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Am J Physiol Heart Circ Physiol 275: H422-H430, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 2, H422-H430, August 1998

Cs+ inhibits spontaneous Ca2+ release from sarcoplasmic reticulum of skinned cardiac myocytes

Makoto Kawai, Munir Hussain, and Clive H. Orchard

Department of Physiology, University of Leeds, Leeds LS2 9NQ, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effect of Cs+ on the function of the cardiac sarcoplasmic reticulum (SR) has been investigated in skinned cardiac myocytes. Isolated rat ventricular myocytes were permeabilized using saponin and then perfused with a solution containing 150 nmol/l Ca2+ and 10 µmol/l fura 2. Fura 2 fluorescence from the skinned cell was monitored to assess SR Ca2+ release. The frequency of spontaneous Ca2+ release from the SR decreased when K+ in the bathing solution was completely replaced with Cs+. Cs+ had little effect on the amplitude of spontaneous release but prolonged both the rise time and decay time. The SR Ca2+ content, assessed by application of caffeine, was reduced in the Cs+ solution. Cyclopiazonic acid produced effects similar to those of Cs+. Extracellular Cs+ (20 mmol/l) increased the amplitude of the Ca2+ transient and the SR Ca2+ content in intact field-stimulated cells but had little effect on the Ca2+ transient when the amplitude and duration of depolarization were kept constant using voltage clamp. These data suggest that Cs+ slows Ca2+ movement across the SR membrane, possibly by blocking the SR K+ channel, but has additional effects in intact cells that overcome its inhibitory effects on the SR.

cesium; cardiac sarcoplasmic reticulum potassium channel; counterion; cyclopiazonic acid; voltage clamp

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

POTASSIUM IONS (K+) are important for the normal electrical activity of cardiac muscle: the transsarcolemmal K+ gradient underlies the resting membrane potential and is necessary for the repolarization of the cell membrane at the end of the action potential. The role of intracellular K+ in excitation-contraction (E-C) coupling is less clear, but there are K+ channels in the cardiac sarcoplasmic reticulum (SR) membrane similar to those in the sarcolemma (13, 22). It has been suggested that K+ crosses the SR membrane via these channels to maintain charge balance during Ca2+ release and uptake by the SR (1, 21, 22). This idea is supported by measurements of ion concentrations in the terminal cisternae (TC) of skeletal muscle SR that showed that the K+ content of the TC increases significantly during tetanic contraction (28).

Cs+ is known to block K+ channels in the squid giant axon (2) and in single heart cells (17). Cs+ also blocks the K+ channel in cardiac SR (13, 27) and skeletal SR (5). Blocking the SR K+ channel with Cs+ leads to inhibition of Ca2+ release (1) and uptake (8) in skeletal SR. Despite these effects of Cs+, K+ is frequently replaced by Cs+ in studies of E-C coupling in single heart cells to block sarcolemmal K+ currents (17). However, such replacement appears to alter the size and voltage dependence of the Ca2+ transient (12, 14, 15, 18), although the mechanism is unclear. In this study, the effect of replacing K+ with Cs+ on cardiac SR function has been investigated in chemically skinned ventricular myocytes. Preliminary results have been published in abstract form (16).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ventricular myocyte isolation. Ventricular myocytes were isolated from adult female Wistar rat hearts as described previously (11). Briefly, the animal was stunned and then killed by cervical dislocation. The heart was quickly removed and mounted on a Langendorff apparatus and retrogradely perfused with a HEPES (Sigma, St. Louis, MO)-Tyrode solution (see Solutions and chemicals and Table 1) containing 0.75 mmol/l Ca2+ for 2-3 min. This solution was then replaced by a Ca2+-free solution (see Table 1), and after 4 min the Ca2+-free solution was replaced by an enzyme solution (see Table 1) containing 1 mg/ml collagenase (type I; Worthington, Freehold, NJ), 0.1 mg/ml protease (type XIV; Sigma), and 50 µmol/l Ca2+ for 6 min. At the end of this perfusion, both ventricles were removed from the heart and cut longitudinally, and the opened ventricles were incubated with the enzyme solution containing 1% bovine serum albumin (Sigma) for 5 min. The cells were then filtered through gauze and collected by centrifugation at 400 rpm for 30 s. Isolated cells were resuspended in HEPES-Tyrode solution until use. This procedure was repeated at 5-min intervals to obtain several batches of cells. These experiments were performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986.

