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(Circulation Research. 2001;88:195.)
© 2001 American Heart Association, Inc.

Cellular Biology

Coordinated Control of Cell Ca²⁺ Loading and Triggered Release From the Sarcoplasmic Reticulum Underlies the Rapid Inotropic Response to Increased L-Type Ca²⁺ Current

A. W. Trafford, M. E. Díaz, D. A. Eisner

From the Unit of Cardiac Physiology, University of Manchester, Manchester, UK.

Correspondence to A.W. Trafford, Unit of Cardiac Physiology, University of Manchester, 1.524 Stopford Bldg, Oxford Rd, Manchester M13 9PT, UK. E-mail trafford{at}man.ac.uk

	Abstract

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Abstract—The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca²⁺ content and systolic Ca²⁺ are controlled when Ca²⁺ entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca²⁺ concentration ([Ca²⁺]_i). Increasing external Ca²⁺ concentration ([Ca²⁺]_o) from 1 to 2 mmol/L increased the amplitude of the systolic Ca²⁺ transient with no effect on SR Ca²⁺ content. This constancy of SR content is shown to result because the larger Ca²⁺ transient activates a larger Ca²⁺ efflux from the cell that balances the increased influx. Decreasing [Ca²⁺]_o to 0.2 mmol/L decreased systolic Ca²⁺ but produced a small increase of SR Ca²⁺ content. This increase of SR Ca²⁺ content is due to a decreased release of Ca²⁺ from the SR resulting in decreased loss of Ca²⁺ from the cell. An increase of [Ca²⁺]_o has two effects: (1) increasing the fraction of SR Ca²⁺ content, which is released on depolarization and (2) increasing Ca²⁺ entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca²⁺ transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca²⁺]_o than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca²⁺ entry and greater fractional release of Ca²⁺ is an important factor in regulating excitation-contraction coupling.

Key Words: calcium • sarcoplasmic reticulum • trigger

	Introduction

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It is now generally accepted that Ca²⁺-induced Ca²⁺ release (CICR) is the major mechanism involved in the release of Ca²⁺ from the sarcoplasmic reticulum (SR) and hence in the activation of cardiac contraction (for reviews, see Bers¹ and Wier and Balke² ). The entry of Ca²⁺ ions into the cell via the L-type Ca²⁺ current produces a local increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) that leads to the opening of the SR Ca²⁺ release channels (ryanodine receptors, RyRs) and Ca²⁺ release from the SR. In this scheme, in addition to its role in triggering Ca²⁺ release, the L-type Ca²⁺ current has another important function in excitation-contraction coupling³ : it loads the cell with Ca²⁺ thereby balancing the efflux of Ca²⁺ from the cell produced by the systolic Ca²⁺ transient. It is well-known that maneuvers which increase the Ca²⁺ entry via the L-type Ca²⁺ current increase the amplitude of the systolic increase of [Ca²⁺]_i and the force of contraction of cardiac muscle.^{4 5 6} This will contribute to the inotropic effects of, for example, catecholamines and calcium channel agonists. The question addressed in this study is how these two roles of the Ca²⁺ current (trigger and loading) contribute to this positive inotropy. Both experimental studies and modeling have suggested that increasing the trigger effect alone would result in a purely transient increase of systolic Ca²⁺. This is because the enhanced release of Ca²⁺ from the SR will increase efflux from the cell and thus decrease the SR content.^{7 8} In contrast, increasing only the loading effect would be expected to increase both SR content and systolic Ca²⁺.⁹

Previous work has investigated the effects of increasing external Ca²⁺ concentration ([Ca²⁺]_o) on the trigger effect alone by holding SR Ca²⁺ content constant. Increasing [Ca²⁺]_o was shown to increase the fraction of SR Ca²⁺ released.^{10 11} Other studies have shown that an increase of SR Ca²⁺ content at constant [Ca²⁺]_o increases the fraction of Ca²⁺ released from the SR.^{11 12 13 14 15} There have, however, been no measurements of SR Ca²⁺ content and systolic Ca²⁺ when [Ca²⁺]_o is changed and SR Ca²⁺ content is allowed to vary in response to the changed Ca²⁺ entry. In particular, there is no information as to how the triggering and loading effects of the L-type Ca²⁺ current are coordinated. In this study, we have examined the effects of increasing the Ca²⁺ current by increasing [Ca²⁺]_o. The results show that the triggering and loading effects are balanced such that changes of [Ca²⁺]_o result in very rapid changes of systolic Ca²⁺ accompanied by little change of SR Ca²⁺ content, suggesting that this concerted regulation of trigger and release is essential for the control of contractility.

