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Journal of Physiology (2002), 538.1, pp. 179-184
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013015

Direct and indirect modulation of rat cardiac sarcoplasmic reticulum function by n-3 polyunsaturated fatty acids

S. C. O'Neill, M. R. Perez *, K. E. Hammond, E. A. Sheader and N. Negretti *

	ABSTRACT

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Measurements were made of trans-sarcolemmal Ca²⁺ fluxes and intracellular [Ca²⁺]_i in rat ventricular myocytes loaded with Indo-1 to determine how the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) suppresses spontaneous waves of Ca²⁺ release. We report that in 10 µM EPA, the Ca²⁺ efflux generated by individual waves increased by 11.3 ± 4.9 % over control levels. However, wave-generated efflux per unit time fell overall by 19 ± 5.3 %. On removal of EPA, wave frequency increased transiently such that Ca²⁺ efflux was greater than normal and the cell lost 28.0 ± 10.6 µmol l^-1 Ca²⁺. This probably represents the loss of extra Ca²⁺ accumulated by the sarcoplasmic reticulum (SR), while Ca²⁺ release was inhibited. These results are evidence of inhibition of the SR Ca²⁺-release mechanism and reduced availability of Ca²⁺ to the SR From the relationship between average intracellular Ca²⁺ and the frequency of spontaneous waves, we have calculated the relative contributions of these different mechanisms to the lower frequency of waves. In EPA, the frequency of spontaneous waves fell by 37.5 ± 8.1 %, the majority of this (29.2 ± 8.8 %) is due to inhibition of the Ca²⁺-release mechanism. In EPA, the rate of fall of Ca²⁺ in the caffeine response (an indicator of surface membrane Ca²⁺ efflux pathway activity) was not altered. We conclude, therefore, that the lower resting level of Ca²⁺ observed in EPA is due to a lower influx of Ca²⁺ across the surface membrane rather than increased activation of efflux pathways. How these effects might contribute to the anti-arrhythmic actions of EPA is discussed.

(Received 19 July 2001; accepted after revision 1 October 2001)
Corresponding author S. C. O'Neill: Department of Medicine, University of Manchester, Manchester M13 9PT, UK. Email: stephen.c.o'neill{at}man.ac.uk

	INTRODUCTION

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Among the many beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) is the protection of cardiac muscle against some of the consequences of ischaemia. During ischaemia cardiac myocytes may be damaged and lose the ability properly to control intracellular Ca²⁺ concentration ([Ca²⁺]_i; Allshire et al. 1987; Smith & Allen, 1988; Camacho et al. 1993). Under such conditions, the normal calcium-induced Ca²⁺ release (CICR) mechanism of the cardiac sarcoplasmic reticulum (SR) can become unstable and allow spontaneous waves of Ca²⁺ release to propagate along the cell. This elevation of Ca²⁺ brings the possibility of triggering an arrhythmic action potential by activating the Na⁺-Ca²⁺ exchanger and depolarising the cell. Another consequence of ischaemia is the activation of phospholipases (Ford et al. 1991; Hazen et al. 1991). These release fatty acids from the phospholipids of the cell membrane. Membranes enriched in n-3 PUFAs (e.g. by supplementation of the diet) would presumably provide more n-3 PUFAs on activation of phospholipases. Such a release of n-3 PUFAs would reduce the electrical excitability of the surface membrane of the cardiac cell, raising the threshold for an action potential (Kang et al. 1995). Depression of electrical excitability is brought about by the combined effect of inhibitions of Na⁺, L-type Ca²⁺ and K⁺ channels (Macleod et al. 1998; Leifert et al. 1999). Therefore, the spontaneous release of Ca²⁺ from the SR in a cell damaged by ischaemia would be less likely to trigger arrhythmias in the presence of n-3 PUFAs.