                              
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  		Table 1.   Cell isolating solutions

Measuring spontaneous Ca2+ release using skinned cells. Isolated ventricular myocytes in the HEPES-Tyrode solution were centrifuged and then resuspended in Ca2+-free relaxing solution (see Solutions and chemicals and Table 2). This solution was then replaced by the skinning solution, which was prepared by adding saponin (50 µg/ml; BDH Laboratory Supplies, Dorset, UK) to the relaxing solution. After the myocytes had been permeabilized (chemically skinned) for 30 min by exposure to this solution (23), they were resuspended in the relaxing solution and stored at 4°C until used. An aliquot of skinned cells was then placed in an experimental bath (vol: 80 µl) on the stage of an inverted microscope (model Diaphot TMD; Nikon, Tokyo, Japan) and perfused with control solution (see Table 2) containing 150 nmol/l Ca2+ and 10 µmol/l fura 2 (pentapotassium salt; Molecular Probes, Eugene, OR) at a rate of 8 µl/s. The single skinned cell under study was held on the bottom of the experimental bath by a glass micropipette. Spontaneous release of Ca2+ from the SR was monitored using fura 2 fluorescence at 510 nm during alternate (every 2 ms) excitation by 340-nm and 380-nm light from a xenon lamp (100 W; Nikon). The fluorescence excited by these two wavelengths was collected by the objective lens (×40 oil immersion Fluor 40, N.A. 1.30; Nikon) and detected by a photomultiplier (Cairn Research, Kent, UK). The ratio of fura 2 fluorescence at 510 nm during excitation at 340 nm to that during excitation at 380 nm (340 nm/380 nm signal) was obtained using an analog divide circuit and used as a measure of Ca2+ within the preparation. Shutters in the light path of the emitted fluorescence were narrowed to almost the same dimensions as the image of the skinned cell to exclude fluorescence from sources other than the cell under study. The fluorescence signals and the fluorescence ratio were stored on videotape [on an SR-330MS video recorder (Victor, Tokyo, Japan) via a DR-890 digitizing unit (Neuro Data Instruments, New York, NY)] and on a microcomputer hard disk (model 486DX2/66; Dan Technology, London, UK) for later off-line analysis. The microcomputer used a 1401 plus analog-to-digital (A/D) interface and SIGAVG software (Cambridge Electronic Design, Cambridge, UK) for data storage and analysis. The signals were also displayed on a sixchannel chart recorder (model 2600S; Gould, Cleveland, OH).

                              
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  		Table 2.   Experimental solutions (skinned cells)

The Ca2+ content of the SR was assessed by monitoring Ca2+ released in response to pressure ejection of 20 mmol/l caffeine (Sigma) from a pipette placed close to, but upstream from, the cell being studied.

Measuring Ca2+ transients in intact cells. The effect of Cs+ on intact cardiac myocytes was also investigated by monitoring Ca2+ transients and caffeine-induced Ca2+ release with the use of fura 2 (cells loaded by 15 min incubation with 3 µmol/l fura 2-AM; Molecular Probes) and almost the same methods and apparatus as for the skinned cells. Intact cells were electrically stimulated at 0.33 Hz via platinum electrodes. Caffeine-induced Ca2+ release was used to assess the SR Ca2+ content as described for skinned cells. Electrical stimulation was stopped 3 s before the caffeine solution was applied to the cell. Electrical stimulation was resumed after the increase of intracellular Ca2+ concentration ([Ca2+]i) evoked by caffeine had recovered to the diastolic level.

Voltage-clamp experiments. In some cells that had been loaded with fura 2, membrane potential was controlled using the perforated patch-clamp technique as described previously (14). This enabled Ca2+ and membrane currents to be recorded simultaneously while the frequency, amplitude, and duration of membrane depolarization were controlled. Whole cell voltage clamp was achieved using a patch-clamp amplifier (AxoPatch 1D; Axon Instruments, Foster, CA) controlled by an A/D interface (1401 Plus, Cambridge Electronic Design) and a microcomputer (PC-433, Elonex PLC, London, UK). Micropipettes made from borosilicate glass (type GC150TF; Clark Electromedical Instruments, Reading, UK) were filled with a K-glutamate-based solution (see Solutions and chemicals) to give a resistance of 0.5-1.0 MOmega .

Perforated patch-clamp recordings were obtained using amphotericin B (250 or 500 µg/ml) as the pore-forming agent. After seal formation, membrane currents and the Ca2+ transient reached a steady state within 5 to 15 min and remained stable for the duration of the experiment. Voltage-clamp pulses were used to depolarize the cell from -40 to 0 mV for 200 ms at 0.5 Hz. The difference between the peak inward current elicited on depolarization and the current remaining at the end of the voltage-clamp pulse was used as a measure of the amplitude of the Ca2+ current (ICa). Voltage and current signals were displayed and recorded as described for the Ca2+ signal.

Solutions and chemicals. The compositions of the solutions used are shown in Tables 1-3. All solutions were made using ultrapure water supplied by a Milli-Q system (Millipore, Bedford, MA).