	Materials and Methods

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Experiments were performed at room temperature (23°C) on single rat and ferret ventricular myocytes (animals killed by stunning and cervical dislocation or pentobarbitone overdose, respectively) as described previously.^{7 13} All animal procedures were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were purchased from Harlan Ltd (UK) and Leeds University (UK). [Ca²⁺]_i was measured using the fluorescent indicator fluo-3 loaded as the acetoxymethyl ester and calibrated as described previously.^{7 16}

Measurement of Sarcolemmal Ca²⁺ Fluxes and SR Ca²⁺ Content
Voltage-clamp control was achieved using the perforated patch technique with amphotericin B and the switch-clamp facility of the Axoclamp 2B amplifier (Axon Instruments) as previously described.⁷ Micropipettes (<2.5 M) contained (mmol/L) KCH₃O₃S 125, KCl 20, NaCl 10, HEPES 10, and MgCl₂ 5 and titrated to pH 7.2 with KOH containing 240 µg/mL amphotericin B. To reduce interference from outward currents, all experiments were performed in the presence of 5 mmol/L 4-aminopyridine and 0.1 mmol/L BaCl₂. With the exception of Figure 2 (action potential clamp), the membrane potential was held at -40 mV and 100-ms-long pulses to 0 mV were applied at 0.5 Hz. Cells were bathed with a modified Tyrode solution containing (mmol/L) NaCl 135, KCl 4, HEPES 10, glucose 10, and MgCl₂ 1 and titrated to pH 7.4 with NaOH. The Ca²⁺ concentration in the superfusate was varied between 0.2 and 2 mmol/L as indicated in the figures.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of [Ca2+]o on systolic and SR Ca2+ in ferret cells. A, Time course. [Ca2+]o was altered as indicated in the figure. B, Action potential clamp action potentials and Ca2+ transients recorded in the [Ca2+]o indicated. C, Dependence of SR Ca2+ content and amplitude of the systolic Ca2+ transient on [Ca2+]o (n=11 cells). D, Data from another cell showing evidence of Ca2+ overload in 0.2 mmol/L [Ca2+]o. The right-hand panel is expanded from the period indicated by the horizontal bar. The cell was stimulated with the action potential waveform at the time indicated by the arrow.

Sarcolemmal Ca²⁺ fluxes were calculated as described previously.^{7 13} SR Ca²⁺ content was measured by applying 10 mmol/L caffeine to the cell to discharge the SR store and then integrating the resulting Na⁺-Ca²⁺ exchange current.¹⁷ The Na⁺-Ca²⁺ exchange current measured either on repolarization or during a caffeine application had to be corrected for Ca²⁺ removal by nonelectrogenic pathways, ie, the sarcolemmal Ca²⁺-ATPase. The value of this correction factor (1.5 in rat and ferret) was obtained as described previously.^{13 17}

All sarcolemmal Ca²⁺ fluxes and measurements of SR Ca²⁺ content are expressed relative to total cell volume. Cell volume was calculated from membrane capacitance measurements assuming a cell volume:capacitance ratio of 6.76 (rat) or 5.39 (ferret) pF/pL.¹⁸

Measurement of Total [Ca] and Time Course of SR Ca²⁺
Changes of [Ca²⁺]_i were converted to changes of total [Ca²⁺] using the Ca²⁺ buffering properties of the cell obtained from the caffeine applications as described previously.^{7 16} The time course of change of SR Ca²⁺ was obtained as follows: change of SR Ca²⁺=net Ca²⁺ entry into the cell-change of total cytoplasmic Ca²⁺.¹⁶

Statistics
All measurements are presented as the mean±SEM for n cells. Tests for significance were performed using paired Student’s t tests.