The purely electrophysiological anti-arrhythmic mechanism outlined above works in parallel with another mechanism that operates at the level of the SR in which the frequency of spontaneous release of Ca²⁺ from the SR is reduced by n-3 PUFAs. This can be attributed to a combination of direct inhibition of the calcium-release channel of the SR (the ryanodine receptor: RyR; Negretti et al. 2000) and lower [Ca²⁺]_i (Kang & Leaf, 1996; Negretti et al. 2000). Both of these effects would be expected to increase the time required to refill the SR between waves, the former by increasing the depletion of the SR by each wave (Overend et al. 1997), and the latter by reducing the availability of Ca²⁺ to the SR for refilling (Díaz et al. 1997a). The lower frequency of spontaneous waves of Ca²⁺ release reduces the risk of arrhythmias.

The two effects of n-3 PUFAs at the level of the SR ought to have different effects on the Ca²⁺ efflux activated by waves of Ca²⁺ release. Studies examining individually the effects of reduced availability of Ca²⁺ to the SR (Díaz et al. 1997a) and inhibition of the RyR (Overend et al. 1997) would suggest that if both mechanisms are contributing to the reduction of wave frequency produced by EPA, the efflux activated by individual waves should be larger (due to the higher SR Ca²⁺ content), but the total efflux activated by waves per unit time should be reduced (if the influx of Ca²⁺ has been reduced). The efflux of Ca²⁺ activated by waves of spontaneous release can be measured under voltage-clamp conditions as the integral of the Na⁺-Ca²⁺ exchange current. We have therefore measured the wave-induced efflux of Ca²⁺ from single ventricular myocytes from rat hearts using the perforated patch-clamp method (Díaz et al. 1997b; Overend et al. 1997), and determined the effect of the n-3 PUFA eicosapentaenoic acid (EPA). We also show here the relative contributions of the two mechanisms to the reduction of spontaneous release frequency by examining the relationship between [Ca²⁺]_i and frequency of release. We find evidence for both mechanisms and conclude that the inhibition of CICR is the more effective.

	METHODS

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Rat ventricular myocytes were isolated using a collagenase and protease technique that has been described previously (Eisner et al. 1989). Rats were killed by stunning and cervical dislocation. For [Ca²⁺]_i measurements, cells were loaded with the membrane-permeant form of Indo-1 at 5 µM for 5 min; 20 min were allowed for de-esterification. Cells were placed in a superperfusion chamber on the stage of an inverted microscope. Indo-1 fluorescence was excited at 360 nm and recorded at 400 and 500 nm (O'Neill & Eisner, 1990) using epi-fluorescence optics. All voltage-clamp experiments were carried out using the perforated patch-clamp technique (Horn & Marty, 1988) using the switch-clamp mode of the Axoclamp 2B amplifier (Axon Instruments). Pipettes were filled with the following solution (mM): KCH₃O₃S 125, KCl 10, NaCl 20, Hepes 10, MgCl₂ 5; titrated to pH 7.2 with KOH, and a final concentration of amphotericin B of 240 µg ml^-1.

The bathing solution was as follows (mM): NaCl 135, KCl 4, Hepes 10, glucose 11, MgCl₂ 1; titrated to pH 7.4 with NaOH. Initially cells were bathed in the above solution at 1 mM CaCl₂. This level was altered to between 2 and 8 mM, as indicated in the figure legends, to induce spontaneous waves of Ca²⁺ release. EPA was prepared in ethanol as a 10 mM stock solution and stored under a N₂ atmosphere before use. Fresh stock solutions were prepared each week. Fatty-acid-free bovine serum albumin (BSA) was added (2 mg ml^-1) to the control solution to ensure the rapid and complete removal of fatty acids from the solution (Kang & Leaf, 1994; Kang et al. 1995). In voltage-clamp experiments, the above solution was modified to contain 5 mM 4-aminopyridine and 0.1 mM BaCl₂. All experiments were carried out at room temperature (25 °C) and in accordance with the provisions of the Animal Procedures Act (1986).

Ca²⁺ fluxes activated by waves were measured from the integral of the Na⁺-Ca²⁺ exchange inward current, as reported previously (Negretti et al. 1995; Díaz et al. 1997a,b). This integral is corrected for non-Na⁺-Ca²⁺ exchange efflux and expressed with respect to the volume of the cell.

All statistics quoted are mean ± S.E.M.; Student's paired t tests were used throughout to test statistical significance.

