I_Na).

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.

	Footnotes

 
Original received February 17, 2006; revision received March 29, 2006; accepted March 30, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. 2nd ed. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2001.

2. Wier WG, Kort AA, Stern MD, Lakatta EG, Marban E. Cellular calcium fluctuations in mammalian heart: direct evidence from noise analysis of aequorin signals in Purkinje fibers. Proc Natl Acad Sci U S A. 1983; 80: 7367–7371.[Abstract/Free Full Text]

3. Orchard CH, Eisner DA, Allen DG. Oscillations of intracellular Ca²⁺ in mammalian cardiac muscle. Nature. 1983; 304: 735–738.[CrossRef][Medline] [Order article via Infotrieve]

4. Wier WG, Cannell MB, Berlin JR, Marban E, Lederer WJ. Cellular and subcellular heterogeneity of [Ca²⁺]_i in single heart cells revealed by fura-2. Science. 1987; 235: 325–328.[Abstract/Free Full Text]

5. Lukyanenko V, Subramanian S, Györke I, Wiesner TF, Györke S. The role of luminal Ca²⁺ in the generation of Ca²⁺ waves in rat ventricular myocytes. J Physiol (Lond). 1999; 518: 173–186.[Abstract/Free Full Text]

6. Ferrier GR, Saunders JH, Mendez C. A cellular mechanism for the generation of ventricular arrhythmias by acetylstrophanthidin. Circ Res. 1973; 32: 600–609.[Abstract/Free Full Text]

7. Lipp P, Pott L. Transient inward current in guinea-pig atrial myocytes reflects a change of sodium-calcium exchange current. J Physiol (Lond). 1988; 397: 601–630.[Abstract/Free Full Text]

8. Lederer WJ, Tsien RW. Transient inward current underlying arrhythmogenic effects of cardiotonic steroids in Purkinje fibers. J Physiol (Lond). 1976; 263: 73–100.[Abstract/Free Full Text]

9. Pogwizd SM. Nonreentrant mechanisms underlying spontaneous ventricular arrhythmias in a model of nonischemic heart failure in rabbits. Circulation. 1995; 92: 1034–1048.[Abstract/Free Full Text]

10. Scoote M, Williams AJ. The cardiac ryanodine receptor (calcium release channel): emerging role in heart failure and arrhythmia pathogenesis. Cardiovasc Res. 2002; 56: 359–372.[Abstract/Free Full Text]

11. Wehrens XH, Lehnart SE, Reiken SR, Deng SX, Vest JA, Cervantes D, Coromilas J, Landry DW, Marks AR. Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2. Science. 2004; 304: 292–296.[Abstract/Free Full Text]

12. Yano M, Kobayashi S, Kohno M, Doi M, Tokuhisa T, Okuda S, Suetsugu M, Hisaoka T, Obayashi M, Ohkusa T, Kohno M, Matsuzaki M. FKBP12.6-mediated stabilization of calcium-release channel (ryanodine receptor) as a novel therapeutic strategy against heart failure. Circulation. 2003; 107: 477–484.[Abstract/Free Full Text]

13. Overend CL, Eisner DA, O’Neill SC. The effect of tetracaine on spontaneous Ca²⁺ release and calcium content in rat ventricular myocytes. J Physiol (Lond). 1997; 502: 471–479.[Abstract/Free Full Text]

14. Overend CL, O’Neill SC, Eisner DA. The effect of tetracaine on stimulated contractions, sarcoplasmic reticulum Ca²⁺ content and membrane current in isolated rat ventricular myocytes. J Physiol (Lond). 1998; 507: 759–769.[Abstract/Free Full Text]

15. Eisner DA, Nichols CG, O’Neill SC, Smith GL, Valdeolmillos M. The effects of metabolic inhibition on intracellular calcium and pH in isolated rat ventricular cells. J Physiol (Lond). 1989; 411: 393–418.[Abstract/Free Full Text]

16. Kameyama M, Hofmann F, Trautwein W. On the mechanism of B-adrenergic regulation of the Ca²⁺ channel in the guinea-pig heart. Pflugers Arch. 1985; 405: 285–293.[CrossRef][Medline] [Order article via Infotrieve]