Three different solutions were used for isolating cells (see Ventricular myocyte isolation and Table 1). The HEPES-Tyrode solution, used during cell isolation, contained 0.75 mmol/l Ca2+, and pH was adjusted to 7.3 with NaOH. The Ca2+-free solution had the same composition as the HEPES-Tyrode solution except that it contained 0.1 mmol/l EGTA (Sigma) and 0 Ca2+. The enzyme solution contained collagenase, protease, and 50 µmol/l Ca2+.

The following solutions were used for measuring Ca2+ release in skinned cells (see also Table 2). The relaxing solution, used for permeabilizing (skinning) ventricular myocytes, contained 0.05 mmol/l EGTA and 0 Ca2+. The control solution, used during the experiments, contained 150 nmol/l Ca2+ and 10 µmol/l fura 2 but was otherwise the same in composition as the relaxing solution. The control solution was prepared by mixing the relaxing solution with the Ca-EGTA solution in proportions necessary to obtain the desired free [Ca2+], which was calculated by solving simultaneous equations using the binding constants listed by Martell and Smith (20). Finally, for K+ replacement experiments, 150 mmol/l Cs+ was used instead of 149 mmol/l K+, and pH was adjusted to 7.1 with CsOH (Sigma).

The solutions for the experiments on intact cells contained 1 mmol/l Ca2+, pH 7.4 (see Table 3), to which CsCl (Sigma) was added to produce a final concentration of 20 mmol/l. The pipette solution used for the voltage-clamp experiments contained (in mmol/l) 120 K-glutamate, 20 KCl, 10 NaCl, 4 MgATP, and 10 HEPES, buffered to pH 7.3 with KOH.

                              
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  		Table 3.   Experimental solutions (intact cells)

Cyclopiazonic acid (CPA; Sigma) was dissolved in DMSO (Sigma) to make a 20 mmol/l stock solution and was added to give a final concentration of 10 or 50 µmol/l (see Table 2). The control solution contained the same concentration of DMSO as the 10 µmol/l CPA solution, and the percentages of added DMSO were 0.05% (vol/vol) at 10 µmol/l and 0.25% (vol/vol) at 50 µmol/l CPA.

All other chemicals were reagent grade. All experiments were carried out at room temperature (22-24°C).

Calibration of [Ca2+]. Calibration of [Ca2+] in the experimental solutions was carried out using solutions containing various [Ca2+], 10 µmol/l fura 2, and 10 mmol/l EGTA. The solutions containing various [Ca2+] were prepared by mixing two complementary solutions: EGTA-calibration solution and Ca-calibration solution. The apparent dissociation constant of EGTA for Ca2+ was 267 nmol/l (for pH 7.1, 1 mmol/l Mg2+, and 20°C). The compositions of the calibration solutions are shown in Table 2.

Statistical analysis. Data are presented as means ± SE, and statistical analysis was performed using paired t-tests. Statistical significance was taken as P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of Cs+ on spontaneous SR Ca2+ release in skinned cells. Figure 1A shows the effect of replacing control (K+ based) solution with Cs+-based solution on spontaneous Ca2+ release in a skinned cardiac myocyte, showing that Cs+ markedly slowed the frequency of spontaneous release and that this effect was reversible on returning to the control solution. Cs+ also significantly decreased the amplitude of spontaneous Ca2+ release, but the decrease in frequency was greater than the decrease in amplitude: frequency decreased from 15.81 ± 0.89 min-1 in the K+ solution to 7.45 ± 0.41 min-1 in the Cs+ solution (P < 0.001, n = 16), and amplitude decreased from 0.134 ± 0.004 fura 2 ratio units in the K+ solution to 0.124 ± 0.004 fura 2 ratio units in the Cs+ solution (P < 0.05, n = 16). Figure 1B shows faster time-base records of spontaneous Ca2+ release (left) and caffeine-induced Ca2+ release (right) in a skinned cardiac myocyte perfused with control (K+ based) solution. Figure 1C shows records from the same cell perfused with the Cs+-based solution, showing that Cs+ also appeared to prolong the duration of spontaneous Ca2+ release. This prolongation is shown more clearly in Fig. 2A, which shows normalized and superimposed records of spontaneous release monitored in the K+ solution and the Cs+ solution. The time course of spontaneous Ca2+ release was assessed using the time taken for Ca2+ to increase from 25% of its peak value to its peak value (TP75) and the decay time from its peak to 25% of peak value (DT75). Figure 2B shows that Cs+ significantly prolonged both the rise time and decay time of spontaneous Ca2+ release: TP75 increased from 0.277 ± 0.012 s in K+ solution to 0.374 ± 0.016 s in Cs+ solution (P < 0.001, n = 16), and DT75 increased from 0.579 ± 0.024 s in the K+ solution to 0.649 ± 0.026 s in the Cs+ solution (P < 0.01, n = 16). These data suggest, therefore, that Cs+ has marked effects on SR function, prolonging the time course and decreasing the frequency of spontaneous Ca2+ release. One possible explanation for these effects is that Cs+ is slowing Ca2+ flux across the SR membrane. This could prolong the time course of each spontaneous Ca2+ release. In addition, if luminal Ca2+ is an important determinant of the frequency of such spontaneous release, this could also decrease frequency (see DISCUSSION), because it would take longer for the critical luminal Ca2+ level to be reached.