	RESULTS

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An example of the effects of EPA on [Ca²⁺]_i in a calcium-overloaded rat ventricular myocyte is shown in Fig. 1. Initially, the calcium-overloaded SR produced spontaneous releases of Ca²⁺ that propagated along the cell. Each wave caused a transient rise of [Ca²⁺]_i. When 10 µM EPA was applied, the frequency of these waves of Ca²⁺ release fell, as did the resting level of Ca²⁺. Both effects were reversed upon removal of EPA and application of BSA to bind the remaining fatty acid. The fall in frequency of waves may be due to either a reduced availability of Ca²⁺ and/or inhibition of the RyR. One way to determine which is involved is to measure the Ca²⁺ efflux activated by waves. If only inhibition of the RyR were present, no change in time-averaged efflux would be measured, as is the case with tetracaine (Overend et al. 1997). A lower total efflux activated by waves (per unit time) would indicate a lower availability of Ca²⁺ to the SR, although it would not rule out simultaneous inhibition of CICR.

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Figure 1. EPA lowers resting Ca2+ and reduces the frequency of spontaneous waves of calcium-induced Ca2+ release (CICR) The cell was bathed in Tyrode solution containing 4 mM Ca2+ to produce a Ca2+ overload and encourage spontaneous release of Ca2+ by the sarcoplasmic reticulum (SR). Application of 10 µM eicosapentaenoic acid (EPA) is indicated by the bar. On removal of EPA from the bathing solution the cell was bathed in 2 mg ml-1 bovine serum albumin (BSA) to assist in removing the fatty acid.

The current trace from a voltage-clamped myocyte shown in Fig. 2 shows several changes when the cell is exposed to EPA. As the frequency of spontaneous waves fell (from about 12 min^-1 to 8 min^-1 in this case), the amplitude of currents activated by each wave increased (on average by 28.8 ± 5.5 %, n = 6, P < 0.005), and there was an inward shift of the holding current. When EPA was removed, the frequency rose to 18 min^-1 before falling again to control levels. The inward shift of the holding current is probably the result of inhibition of the steady-state outward current that EPA has been shown to produce (Macleod et al. 1998). The measurement of wave-activated efflux was not affected by this changing baseline. The changes in wave-activated efflux and frequency are summarised in the histograms in Fig. 3 for six cells. EPA significantly reduced the frequency of waves, and there was a transient increase of frequency on its removal. This is similar to the changes of frequency incurred on application and removal of the RyR inhibitor tetracaine (Overend et al. 1997). At the same time, there was a significant increase in the efflux of Ca²⁺ activated by individual waves. Overall, however, the increase in efflux activated by individual waves was not sufficient to compensate for the reduction in frequency and total wave-activated efflux of Ca²⁺ per unit time falls. Importantly, on removal of EPA, both the frequency and total efflux per unit time transiently overshot the control value, as is seen on removal of the RyR inhibitor tetracaine (Overend et al. 1997). In other words, the cell was losing Ca²⁺. The sum of this 'extra' efflux was 28.0 ± 10.6 µmol l^-1 of Ca²⁺. This represents the Ca²⁺ gained by the SR in the presence of EPA and is similar to that obtained in our previous direct measurement (Negretti et al. 2000).

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Figure 2. The effect of EPA on the size and frequency of transient inward currents that accompany spontaneous waves of CICR The cell was voltage clamped at -40 mV throughout the record using the perforated patch-clamp technique. The pipette solution contained 20 mM Na+ to encourage spontaneous release of Ca2+ by the SR; the bathing solution contained 2 mM Ca2+.

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Figure 3. Summary of the effects of EPA on wave frequency, and individual and time-averaged wave-activated Ca2+ efflux The histograms show summary data for six cells. All data are normalised with respect to the control levels. The data labelled 'Total Efflux' were calculated from the values of efflux in units of µmol (l cell volume)-1 s-1. This was calculated from the sum of individual wave effluxes over a period of 1-2 min in each condition. * P < 0.05, ** P < 0.03, *** P < 0.01.