	Results

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The experiment in Figure 1A shows the effects of changing [Ca²⁺]_o on systolic [Ca²⁺]_i in a rat myocyte. Decreasing [Ca²⁺]_o from 1 to 0.2 mmol/L produced a large decrease of systolic [Ca²⁺]_i, which was followed by a small and slow recovery. Increasing [Ca²⁺]_o to 2 mmol/L resulted in an increase of the amplitude of the systolic Ca²⁺ transient followed by a decay to a level still greater than that in 1 mmol/L. The mean steady-state levels of systolic Ca²⁺ are shown in Figure 1C. On average, the amplitude of the Ca²⁺ transient was (13 cells) 76±10, 476±80, and 816±140 nmol/L in, respectively, 0.2, 1, and 2 mmol/L [Ca²⁺]_o. In other words, the amplitude of the Ca²⁺ transient was roughly proportional to [Ca²⁺]_o. This experiment also provides data about the SR Ca²⁺ content. This was investigated by adding caffeine (10 mmol/L) to release SR Ca²⁺. A qualitative estimate of SR Ca²⁺ content is provided by the resulting increase of [Ca²⁺]_i. This shows that the caffeine-evoked increase of [Ca²⁺]_i was slightly greater in 0.2 mmol/L than in 1 or 2 mmol/L Ca²⁺. This is confirmed by the quantitative measurement obtained from the integral of the Na⁺-Ca²⁺ exchange current (Figure 1B). On average, as shown in Figure 1C (13 cells), the SR Ca²⁺ content is the same (P>0.4) in 1 mmol/L (64±6 µmol/L) and 2 mmol/L (66±5 µmol/L) Ca²⁺ but is greater in 0.2 mmol/L (79±7 µmol/L, P<0.0005 versus 1 and 2 mol/L [Ca²⁺]_o).

View larger version (25K): [in this window] [in a new window]   Figure 1. Effects of elevation of [Ca2+]o on systolic Ca2+ and SR Ca2+ content in a rat ventricular myocyte. A, Time course of [Ca2+]i. Depolarizing pulses (100 ms) were applied at 0.5 Hz from a holding potential of -40 to 0 mV. [Ca2+]o was changed as indicated in the figure (in mmol/L). B, SR Ca2+ content. Traces show (from top to bottom): [Ca2+]i, membrane current, and integral of current (a measure of SR Ca2+ content). Caffeine (10 mmol/L) was applied as indicated above the bars. Records were obtained from the same cell as used for panel A after 2 minutes of stimulation in the Ca2+ concentrations indicated. C, Mean values for the amplitude of the Ca2+ transient and SR content. Error bars represent SEM for 13 experiments.

The experiment in Figure 1 was obtained from rat ventricular cells using brief, square voltage-clamp pulses. The use of such pulses is convenient as it facilitates measurement of Ca²⁺ influx during depolarization and efflux on repolarization. However, it leaves unanswered the question of what happens with the physiological action potential. It might also be argued that one cannot generalize from experiments on rat myocytes because this species has a very brief action potential. We have therefore also performed experiments on ferret ventricular myocytes stimulated to produce action potentials. To be able to use the voltage-clamp technique to measure SR Ca²⁺ content, we used the action potential clamp technique. The waveform of the action potential was obtained by depolarizing the cell with a brief (4-ms long) current pulse in 1 mmol/L [Ca²⁺]_o (solution not containing 4-aminopyrridine or BaCl₂). Figure 2 shows that decreasing [Ca²⁺]_o from 1 to 0.2 mmol/L again decreased the amplitude of the systolic Ca²⁺ transient followed by an increase on elevating [Ca²⁺]_o to 2 mmol/L. Specimen records of the steady-state Ca²⁺ transients are shown in Figure 2B. Figure 2C shows mean data for the SR Ca²⁺ contents and amplitude of the systolic Ca²⁺ transient plotted as a function of [Ca²⁺]_o (data from 11 cells). As was the case for the rat, increasing [Ca²⁺]_o from 0.2 to 1 to 2 mmol/L had a much larger fractional effect on the amplitude of the Ca²⁺ transient than on SR content. Specifically, elevating [Ca²⁺]_o from 1 to 2 mmol/L had no significant effect on SR content (52.0±2 versus 52.7±2 µmol/L, P>0.5) whereas decreasing it from 1 to 0.2 mmol/L produced an increase (61.3±3 µmol/L, P<0.001 versus 1 and 2 [Ca²⁺]_o). Figure 2D shows another indication that in this cell the SR Ca²⁺ content has increased. Reducing [Ca²⁺]_o from 2 to 0.2 mmol/L produced an abrupt decrease in [Ca²⁺]_i; however, after several pulses in low [Ca²⁺]_o, a delayed large Ca²⁺ release was triggered.