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  		Fig. 1.   Effects of Cs+ on spontaneous and caffeine-induced Ca2+ release from a skinned cardiac myocyte. A: representative slow time-base chart recording of fura 2 fluorescence from a skinned rat ventricular myocyte showing the effect on spontaneous Ca2+ release of changing from a K+-based solution to a Cs+-based solution and back, as indicated above the recording. B and C: typical faster time-base recordings of spontaneous Ca2+ release (left) and caffeine-induced Ca2+ release (right) from a representative myocyte in K+- and Cs+-based solutions, respectively. Caffeine solution was applied at the time indicated by arrows. [Ca2+], Ca2+ concentration.


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  		Fig. 2.   Effects of Cs+ on time course of spontaneous Ca2+ release. Averaged (n = 8), superimposed, and normalized fast time-base recordings of spontaneous Ca2+ release in presence of K+ and Cs+ (A) and time courses of such spontaneous increases of [Ca2+] (B). Time course was assessed using time from 25% peak value to peak (TP75, open circle ) and decay time from peak to 25% of peak value (DT75, ). Data are means ± SE; n = 16. ** P < 0.01, *** P < 0.001 compared with control.

To test whether these changes were graded with Cs+ concentration ([Cs+]), we investigated the effect of replacing only 50% of the K+ in the control solution with Cs+. Figure 3 shows data from another group of cells, showing that these changes were graded with [Cs+] and that similar changes were produced using a solution containing 50% Cs+-50% K+, although they were less marked than the changes observed with complete replacement.


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  		Fig. 3.   Effects of graded increases of Cs+ on spontaneous Ca2+ release. Effects of Cs+ (50 and 100% replacement of K+ with Cs+) on frequency (A), amplitude (B), and time course (C) of spontaneous Ca2+ release. Time course was assessed using TP75 (open circle ) and DT75 (). Data are means ± SE; n = 10. NS, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control.

Effects of Cs+ on caffeine-induced Ca2+ release in skinned cells. Because the experiments describing the Effects of Cs+ on spontaneous SR Ca2+ release in skinned cells suggested that Cs+ alters SR function, caffeine was used to investigate further the role of the SR in the response to Cs+.

Figure 1, B and C (right), shows caffeine-induced Ca2+ release in the presence of K+ and Cs+. In the presence of Cs+, the SR Ca2+ content, assessed using caffeine, was slightly decreased. The amplitude of the caffeine-induced release was 0.173 ± 0.010 fura 2 ratio units in the K+ solution and 0.152 ± 0.007 fura 2 ratio units in the Cs+ solution (P < 0.01, n = 10, Fig. 4A). Thus it appears that Cs+ slightly decreases the apparent SR Ca2+ content. However, it has previously been shown (7) that the Ca2+ content of the SR assessed in this way depends on the time since the previous (spontaneous) Ca2+ release: as the interval from the preceding release is prolonged, the SR Ca2+ content increases. Because the caffeine-induced release could not be synchronized to the spontaneous release, the time between the last spontaneous release before the caffeine-induced release and the caffeine-induced release itself was measured in the K+ and Cs+ solutions. Figure 4B shows that this loading time was significantly longer in the presence of Cs+, presumably because of the decrease in the rate of spontaneous release: the loading time increased from 1.305 ± 0.148 to 3.082 ± 0.360 s (P < 0.01, n = 10). This would be expected to increase SR Ca2+ content. The observation that the SR Ca2+ load was decreased in the presence of Cs+ (Fig. 4A) is compatible with the idea that Cs+ slows the rate of Ca2+ uptake into the SR, thus decreasing SR content.


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  		Fig. 4.   Effects of Cs+ on caffeine-induced Ca2+ release. A: amplitude of caffeine-induced Ca2+ release in presence of K+ or Cs+. B: time between last spontaneous release and application of caffeine (loading time, open circle ) and time between recovery of [Ca2+] after application of caffeine and first spontaneous release (recovery time, ). Data are means ± SE; n = 10. * P < 0.05, ** P < 0.01 compared with control.

Figure 4B also shows that the time between the caffeine-induced release and the first subsequent spontaneous release was significantly prolonged from 7.555 ± 1.585 s in K+ to 14.355 ± 1.075 s in the presence of Cs+ (P < 0.05, n = 10). This is also consistent with the hypothesis that Cs+ slows Ca2+ uptake by the SR so that a longer time is required following the application of caffeine for the SR Ca2+ content to increase sufficiently to generate a spontaneous Ca2+ release.