As mentioned above, the current activated by Ca²⁺ waves increased in amplitude by about 30 %; however, efflux activated by individual waves increased by only 12 %. The data shown in Fig. 4 suggest why this might be. Individual currents activated by propagating waves of CICR (from the same data as in Fig. 2) are shown in the upper panel under control conditions and in EPA. Although the current amplitude clearly increased, the lower panel shows that the duration of the wave decreased in EPA. Therefore, the integral of the current in EPA was not increased in proportion to the increase of current amplitude. On average, the half-time of transient inward currents in EPA was 83.2 ± 7.2 % of the control value (n = 6, P < 0.01).

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Figure 4. The time course of the transient inward current is altered by EPA The upper trace shows individual currents. In the lower panel, the control current is shown superimposed on the current in EPA, emphasising the reduced duration of the latter. The filled circle identifies the current trace recorded in the presence of EPA.

The reduced overall wave-activated efflux of Ca²⁺ observed in the presence of EPA indicates that the fall in resting Ca²⁺ is indeed partly responsible for the inhibition of wave frequency. However, the increased frequency and efflux observed immediately following the removal of EPA suggest an increased SR Ca²⁺ content. This could only take place if EPA is also producing inhibition of the RyR (Overend et al. 1997). The experiment shown in Fig. 5 attempts to separate these two effects. In panel A, the two traces show spontaneous waves in the absence (left) and presence (right) of 10 µM EPA; external Ca²⁺ concentration ([Ca²⁺]_o) was 4 mM in both cases. In EPA, the frequency of waves and resting Ca²⁺ levels were lower. When [Ca²⁺]_o was increased (not shown), the frequency of spontaneous waves and resting Ca²⁺ increased. This represents a new balance of Ca²⁺ influx and efflux; the higher average level of [Ca²⁺]_i activated sufficient Ca²⁺ efflux to balance the increased influx due to the increased driving force for Ca²⁺ entry. An indirect measure of the level of influx, therefore, can be obtained from the average level of Ca²⁺. The relationship between frequency and the average level of Ca²⁺ in panel B shows the change in frequency due to a simple change of [Ca²⁺]_i (i.e. Ca²⁺ influx). Inhibition of CICR would give a lower frequency of waves for any given influx of Ca²⁺. The plot in Fig. 5B shows that the fall of Ca²⁺ in EPA is not sufficient to explain all of the fall in frequency we observed. The remaining part of the inhibition of frequency must be due to the inhibition of CICR by EPA. Similar results were found in another five cells. Inhibition of CICR in these cells lowered the frequency of waves by 29.2 ± 8.8 % (n = 6, P < 0.03) out of a total fall of 37.5 ± 8.1 % (i.e. inhibition of CICR appears to be the larger effect).

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Figure 5. EPA changes the relationship between the frequency of spontaneous release of Ca2+ and the mean cytoplasmic [Ca2+] A, two sample records of spontaneous waves of CICR in control conditions and in EPA; in both cases the cell was bathed in 4 mM [Ca2+]o. In these experiments the cells were exposed in turn to 2, 4 and 8 mM [Ca2+]o to alter the mean [Ca2+]i and frequency of release. B, the change in frequency of spontaneous release as a function of mean [Ca2+]i (indicated by the Indo-1 ratio) in the absence () and presence () of EPA (4 mM [Ca2+]o).

The fall of resting Ca²⁺ in the presence of EPA does, however, make a contribution to the lower frequency of spontaneous waves. Resting [Ca²⁺]_i may be lowered by reduced influx or greater efflux of Ca²⁺. If the efflux of Ca²⁺ is increased we might expect the rate of fall of the caffeine response to change as well. Caffeine releases the SR Ca²⁺ content into the cytoplasm. In the maintained presence of caffeine, the SR cannot re-sequester Ca²⁺ and so [Ca²⁺]_i falls due to the action of the surface membrane efflux pathways alone. An example is shown in Fig. 6, where it may be seen that the control caffeine response (left) is, as expected, smaller than that in EPA (right), but that EPA has no effect on the rate of fall of [Ca²⁺]_i. The middle panel emphasises this, showing the control and EPA caffeine responses normalised and superimposed on each other. Single exponentials were fitted to data such as that shown in the upper panel; the histogram shows average values for 12 cells. The efflux pathways appear to be operating normally, the fall of resting Ca²⁺ in the presence of EPA is therefore likely to be due to a reduced influx of Ca²⁺.