The results of experiments such as those illustrated in Figures 1 and 2 show that, over the range of [Ca²⁺]_o examined, a 10-fold increase of systolic Ca²⁺ is accompanied by very little change of SR Ca²⁺ content. At the extremes of the range, there is a 20% decrease of SR Ca²⁺ content. Therefore, in subsequent experiments, we addressed two questions. (1) What is the origin of the paradoxical increase of SR content on decreasing [Ca²⁺]_o? (2) How is it that, when increasing [Ca²⁺]_o, despite the increased amplitude of systolic Ca²⁺ fluxes, SR Ca²⁺ content alters very little? In Figure 3A, [Ca²⁺]_o was decreased from 2 to 0.2 mmol/L. The [Ca²⁺]_i record shows that the decrease of [Ca²⁺]_o decreased systolic [Ca²⁺]_i. On return to 2 mmol/L [Ca²⁺]_o, there was a marked overshoot in the amplitude of the systolic Ca²⁺ transient, again indicating a gain of Ca²⁺ by the SR during stimulation in low [Ca²⁺]_o. The specimen records of Figure 3B show the accompanying currents and Ca²⁺ fluxes. Trace a shows the L-type Ca²⁺ current during depolarization and the Na⁺-Ca²⁺ exchange current on repolarization. The record of calculated net sarcolemmal flux shows, in agreement with previous work,¹³ that Ca²⁺ influx and efflux were identical in magnitude. The bottom trace shows the calculated value of SR Ca²⁺ content during the systolic Ca²⁺ transient. The initial value of SR content was obtained previously after steady-state stimulation in 2 mmol/L [Ca²⁺]_o from the application of 10 mmol/L caffeine.⁷ The change of SR Ca²⁺ content during the pulse was calculated by adding the net Ca²⁺ movement to the SR content (see Materials and Methods). In the steady state in 2 mmol/L [Ca²⁺]_o (trace a), the SR content decreases from 92 to 40 µmol/L whereas in the steady state in 0.2 mmol/L [Ca²⁺]_o (trace b), the initial level of SR Ca²⁺ is greater but the calculated decrease is much smaller (103 to 96 µmol/L). Importantly, in both traces a and b, SR Ca²⁺ content has returned to its initial level by the end of the 1.5-second measurement period. Therefore, in the steady state, in 0.2 mmol/L [Ca²⁺]_o, the decrease of Ca²⁺ entry is compensated for by a decreased Ca²⁺ efflux. Trace c was obtained from the first stimulus on return to 2 mmol/L Ca²⁺ (after steady-state stimulation in 0.2 mmol/L [Ca²⁺]_o). The large Na⁺-Ca²⁺ exchange current on repolarization corresponds to an efflux of 10.2 µmol/L compared with an entry of 2.8 µmol/L, resulting in a net loss of Ca²⁺ from the cell and reduction of SR Ca²⁺ content as shown in trace c. The sarcolemmal Ca²⁺ fluxes for each pulse are shown in the middle panel of Figure 3A. Influx and efflux initially balance. On reduction of [Ca²⁺]_o, the efflux initially decreases by more than the influx, leading to a predicted gain of SR Ca²⁺ content (Figure 3A, bottom trace). This accompanies the partial recovery of the systolic Ca²⁺ transient. On restoration of 2 mmol/L [Ca²⁺]_o, the efflux is initially greater than the influx, resulting in a loss of Ca²⁺ that parallels the decay of systolic [Ca²⁺]_i. The Ca²⁺ fluxes can therefore account not only for the measured changes of SR Ca²⁺ content but, in addition, for the overshoot in systolic Ca²⁺ seen on raising [Ca²⁺]_o.