Effects of CPA on skinned cells. This series of experiments was designed to test the hypothesis that the effects of Cs+ could be due, at least in part, to slowed Ca2+ uptake by the SR. If this hypothesis is correct, it might be expected that the effects of Cs+ could be mimicked by drugs such as CPA that inhibit SR Ca2+ uptake by inhibiting the SR Ca2+ ATPase.

Figure 5 shows original chart records of spontaneous Ca2+ release in a skinned myocyte in control solution (A) and in the presence of 10 µmol/l (B) and 50 µmol/l (C) CPA. These traces show that CPA decreased the frequency of spontaneous Ca2+ release. Figure 5D shows superimposed normalized records of spontaneous Ca2+ releases obtained in control conditions and in the presence of CPA, showing that this inhibitor also slowed the declining phase of the spontaneous Ca2+ release. Figure 6 shows mean data: CPA significantly decreased the frequency of spontaneous Ca2+ release from 10.27 ± 1.75 min-1 in control to 2.41 ± 0.33 and 0.68 ± 0.06 min-1 in 10 and 50 µmol/l CPA, respectively (P < 0.01 and P < 0.001, respectively, n = 8). In addition, the amplitude of spontaneous Ca2+ release was slightly decreased to 89.8 ± 3.6% of control in 10 µmol/l CPA and to 93.8 ± 3.5% in 50 µmol/l CPA (both P < 0.05, n = 8; Fig. 6B). CPA also prolonged the decay time of the spontaneous Ca2+ release (DT75 in Fig. 6C) from 0.655 ± 0.045 to 1.307 ± 0.077 s in 10 µmol/l CPA and to 3.037 ± 0.295 s in 50 µmol/l CPA, suggesting that the Ca2+ uptake rate of the SR was reduced. The rise time of Ca2+ release (TP75 in Fig. 6C) was also slightly prolonged by CPA: TP75 increased from 0.325 ± 0.03 s in control to 0.483 ± 0.04 s in 10 µmol/l CPA and to 0.506 ± 0.03 s in 50 µmol/l CPA (P < 0.01 and P < 0.001, respectively, n = 8). Thus the effect of CPA on skinned cells was to decrease both the frequency and amplitude of spontaneous Ca2+ release and to prolong both the rise time and decay time of such release, although the prolongation was more pronounced for the decay than for the rise time. These effects, therefore, are similar to those produced by Cs+.


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  		Fig. 5.   Effects of cyclopiazonic acid (CPA) on spontaneous Ca2+ release. Representative chart recordings of spontaneous Ca2+ release in a skinned rat ventricular myocyte in control conditions (A) and in presence of 10 (B) and 50 (C) µmol/l CPA. D: superimposed normalized fast time-base recordings of spontaneous Ca2+ release in control conditions and in presence of 10 and 50 µmol/l CPA.


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  		Fig. 6.   Mean effects of CPA on spontaneous Ca2+ release. Effects of 10 and 50 µmol/l CPA on frequency (A), amplitude (B), and time course (C) of spontaneous Ca2+ release. Time course was assessed using TP75 (open circle ) and DT75 (). Data are means ± SE; n = 8. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control.

Effects of Cs+ on intact cells. To test whether Cs+ could also alter Ca2+ release in intact myocytes, the effects of extracellular Cs+ were investigated in field-stimulated and voltage-clamped ventricular myocytes. Figure 7A shows a typical slow time-base fluorescence record from an intact cell that was being field stimulated before, during, and after the addition of 20 mmol/l CsCl to the bathing solution. Cs+ caused a reversible increase in both systolic and diastolic [Ca2+]i (Fig. 7A). Figure 7, B and C, shows averages of 15 individual Ca2+ transients recorded in each solution. Surprisingly, in the intact cells the amplitude of the Ca2+ transient (Fig. 7, A-C) and caffeine-induced Ca2+ release (Fig. 7A) were both slightly increased in the presence of Cs+. The Ca2+ transient was 109.0 ± 3.4% of control (P < 0.05, n = 11), and the caffeine-induced Ca2+ release was 109.5 ± 2.7% of control (P < 0.01, n = 11). The rise and decay times of the Ca2+ transient were not significantly affected by Cs+ (n = 11; see Fig. 7B for normalized averages of Ca2+ transients). Thus the effects of Cs+ on the intact cell are different from those expected for inhibition of the SR. These results suggest that Cs+ has additional effects in the intact cell that overcome the inhibitory effects on the SR. To test whether these additional effects could be due to changes in the configuration of the action potential, 20 mmol/l extracellular Cs+ was added to cells in which the duration and amplitude of depolarization were kept constant by voltage clamp using the perforated patch-clamp technique (see MATERIALS AND METHODS for details).