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Figure 6. EPA does not alter surface membrane Ca2+ efflux pathways The upper panel shows Indo-1 records of [Ca2+]i. In each trace the cell was electrically stimulated before application of 10 mM caffeine; [Ca2+]o was 1 mM throughout. The middle panel shows the caffeine responses normalised and superimposed on each other. Exponentials were fitted to the decay phase of each caffeine response. The histogram below shows average rate constants from such fits made in 12 cells. The average rate constants were 0.31 ± 0.03 s-1 in control solution and 0.29 ± 0.02 s-1 in EPA (P > 0.1).

	DISCUSSION

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The appearance in cardiac muscle of spontaneous waves of CICR from the SR under conditions of Ca²⁺ overload can lead to the generation of spontaneous action potentials (i.e. arrhythmogenesis; Capogrossi et al. 1987). It has been shown that n-3 PUFAs are protective against cardiac arrhythmias (Billman et al. 1994) and that they reduce the frequency of spontaneous release of Ca²⁺ from the SR (Kang & Leaf, 1996; Negretti et al. 2000). It is now known that at least two mechanisms are involved in this lowering of SR spontaneous activity: the resting level of [Ca²⁺]_i is lowered and the CICR mechanism is inhibited (Kang & Leaf, 1996; Negretti et al. 2000).

Lower [Ca²⁺]_i and inhibition of CICR inhibit spontaneous waves

These two mechanisms are able to reduce the frequency of spontaneous waves of Ca²⁺ release because for each wave, the rise in [Ca²⁺]_i activates efflux pathways and the cell loses Ca²⁺. Therefore, following each wave, the SR contains less Ca²⁺ than before the wave began. As a propagating wave can only proceed when the Ca²⁺ sensitivity of Ca²⁺ release is high (Trafford et al. 1995), and as the sensitivity is reduced at low SR Ca²⁺ content, another wave cannot propagate until the lost Ca²⁺ has been replaced. This will take longer at lower cytoplasmic Ca²⁺ levels, as the SR Ca²⁺-ATPase is less active. On the other hand, wave propagation following inhibition of the RyR requires a greater load of Ca²⁺ in the SR to provide a compensatory increase of RyR sensitivity. The higher SR content means that when a wave of release takes place more Ca²⁺ is released and more efflux is activated, therefore more time is required for SR refilling. The purpose of the present study was to demonstrate the contribution of each of these mechanisms to the effect of EPA on the spontaneous release of Ca²⁺ from the SR.

Individual waves activate more efflux in EPA

When CICR is depressed by tetracaine, the increase of SR Ca²⁺ content ensures that the lower frequency of waves is compensated for by increased transient inward current and, therefore, efflux (Overend et al. 1997). Although in EPA individual waves do activate more efflux than control waves, it seems that the shorter duration of the transient inward current in EPA prevents a complete compensatory increase in efflux. We have reported previously that waves in EPA propagate faster than in a control solution (Negretti et al. 2000), all other things being equal, therefore the wave should terminate sooner, as indicated by the transient inward currents (Fig. 4). The shorter duration of the waves in EPA seems to limit the time available for activation of Ca²⁺ efflux. Although this explains why the current integral is limited, it is not clear why faster propagation should limit the efflux when currents are larger in EPA but not in tetracaine. Inhibition of CICR by EPA is, however, indicated by the greater Ca²⁺ efflux activated by individual waves.