View larger version (26K): [in this window] [in a new window]   Figure 3. Ca2+ fluxes during alteration of [Ca2+]o concentration. A, Time course. [Ca2+]o was changed as indicated on the figure. Traces show (top to bottom): [Ca2+]i, Ca2+ entry (integral of the L-type Ca2+ current) and exit (calculated from the corrected [see Materials and Methods]) Na+-Ca2+ exchange tail current), and predicted change of SR Ca2+ content. B, Specimen records. Traces show (from top to bottom): [Ca2+]i, current, net sarcolemmal Ca2+ flux, and calculated SR Ca2+ content. Records were obtained at the times shown in panel A. Traces a and b are the mean of 5 stimuli, and trace c is from a single stimulus.

In related experiments, we have examined the effects of increasing [Ca²⁺]_o from 1 to 2 mmol/L. In the experiment illustrated in Figure 4, in agreement with the mean data, this produced no change of SR Ca²⁺ content (84.5 versus 85.6 µmol/L in 1 and 2 mmol/L [Ca²⁺]_o; not shown). Figure 4A shows that increasing [Ca²⁺]_o produces an increase of the amplitude of the systolic Ca²⁺ transient. As expected, this is also accompanied by an increase of the amplitude of the systolic increase of total Ca²⁺ (Figure 4B). Figure 4C shows measurements of sarcolemmal Ca²⁺ influx (the integral of the I_Ca-L) and efflux (the integral of I_Na-Ca, corrected for nonelectrogenic fluxes). These fluxes are in balance in 1 mmol/L [Ca²⁺]_o and increase equally in 2 mmol/L [Ca²⁺]_o. By summing Ca²⁺ influx and efflux, the change of cell or SR Ca²⁺ can be calculated (Figure 4D), and, as expected, from the equal changes in Ca²⁺ entry and efflux on raising [Ca²⁺]_o, no change of SR Ca²⁺ content is predicted. This agrees with the quantitative measures of SR Ca²⁺ obtained by applying 10 mmol/L caffeine. The bottom trace shows the fractional release of Ca²⁺ from the SR on each pulse. This was obtained by calculating the increase of total Ca²⁺ less the sarcolemmal Ca²⁺ entry and dividing this value by diastolic SR Ca²⁺ content. Again, this analysis shows an abrupt and sustained increase in fractional release on raising [Ca²⁺]_o.

View larger version (23K): [in this window] [in a new window]   Figure 4. Analysis of Ca2+ fluxes produced by elevation of [Ca2+]o. Traces show [Ca2+]i (A), time course of the change of total Ca2+ (B), calculated Ca2+ influx and efflux on each pulse (C), calculated SR content (D) and fraction of the SR Ca2+ content released on each beat (E).

The above work reveals that an increase of Ca²⁺ current produces a rapid increase of the amplitude of the systolic Ca²⁺ transient with either no or a very small effect on SR Ca²⁺ content. Thus, a combined increase of trigger and influx results in a rapid and largely maintained increase of systolic [Ca²⁺]_i. The experiment of Figure 5A compares this situation with the inotropic effects of only increasing SR Ca²⁺ content. In this experiment, the SR has been depleted by the addition of 10 mmol/L caffeine. On resumption of stimulation, there is a slow increase in the amplitude of the systolic Ca²⁺ transient, reflecting the time taken for the SR Ca²⁺ content to increase. This is emphasized by the traces in Figure 5B. In agreement with previous work,¹³ when the SR is empty, stimulation produces a large I_Ca-L and a small I_Na-Ca. As the amplitude of the systolic Ca²⁺ transient increases, these currents respectively decrease and increase until the Ca²⁺ fluxes through them are identical and the cell is therefore in Ca²⁺ flux balance.^{13 19} The bottom panel of Figure 5B shows the cumulative Ca²⁺ gain by the cell obtained by summing the Ca²⁺ entry and efflux on each pulse.^{7 13} It is clear that the amplitude of systolic Ca²⁺ increases along with that of SR content and that (Figure 5D) there is a very steep relationship between SR Ca²⁺ content and the amplitude of the systolic Ca²⁺ transient.¹¹ A comparison of the inotropic effect of simply increasing SR Ca²⁺ content or the trigger for Ca²⁺ release (I_Ca-L) is provided by the immediate and sustained (after a single potentiated beat) increase of [Ca²⁺]_i when [Ca²⁺]_o is increased from 0.2 to 2 mmol/L in the last part of the experimental record. Figure 5C shows Ca²⁺ efflux as a function of the amplitude of systolic [Ca²⁺]_i.