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  		Fig. 7.   Effects of Cs+ on the Ca2+ transient and caffeine-induced Ca2+ release in field-stimulated intact cells. A: slow time-base chart recording showing Ca2+ transients and caffeine-induced Ca2+ release (indicated by arrows) in control and Cs+-containing (20 mmol/l) solutions in a representative myocyte. B: superimposed Ca2+ transients recorded in control solution and in presence of Cs+. C: representative averaged (n = 15) Ca2+ transients from A before, during, and after exposure to Cs+.

Figure 8, A and B, shows original chart recordings of membrane current (top) and Ca2+ transients (bottom) from a representative cell in which membrane potential was controlled before and during the application of Cs+. Under these conditions Cs+ had no significant effect on the amplitude of ICa or the Ca2+ transient but caused an inward shift of holding current that is compatible with inhibition of outward current. The amplitude of the Ca2+ transient was 1.50 ± 0.53 and 1.56 ± 0.16 fura 2 ratio units in the absence and presence of 20 mmol/l Cs+, respectively [n = 6, not significant (NS)]. Figure 8C shows representative fast time-base recordings of averaged ICa and Ca2+ transients in the absence and presence of Cs+, showing that Cs+ also had no significant effect on the time course of these variables. The time to peak of the Ca2+ transient was 55.7 ± 4.7 ms during control and 54.6 ± 5.3 ms in Cs+ (NS). The decay of the Ca2+ transient was also unaffected by Cs+. These data suggest that the changes in the Ca2+ transient produced by Cs+ in field-stimulated cells were due to changes in the resting potential and/or action potential.


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  		Fig. 8.   Effects of Cs+ on Ca2+ transient and Ca2+ current in isolated ventricular myocytes during voltage clamp. A and B: slow time-base chart recordings of membrane current (Im; top) and Ca2+ transients (bottom) are shown in control solution and in presence of 20 mmol/l Cs+, respectively. C: fast time-base averaged records of Im (top) and Ca2+ transient (bottom) in absence and presence of Cs+.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Spontaneous Ca2+ release from SR. The present paper describes a method that enables Ca2+ release from the SR to be monitored in a single skinned myocyte. This method has been used to investigate the effect of Cs+ on spontaneous Ca2+ release and SR Ca2+ content, assessed using caffeine. Previous studies of SR Ca2+ release have used SR Ca2+ channels incorporated in planar phospholipid bilayers (see, e.g., Ref. 26), mechanically skinned single cells (10), and chemically skinned (29) or intact (24) multicellular preparations of cardiac muscle. The present technique has the advantage that it enables the function of the SR to be monitored in situ while the composition of the solution bathing the SR in a single cell is controlled, thus overcoming the problems of an unknown solution composition around the SR, inhomogeneity between cells, and diffusion delays, but without the technical difficulties associated with mechanical skinning. Although it is possible that SR function is altered by skinning, the solutions used were designed to mimic the normal intracellular environment, and previous studies using such cells have provided useful information about SR function. In addition, the data obtained in the present study are consistent with previous observations of the effects of Cs+ on intact cells (see Inhibitory effects of Cs+ on spontaneous Ca2+ release), supporting the idea that the SR of the skinned cells is responding to Cs+ in the same way as in intact cells.

The amplitude of the rise of [Ca2+] observed during SR Ca2+ release in a skinned cell will depend on the volume of solution into which the Ca2+ is released and the rate of flow of this solution; for example, increasing perfusion rate will truncate the observed rise of Ca2+. This makes absolute calibration difficult. However, because these variables will be constant in a particular cell, it is possible to study changes in [Ca2+]. Diffusion and washout of Ca2+ following release will also influence the rate of rise and decline of the observed Ca2+ release. An increase in the rate of solution flow, for example, will result in a more rapid decline of [Ca2+] as Ca2+ is washed out. Thus the observed rate of decline of Ca2+, which is due to both resequestration of Ca2+ by the SR (see below) and washout by the perfusing solution, is the fastest decline of Ca2+ that could be attributed to SR-pump activity alone. However, the variables that will affect diffusion and washout of Ca2+ will remain constant in a particular cell. Thus it seems likely that changes in time course will reflect changes in cell function rather than technical artifacts. The observation that the SR Ca2+ ATPase inhibitor CPA slows the rate of decline of spontaneous Ca2+ release (Fig. 6) is also consistent with the suggestion that changes in SR function can influence the time course of the observed changes in [Ca2+].

The characteristics of the spontaneous Ca2+ release observed in this preparation are consistent with those reported previously for spontaneous Ca2+ release from the SR of cardiac cells. The fraction of the SR Ca2+ content released during each spontaneous release was estimated by comparing the area of a spontaneous release with that of a caffeine-induced release. This gave a value of 14.5 ± 2.1%, similar to the value of 13.6% reported by Díaz et al. (6) for the intact cell. In addition, the frequency of spontaneous release increased as the Ca2+ in the bathing solution increased: frequency was 5.78 ± 0.63 min-1 with 100 nmol/l [Ca2+], 8.83 ± 0.69 min-1 with 140 nmol/l [Ca2+], 13.25 ± 1.04 min-1 with 180 nmol/l [Ca2+], and 17.79 ± 1.60 min-1 with 240 nmol/l [Ca2+] (each P < 0.05, n = 10). These values are within the range reported previously in intact preparations (3, 4, 6, 10).