17. Allen DG, Kurihara S. Calcium transients in mammalian ventricular muscle. Eur Heart J. 1980; 1: 5–15.[Free Full Text]

18. Carmeliet E, Morad M, Van der Heyden G, Vereecke J. Electrophysiological effects of tetracaine in single guinea-pig ventricular myocytes. J Physiol (Lond). 1986; 376: 143–161.[Abstract/Free Full Text]

19. Díaz ME, Trafford AW, O’Neill SC, Eisner DA. Measurement of sarcoplasmic reticulum Ca²⁺ content and sarcolemmal Ca²⁺ fluxes in isolated rat ventricular myocytes during spontaneous Ca²⁺ release. J Physiol (Lond). 1997; 501: 3–16.[Abstract/Free Full Text]

20. Negretti N, Varro A, Eisner DA. Estimate of net calcium fluxes and sarcoplasmic reticulum calcium content during systole in rat ventricular myocytes. J Physiol (Lond). 1995; 486: 581–591.[Abstract/Free Full Text]

21. Valdeolmillos M, Eisner DA. The effects of ryanodine on calcium-overloaded sheep cardiac Purkinje fibers. Circ Res. 1985; 56: 452–456.[Abstract/Free Full Text]

22. Allen DG, Eisner DA, Pirolo JS, Smith GL. The relationship between intracellular calcium and contraction in calcium-overloaded ferret papillary muscles. J Physiol (Lond). 1985; 364: 169–182.[Abstract/Free Full Text]

23. Capogrossi MC, Stern MD, Spurgeon HA, Lakatta EG. Spontaneous Ca²⁺ release from the sarcoplasmic reticulum limits Ca²⁺-dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium. J Gen Physiol. 1988; 91: 133–155.[Abstract/Free Full Text]

24. Cheng H, Lederer MR, Lederer WJ, Cannell MB. Calcium sparks and [Ca²⁺]_i waves in cardiac myocytes. Am J Physiol. 1996; 270: C148–C159.[Medline] [Order article via Infotrieve]

25. Eisner DA, Trafford AW, Díaz ME, Overend CL, O’Neill SC. The control of Ca release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation. Cardiovasc Res. 1998; 38: 589–604.[Abstract/Free Full Text]

26. Maier LS, Bers DM. Calcium, calmodulin, and calcium-calmodulin kinase II: heartbeat to heartbeat and beyond. J Mol Cell Cardiol. 2002; 34: 919–939.[CrossRef][Medline] [Order article via Infotrieve]

27. Díaz ME, Eisner DA, O’Neill SC. Depressed ryanodine receptor activity Increases variability and duration of the systolic Ca²⁺ transient in rat ventricular myocytes. Circ Res. 2002; 91: 585–593.[Abstract/Free Full Text]

28. Györke S, Lukyanenko V, Györke I. Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes. J Physiol (Lond). 1997; 500: 297–309.[Abstract/Free Full Text]

29. Inesi G, De Meis L. Regulation of steady state filling in sarcoplasmic reticulum. Roles of back-inhibition, leakage and slippage of the calcium pump. J Biol Chem. 1989; 264: 5929–5936.[Abstract/Free Full Text]

30. Choi HS, Trafford AW, Eisner DA. Measurement of calcium entry and exit in quiescent rat ventricular myocytes. Pflugers Arch. 2000; 440: 600–608.[Medline] [Order article via Infotrieve]

31. Viatchenko-Karpinski S, Terentyev D, Jenkins LA, Lutherer LO, Gyorke S. Synergistic interactions between Ca²⁺ entries through L-type Ca²⁺ channels and Na⁺-Ca²⁺ exchanger in normal and failing rat heart. J Physiol (Lond). 2005; 567: 493–504.[Abstract/Free Full Text]

32. Boyett MR, Kirby MS, Orchard CH. Rapid regulation of the "second inward current" by intracellular calcium in isolated rat and ferret ventricular myocytes. J Physiol (Lond). 1988; 407: 77–102.[Abstract/Free Full Text]

33. Györke S, Fill M. Ryanodine receptor adaptation: control mechanism of Ca²⁺-induced Ca²⁺ release in heart. Science. 1993; 260: 807–809.[Abstract/Free Full Text]