View larger version (24K): [in this window] [in a new window]   Figure 5. Comparison of the effects of SR refilling alone with those of changing [Ca2+]o. A, Time course. Before the record began, the cell had been stimulated. Stimulation was discontinued and caffeine (10 mmol/L) applied. After removing caffeine, stimulation was recommenced. [Ca2+]o was then lowered to 0.2 mmol/L and subsequently elevated to 2 mmol/L. B, Time course of change of the L-type Ca2+ current and Na+-Ca2+ exchange tail current (top) and calculated gain of Ca2+ by the cell (bottom). The traces have been temporally aligned with those in panel A. C, Relationship between Ca2+ efflux and amplitude of the systolic Ca2+ transient obtained from the period of SR refilling after caffeine removal. D, Relationship between SR Ca2+ content and amplitude of the systolic Ca2+ transient. The curve is a fit to the Equation (see text) Ca2+ transient=a+bx(SR)n, where the parameters have the values: a=0.0691, b=8.9x10–7, and n=3.3, with dimensions such that Ca2+ transient and SR are in µmol/L.

	Discussion

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The major finding of this study is that the increase of systolic [Ca²⁺]_i produced by elevating [Ca²⁺]_o does not result from an increase of SR Ca²⁺ content. Indeed, over a certain (10-fold) range of [Ca²⁺]_o, either no change or a decrease of SR Ca²⁺ content accompanies the increase of systolic [Ca²⁺]_i. Before discussing these results further, it is important to briefly consider the validity of the methods used. The SR Ca²⁺ content is measured from the integral of the caffeine-evoked Na⁺-Ca²⁺ exchange current.¹⁷ This involves correcting for the fraction of Ca²⁺, which is pumped out of the cell by the electroneutral Ca²⁺-ATPase. A concern might be that this fraction could change at different [Ca²⁺]_o. However, over a wide range of [Ca²⁺]_o (0 to 5 mmol/L) the fraction has been found to be constant.²⁰

The relative constancy of SR Ca²⁺ content in the face of changes of [Ca²⁺]_o can be explained as follows. An increase of the L-type Ca²⁺ current will have two effects on excitation-contraction coupling. (1) It will increase Ca²⁺ entry into the cell and thereby SR Ca²⁺ content. This will increase systolic release until a steady state is reached when the increased Ca²⁺ entry on each beat is balanced by increased efflux on Na⁺-Ca²⁺ exchange. (2) It will increase the trigger for SR Ca²⁺ release and the fraction of SR Ca²⁺ that is released. This latter effect alone is similar to that produced by low concentrations of caffeine^{7 8 21} and would be expected to produce a transient increase of systolic [Ca²⁺]_i and a maintained decrease of SR Ca²⁺ content (eg, Figure 6B).

View larger version (26K): [in this window] [in a new window]   Figure 6. Modeling of the effects of increasing Ca2+ entry without affecting the trigger for Ca2+ release from the SR. A, Time course. Top trace shows systolic Ca2+ transients. [Ca2+]o was elevated as shown. Filled symbols in the two bottom traces show the measured SR Ca2+ contents and amplitude of the systolic Ca2+ transients. The SR content was measured as shown in Figure 2. Open symbols show the values of these parameters, assuming that the increase of [Ca2+]o affects only Ca2+ influx and not the trigger for Ca2+ release. For the model (see text), the following parameters were used: a=0.077, b=1.41x10–4, n=2.02, and c=6.32. B, Comparison with the effects of applying caffeine (500 µmol/L) to a different cell.

The net direction of the change of SR Ca²⁺ content will depend on the balance between these two factors. The increased trigger will increase the amount of Ca²⁺ released from the SR and therefore the amount pumped out of the cell. If this extra amount pumped out exactly balances the increased influx on the L-type current, then an abrupt and maintained increase of systolic [Ca²⁺]_i with no change of SR Ca²⁺ content will be expected. That this can occur is demonstrated directly by Figure 4. For this balance to occur, all that is required is that a given increase of L-type Ca²⁺ current increases the fraction of the SR Ca²⁺ content, which is released and hence the Ca²⁺ efflux from the cell by the same fraction. The relationship between Ca²⁺ influx on the L-type channel and the resulting Ca²⁺ efflux from the cell due to SR release must be linear.