It has long been known that increasing the [Ca2+] around the SR of skinned cardiac cells leads to cyclic Ca2+ release from the SR (10) and that the frequency of such release increases with bathing [Ca2+] (10). It is less clear whether such spontaneous release depends on the [Ca2+] around the SR triggering Ca2+-induced Ca2+ release or on the [Ca2+] within the lumen of the SR (7, 25). However, because Ca2+-induced Ca2+ release and spontaneous Ca2+ release have different characteristics, and because the latter can be observed in conditions that inhibit the former (9), it has been suggested that spontaneous Ca2+ release is not due to Ca2+-induced Ca2+ release (7, 9). The present data support this suggestion. First, because the frequency of spontaneous release decreases in the presence of Cs+, even though bathing [Ca2+] is constant, it is suggested that some factor other than bathing [Ca2+] determines the frequency of release. Second, because the amplitude of release is relatively constant, it is suggested that luminal [Ca2+] has to reach a certain critical threshold before Ca2+ release occurs (7). Recent reports that an increase in the [Ca2+] at the luminal face of the ryanodine receptor increases the open probability of this channel (19) suggest a possible mechanism whereby an increase in luminal [Ca2+] leads to spontaneous Ca2+ release. It is possible that the released Ca2+ then acts on the cytoplasmic face of the ryanodine receptor to increase open probability further and trigger further release, leading to a rapid and large Ca2+ release before stochastic attrition results in closure of the release channels.

Inhibitory effect of Cs+ on spontaneous Ca2+ release. It has previously been reported that intracellular Cs+ reduces Ca2+ transient amplitude by ~40-50% in guinea pig myocytes (12, 15) and reduces "phasic" contraction amplitude by 40-45% in rabbit myocytes (18). In the present study, replacement of K+ in the bathing solution by Cs+ decreased both the frequency and amplitude of spontaneous Ca2+ release and prolonged the rise time and decay time. The SR Ca2+ content, estimated by the application of caffeine, decreased slightly, and the time before the next spontaneous Ca2+ release (recovery time) was prolonged in the Cs+ solution. Similar but less marked effects were observed when only 50% of K+ was replaced with Cs+.

Because the bathing [Ca2+] was constant, it appears likely that changes in luminal, rather than "cytoplasmic," [Ca2+] underlie the observed changes in spontaneous Ca2+ release in the presence of Cs+ (see Spontaneous Ca2+ release from SR). One possible mechanism whereby Cs+ could alter luminal Ca2+ is by slowing the rate of Ca2+ flux across the SR membrane; this could explain the present data as follows. 1) Slower Ca2+ uptake by the SR would prolong the time required for luminal [Ca2+] to reach a critical level required for spontaneous Ca2+ release to occur, thus slowing the frequency of spontaneous Ca2+ release, with little change in amplitude (Fig. 1). 2) Slower SR Ca2+ uptake would also be expected to decrease SR Ca2+ content, assessed using caffeine (Fig. 4). The observed decrease would presumably have been greater if the loading time had not also been increased in the presence of Cs+ (Fig. 4; see RESULTS). 3) Slowed SR Ca2+ uptake would also account for the increased time between depletion of the SR using caffeine and the first subsequent spontaneous Ca2+ release (Fig. 4), because the SR would take longer to refill sufficiently to generate another spontaneous release. 4) Slower movement of Ca2+ across the SR membrane could also account for the slower rise and fall of the spontaneous Ca2+ release (Fig. 2).

In support of this idea, the SR Ca2+ ATPase inhibitor CPA mimicked many of the effects of Cs+, decreasing the frequency of spontaneous release with little change in amplitude and slowing the time course of the spontaneous Ca2+ release (Figs. 5 and 6). Although our hypothesis could explain why CPA might be expected to decrease the frequency of spontaneous release with little effect on amplitude (by increasing the time required for luminal Ca2+ to reach the critical level required for spontaneous release to occur) and to prolong the declining phase of the spontaneous Ca2+ release (by slowing reuptake into the SR), it is less clear why CPA should prolong the rising phase of the Ca2+ release. It is unlikely that this change is caused by a change in SR Ca2+ content, because the amplitude of the Ca2+ release is almost the same as in the absence of CPA. This suggests, therefore, that Ca2+ uptake occurring during the release phase can alter the time course of release. However, it appears unlikely that the effects of Cs+ on the rising phase of the Ca2+ release can be completely explained by this mechanism, because Cs+ has smaller effects than CPA on the frequency and rate of decline of spontaneous Ca2+ releases (suggesting a smaller effect on rate of Ca2+ uptake) but a larger effect than CPA on the rise of the spontaneous Ca2+ release (compare Figs. 3 and 6). This suggests, therefore, that Cs+ has effects on Ca2+ release in addition to its effects on Ca2+ uptake.