Total wave-activated efflux is reduced in EPA

It is clear from Fig. 3 that the total wave-activated efflux of Ca²⁺ per unit time is reduced in EPA. Inhibition of CICR alone does not reduce the amount of efflux generated by spontaneous waves of Ca²⁺ release (Overend et al. 1997). The efflux of Ca²⁺ generated by waves of CICR has to balance influx; if influx remains unchanged (as is the case with tetracaine), the requirement for efflux also remains unchanged. EPA must also, therefore, be affecting other mechanisms in addition to inhibition of CICR. If the fall of resting Ca²⁺ is brought about by reduced Ca²⁺ influx, the need for efflux is reduced. Alternatively, if the Ca²⁺ efflux pathways are operating faster, this may also reduce the resting level of [Ca²⁺]_i. As the rate of fall of Ca²⁺ in the caffeine response is unchanged by EPA (Fig. 6), we may conclude the Ca²⁺ efflux pathways are operating normally in the presence of EPA; if there were a higher rate of Ca²⁺ extrusion at resting levels of [Ca²⁺]_i (as would be required to lower resting [Ca²⁺]), we would also see a faster fall of [Ca²⁺] following application of caffeine.

If efflux pathways are not operating faster than normal, we conclude that the influx of Ca²⁺ is reduced in the presence of EPA, and that this is partly responsible for lowering the frequency of spontaneous waves of CICR. Studies examining the electrophysiological effects of n-3 PUFAs have shown that they inhibit the L-type Ca²⁺ current. This would reduce Ca²⁺ influx in the range of membrane potentials where this current is active, but may not be so important at the resting potential (e.g. in Fig. 1). The reduced influx of Ca²⁺ can only be partly responsible for inhibiting spontaneous waves as it could not also cause the increase in SR Ca²⁺ content that has been previously reported (Negretti et al. 2000). In this study, such an increase of SR Ca²⁺ content is indicated by the greater Ca²⁺ efflux activated by individual waves and the transient increase of efflux activated by waves on removal of EPA (Fig. 3).

Increased Ca²⁺ efflux on EPA removal: evidence of increased SR Ca²⁺

The transient increase of wave-activated efflux of Ca²⁺ following the removal of EPA (Fig. 3) is a consequence of the removal of the inhibitory effect of EPA on CICR. In EPA, the SR has been able to accumulate a higher than normal load of Ca²⁺ due to inhibition of the leak (i.e. waves) that normally sets the upper limit on content. However, once the inhibition of release is relieved by removal of EPA, the SR can no longer sustain this higher load and there follows a period where it offloads Ca²⁺ by means of a higher than normal frequency of waves. In the similar situation of removal of tetracaine, this takes the form of a clear burst of high-frequency waves (Overend et al. 1997). The burst in the case of removal of EPA is less clear than that seen following the removal of tetracaine, probably because of the relatively slow removal of the fatty acid. The effect is clear, however, when efflux is summed over the 1st min following removal of EPA from the bathing solution. The amount of Ca²⁺ lost in this period over and above that expected during the control period, is on average, similar to the increase in SR Ca²⁺ content that we have reported previously (Negretti et al. 2000).

Contributions of inhibition of release and lower [Ca²⁺]_i to inhibition of waves

The evidence shown so far supports a role for inhibition of CICR and lower availability of Ca²⁺ to the SR in inhibiting spontaneous waves of Ca²⁺ release by the SR The experiments reported here also allow us to determine the contributions of each of these factors. We have used the average level of [Ca²⁺]_i as an indirect measure of Ca²⁺ influx. If we assume that there is no saturation of the dye during Ca²⁺ release from the SR, the average [Ca²⁺]_i should rise as influx rises. This increase of [Ca²⁺]_i activates greater efflux of Ca²⁺ to balance the increased influx. In the plot of Fig. 5B, the point taken in the presence of EPA lies to the right of the control relationship between average [Ca²⁺]_i and oscillation frequency. Thus, for a given influx of Ca²⁺ in EPA, there is a lower frequency of oscillations. In the example shown in Fig. 5B, the control relationship predicts that a fall in frequency from about 16 to 12 min^-1 would result from the EPA-induced fall of [Ca²⁺]_i. The remainder of the fall in frequency to about 9 min^-1 is, therefore, due to inhibition of the RyR by EPA. Although in this example both mechanisms are contributing roughly equally to the lower frequency of waves in EPA, on average the inhibition of CICR seems to be the more powerful influence, producing about 75 % of the effect.

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Acknowledgements

This work was supported by a Wellcome Trust Collaborative Research Initiative Grant.

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