Decreasing [Ca²⁺]_o from 1 to 0.2 mmol/L increases SR Ca²⁺ content. The most likely explanation for this observation is that, over this range, a decrease of [Ca²⁺]_o has a fractionally larger effect on the amount of Ca²⁺ released from the SR than it does on the Ca²⁺ entry into the cell. Direct evidence of this is provided by Figure 3B where a decrease of [Ca²⁺]_o has a larger fractional effect on the amount of Ca²⁺ released from the SR than on the Ca²⁺ influx into the cell. It is also consistent with the transient nature of the changes of systolic [Ca²⁺]_i to increasing [Ca²⁺]_o from 0.2 to 2 mmol/L. On the first pulse, the stimulation of Ca²⁺ release results in a Ca²⁺ transient that produces a larger Ca²⁺ efflux than even the increased entry. The cell therefore loses Ca²⁺ until a new steady state is reached.

It is important to compare our work with previous studies on the effects of [Ca²⁺]_o on SR content. Two studies have used the magnitude of the rapid cooling contraction (RCC) as a measure of SR content. In rabbit ventricular muscle, one study found that decreasing [Ca²⁺]_o from 2 to 0.2 mmol/L decreased the twitch to 23±2% but the RCC to 90±8%.¹⁰ In another study, decreasing [Ca²⁺]_o from 2.5 to 0.5 mmol/L decreased the twitch to <50% but had no effect on the RCC.²² Even with the use of a qualitative measure, SR content changes by much less than the Ca²⁺ transient. Interestingly, one of these reports found that "the RCC amplitude in 0.2 mmol/L [Ca²⁺]_o is sometimes larger than at 2 mmol/L [Ca²⁺]_o" (page C110).¹⁰ Other studies have provided different results. For example, increasing [Ca²⁺]_o from 1 to 6 mmol/L in rat ventricular myocytes²³ or from 1 to 8 mmol/L in rat ventricular trabeculae²⁴ found an increase of the amplitude of the caffeine-evoked increase of [Ca²⁺]_i attributed to an increase of SR content. At these higher [Ca²⁺]_o, it is possible that there is an increase in diastolic [Ca²⁺]_i and that this stimulates Ca²⁺ uptake into the SR during diastole and thereby elevates SR CaCa²⁺.²³ In a separate study, measurements of caffeine-releasable ⁴⁵Ca²⁺ in cultured neonatal rat myocardium found that increasing [Ca²⁺]_o (0.25 to 4 mmol/L) increased SR content.²⁵ The alterations in the frequency of spontaneous beating in this preparation make it more difficult to compare this study with the present one.

It is also worth comparing the effects of [Ca²⁺]_o in the present work with that in our previous study of nonstimulated cells where we found that increasing [Ca²⁺]_o increased SR content.²⁰ In the absence of stimulation, an increase of [Ca²⁺]_o will simply increase Ca²⁺ entry, and such an increase of content is to be expected.

Coordinated Stimulation of Trigger and Loading Produces Rapid Inotropic Responses
The data in the present study allow the comparison of two different inotropic interventions: (1) recovery after emptying the SR and (2) increasing [Ca²⁺]_o. During the recovery from an empty SR, the increase of systolic Ca²⁺ is due to an increase of SR Ca²⁺ content.¹³ The slow nature of this response is due to the fact that on each beat, the extra Ca²⁺ that can be taken up into the SR is limited by the difference between the amount entering and leaving the cell. In contrast, when [Ca²⁺]_o is elevated, the fact that trigger and loading components of the increased I_Ca-L are increased proportionately means that systolic [Ca²⁺]_i can reach a steady-state level almost immediately. Previous work has shown that the fractional release of Ca²⁺ from the SR is increased by elevating SR Ca²⁺ content.^{11 12 14} The present work shows, however, that elevation of [Ca²⁺]_o does not necessarily lead to a parallel increase of SR Ca²⁺ content, and, therefore, the increased fractional release observed when I_Ca-L is increased under these conditions is entirely due to the increased trigger (I_Ca-L).