The hypothesis that Cs+ slows Ca2+ uptake and release by the SR could also explain earlier observations that, in cells dialyzed with Cs+-containing solutions, the size of the Ca2+ transient and, hence, contraction, appears to decrease: slower SR Ca2+ uptake would tend to decrease the amount of Ca2+ sequestered by the SR and, hence, SR Ca2+ content so that during regular stimulation the amount of Ca2+ available for release in response to a stimulus would be decreased. Thus the amount of Ca2+ released and, hence, the size of the Ca2+ transient would be decreased.

Unexpectedly, however, in field-stimulated intact cells, Cs+ increased the amplitude of the Ca2+ transient. There are a number of possible explanations for this result. First, Cs+ was applied at a relatively low concentration and extracellularly. Thus it is not clear that sufficient Cs+ entered the cell to inhibit SR function significantly. Second, K+-channel blockade by Cs+ would be expected to prolong the action potential and, hence, increase the Ca2+ content of the cell, which might offset the inhibitory effects of Cs+ on the SR. Finally, the inhibition of outward current by Cs+ observed in voltage-clamped cells (Fig. 8) would cause depolarization of the resting membrane potential in field-stimulated cells. This could be expected to produce an increase in resting [Ca2+], as observed in intact cells in the presence of Cs+ (Fig. 7), that would increase the Ca2+ available for uptake by the SR Ca2+ ATPase and, hence, increase the SR Ca2+ content (11). This would offset the direct inhibitory effects of Cs+ on the SR. In support of the hypothesis that changes in the resting potential and/or action potential underlie the observed changes in the Ca2+ transient in the intact cell the data in Fig. 8 show that such changes were not observed when the resting potential and the duration and amplitude of depolarization were kept constant. Because ICa is not altered by Cs+ (Fig. 8), it is unlikely that changes in ICa modulate the Ca2+ transient during exposure to Cs+. These data, therefore, are consistent with the idea that the addition of 20 mmol/l Cs+ to the bathing solution alters the resting and/or action potential and that it is these changes that alter the Ca2+ transient, possibly because under these conditions, unlike those in the skinned or dialyzed cell, intracellular Cs+ does not increase sufficiently to inhibit SR function significantly.

Possible mechanisms for action of Cs+ on SR. Cs+ is well known as an inhibitor of K+ channels (see introduction), which are also present within the SR membrane. Although the role of these channels is unknown, it has been suggested that K+ moves in the opposite direction to Ca2+ across the SR membrane during uptake and release of Ca2+ by the SR to avoid the development of large charge imbalances that could inhibit Ca2+ movement. The present data are consistent with this suggestion. The blockade of the SR K+ channels by Cs+ (27) may result in the development of a potential across the SR membrane during Ca2+ uptake or release that would tend to inhibit further Ca2+ movement.

Although other ions may also cross the SR membrane to help compensate for the charge movement associated with Ca2+ movement, the blockade of K+ flux will slow the rate at which charge movement can be compensated, thus slowing the rate of Ca2+ movement.

In summary, the present data suggest that Cs+ slows the flux of Ca2+ across the SR membrane, possibly by blocking the SR K+ channel, thus inhibiting compensation for the charge movement associated with Ca2+ uptake and release. In the intact cell this could decrease the Ca2+ content of the SR and, hence, the amount of Ca2+ released in response to stimulation. Although changes in the electrical activity of the cell may help to compensate for the direct inhibitory effects of Cs+ on the SR in the intact cell, it seems likely that the high concentrations of intracellular Cs+ achieved during whole cell clamp and the subsequent dialysis of the cytoplasm can inhibit the SR sufficiently to decrease SR Ca2+ content and, hence, the size of the Ca2+ transient (12, 15). This may have important consequences for studies of E-C coupling in which Cs+ has been used to block sarcolemmal K+ currents. The present data also suggest that SR luminal [Ca2+] is an important determinant of spontaneous Ca2+ release and that such release occurs when luminal [Ca2+] reaches a critical level (7).

    ACKNOWLEDGEMENTS

We thank Dr. Derek Steele and Dr. Simon Harrison for useful discussion.

    FOOTNOTES

This work was supported by the Japan Heart Foundation and the British Heart Foundation.

Address for reprint requests: M. Kawai, Dept. of Physiol., Univ. of Leeds, Leeds LS2 9NQ, UK.

Received 4 December 1997; accepted in final form 30 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(2):H422-H430
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