34. Sitsapesan R, Montgomery RAP, Williams AJ. New insights into the gating mechanisms of cardiac Ryanodine receptors revealed by rapid changes in ligand concentration. Circ Res. 1995; 77: 765–772.[Abstract/Free Full Text]

35. DelPrincipe F, Egger M, Niggli E. Calcium signalling in cardiac muscle: refractoriness revealed by coherent activation. Nat Cell Biol. 1999; 1: 323–329.[CrossRef][Medline] [Order article via Infotrieve]

36. Callewaert G, Vereecke J, Carmeliet E. Existence of a calcium-dependent potassium channel in the membrane of cow cardiac Purkinje cells. Pflugers Arch. 1986; 406: 424–426.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				M. Hirose, B. D. Stuyvers, W. Dun, H. E.D.J. ter Keurs, and P. A. Boyden Function of Ca2+ Release Channels in Purkinje Cells That Survive in the Infarcted Canine Heart: A Mechanism for Triggered Purkinje Ectopy Circ Arrhythm Electrophysiol, December 1, 2008; 1(5): 387 - 395. [Abstract] [Full Text] [PDF]

				G. Antoons and K. R. Sipido Targeting calcium handling in arrhythmias Europace, December 1, 2008; 10(12): 1364 - 1369. [Abstract] [Full Text] [PDF]

				L. A. Venetucci and D. A. Eisner Calsequestrin Mutations and Sudden Death: A Case of Too Little Sarcoplasmic Reticulum Calcium Buffering? Circ. Res., August 1, 2008; 103(3): 223 - 225. [Full Text] [PDF]

				N. Liu and S. G. Priori Disruption of calcium homeostasis and arrhythmogenesis induced by mutations in the cardiac ryanodine receptor and calsequestrin Cardiovasc Res, January 15, 2008; 77(2): 293 - 301. [Abstract] [Full Text] [PDF]

				L. A. Venetucci, A. W. Trafford, S. C. O'Neill, and D. A. Eisner The sarcoplasmic reticulum and arrhythmogenic calcium release Cardiovasc Res, January 15, 2008; 77(2): 285 - 292. [Abstract] [Full Text] [PDF]

				D. R. Gonzalez, F. Beigi, A. V. Treuer, and J. M. Hare Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes PNAS, December 18, 2007; 104(51): 20612 - 20617. [Abstract] [Full Text] [PDF]

				C.M. Loughrey, N. Otani, T. Seidler, M.A. Craig, R. Matsuda, N. Kaneko, and G.L. Smith K201 modulates excitation-contraction coupling and spontaneous Ca2+ release in normal adult rabbit ventricular cardiomyocytes Cardiovasc Res, November 1, 2007; 76(2): 236 - 246. [Abstract] [Full Text] [PDF]

				K. R. Sipido CaM or cAMP: Linking {beta}-Adrenergic Stimulation to 'Leaky' RyRs Circ. Res., February 16, 2007; 100(3): 296 - 298. [Full Text] [PDF]

				J. Curran, M. J. Hinton, E. Rios, D. M. Bers, and T. R. Shannon {beta}-Adrenergic Enhancement of Sarcoplasmic Reticulum Calcium Leak in Cardiac Myocytes Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase Circ. Res., February 16, 2007; 100(3): 391 - 398. [Abstract] [Full Text] [PDF]

				L. A. Venetucci, A. W. Trafford, and D. A. Eisner Increasing Ryanodine Receptor Open Probability Alone Does Not Produce Arrhythmogenic Calcium Waves: Threshold Sarcoplasmic Reticulum Calcium Content Is Required Circ. Res., January 5, 2007; 100(1): 105 - 111. [Abstract] [Full Text] [PDF]

				C. Pott and J. I. Goldhaber Is the Ryanodine Receptor a Target for Antiarrhythmic Therapy? Circ. Res., May 26, 2006; 98(10): 1232 - 1233. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 98/10/1299    most recent 01.RES.0000222000.35500.65v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Venetucci, L.A. Articles by Eisner, D.A. Search for Related Content PubMed PubMed Citation Articles by Venetucci, L.A. Articles by Eisner, D.A. Related Collections Contractile function Arrythmias-basic studies Calcium cycling/excitation-contraction coupling