It is instructive to consider what the response to elevation of [Ca²⁺]_o would be if there was no effect on the trigger and the only role for increased Ca²⁺ entry is to increase SR loading. This can be estimated as follows:

At a constant I_Ca-L, the magnitude of the systolic Ca²⁺ transient is given by the following:

(1)

where a is the amplitude of the Ca²⁺ transient when the SR is empty (presumably reflecting the contribution due to Ca²⁺ entering via the L-type current), b is a constant, and n determines the steepness of the relationship; b is related to the fraction of Ca²⁺ released from the SR. In other words, the value of b will be greater in 2 than in 0.2 mmol/L [Ca²⁺]_o. This relationship is shown in Figure 5D for 2 mmol/L [Ca²⁺]_o. We calculated the value of b in 0.2 mmol/L [Ca²⁺]_o by multiplying the value in 2 mmol/L by the ratio (amplitude of Ca²⁺ transient in 0.2 mmol/L:amplitude in 2 mmol/L) at a constant SR content. This is obtained from the last transient on 0.2 mmol/L and the first transient in 2 mmol/L. It was also necessary to know the relationship between Ca²⁺ efflux and the amplitude of the systolic Ca²⁺ transient. As illustrated in Figure 5C, this relationship is linear over the range of [Ca²⁺]_i studied and can therefore be represented as follows:

(2)

where c is a constant.

Similar relationships have been published previously.⁷ On any beat, using Equation 1, we can calculate the expected Ca²⁺ transient from the calculated SR content. The efflux can then be calculated from the Ca²⁺ transient (Equation 2). Subtracting this Ca²⁺ efflux from the measured Ca²⁺ influx gives the change of SR Ca²⁺ content. We have used a constant Ca²⁺ influx (measured in the steady state in 2 mmol/L [Ca²⁺]_o). The open symbols in Figure 6A show the results of this calculation. The predicted increase of systolic Ca²⁺ transient is much slower than that observed and is similar to that observed after emptying the SR (Figure 5). In addition, there is a calculated large increase of SR Ca²⁺ content whereas, experimentally, a small decrease is seen. This model assumes that the amplitude of Ca²⁺ entry depends only on [Ca²⁺]_o and not on the amplitude of the systolic Ca²⁺ transient. Although an increase of systolic Ca²⁺ can decrease the Ca²⁺ current (Figure 5B), this effect is less obvious when [Ca²⁺]_o is changed (see Figure 3A), and we have therefore disregarded it. Finally, the data of Figure 6B show the effects of simply increasing the efficacy of the trigger (by applying 500 µmol/L caffeine). In agreement with previous work,⁷ this results in a purely transient increase of systolic Ca²⁺. The rapidly developing and maintained increase of systolic Ca²⁺ (Figure 6A, filled symbols) can therefore be seen to result from a combination of the delayed development (Figure 5A, open symbols) and a transient stimulation (Figure 6B).

	Conclusions

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

 
The results show that the two effects of increased Ca²⁺ influx (increased triggering of SR Ca²⁺ release and increased loading of the SR) balance such that an increase of systolic Ca²⁺ transient is obtained with little change or even a decrease of SR Ca²⁺ content. This will contribute physiologically during, for example, the positive inotropic effects of catecholamines. Although, in this case, there will be additional effects due to phosphorylation of the SR Ca²⁺-ATPase and RyRs, the present data detail the role that increasing I_Ca-L during such stimulation would be expected to play.

The concerted increase of trigger and loading results in rapid increases of systolic Ca²⁺ transient with no need to increase SR Ca²⁺ content. If it were not for the increased trigger, then positive inotropy would develop slowly and require an increased SR Ca²⁺ content. This would be energetically wasteful (in terms of the demands made on the SR Ca²⁺-ATPase) and would also predispose the cell to spontaneous Ca²⁺ release.^{20 26 27} Finally, although the present data show that the positive inotropic effects of elevated [Ca²⁺]_o are not accompanied by an increase of SR Ca²⁺ content, the increased Ca²⁺ loading of the cell is essential to balance the extra Ca²⁺ efflux. If it did not occur, then the increased fractional release would result in a loss of Ca²⁺ from the SR and a purely transient increase of contraction.

	Acknowledgments

 
This work was supported by the British Heart Foundation and The Wellcome Trust.

	Footnotes

 
Original received August 4, 2000; revision received December 4, 2000; accepted December 5, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

 
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