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(Circulation Research. 2003;93:4.)
© 2003 American Heart Association, Inc.

Editorials

Illuminating Sarcoplasmic Reticulum Calcium

L. Venetucci, A.W. Trafford, D.A. Eisner

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

Correspondence to D.A. Eisner, Unit of Cardiac Physiology, 1.524 Stopford Bldg, University of Manchester, Oxford Road, Manchester M13 9PT. E-mail eisner{at}man.ac.uk

Key Words: calcium • sarcoplasmic reticulum • fluorescence

The bulk of the Ca²⁺ that activates contraction in the heart comes from the sarcoplasmic reticulum (SR). Calcium is released by the process of Ca²⁺-induced Ca²⁺ release (CICR) in which the entry of a small amount of Ca²⁺ across the cell membrane triggers the release of much more from the SR. This mechanism depends on the fact that Ca²⁺ entry from the extracellular fluid (via the L-type Ca²⁺ current) increases the probability that the SR Ca²⁺ release channel (ryanodine receptor, RyR) is open. The greater the open probability of the RyR, the greater the release of Ca²⁺ from the SR and therefore the larger the contraction of the heart. A major factor determining the contractility of the heart is the Ca²⁺ content of the SR. As one might expect, the more Ca²⁺ that there is in the SR, the more is released on each contraction. However the degree of filling of the SR has other important implications for cardiac physiology and pathology. Excessive filling of the SR (Ca²⁺ overload) results in Ca²⁺ release even in the absence of a trigger. When this Ca²⁺ release occurs in diastole, it can activate inward membrane currents and produce afterdepolarizations. In the field of heart failure, the decrease of the systolic Ca²⁺ transient is generally associated with a decrease of SR Ca²⁺ content, although the precise mechanisms responsible for this remain controversial.^1,2

Given the importance of SR Ca²⁺ content, it is essential to be able to measure it. Many previous studies have used indirect methods. A convenient way is to release the SR Ca²⁺ into the cytoplasm (either by applying caffeine or rapid cooling^3,4) and then measure the amplitude of the resulting contraction or increase of [Ca²⁺]_i. This method produces a qualitative measure of SR Ca²⁺ content, although, if the Ca²⁺ buffering properties of the cytoplasm are known, the total amount of Ca²⁺ released from the SR can be calculated from the increase of [Ca²⁺]_i.⁵ If one assumes that all the SR Ca²⁺ is released, then the calculated release of total Ca²⁺ is equivalent to the amount originally stored in the SR. Another way to quantify the amount released depends on the fact that the Ca²⁺ released from the SR is largely pumped out of the cell by the electrogenic Na⁺-Ca²⁺ exchange (NCX). Therefore, if the experiment is performed under voltage-clamp conditions, the integral of the NCX current gives a measure of the amount of Ca²⁺ originally stored in the SR.⁶

The above methods give a measure of the total amount of Ca²⁺ in the SR rather than the free concentration. While the total amount is a useful parameter, it is often important to know the free concentration. Intra-SR Ca²⁺ is buffered by various proteins including calsequestrin, and it is presumably the free concentration that influences, for example, transport by SERCA and the RyR. Another problem with these other methods is that they do not provide a continuous "readout" of SR content.

In other tissues, SR (and ER) Ca²⁺ concentrations have been measured by putting Ca²⁺-sensitive indicators into the SR. In cardiac cells, there have been no previous reports with this approach, although anecdotally several laboratories have tried. The only success we are aware of in cardiac muscle used an NMR probe⁷ and, as a consequence, the data represent an average of the whole heart with limited temporal resolution. Shannon et al⁸ in this issue of Circulation Research have successfully used the low-affinity Ca²⁺ indicator fluo-5N to measure free SR Ca²⁺ ([Ca²⁺]_SR) continuously during stimulation. The data show that each systolic contraction is accompanied by the expected decrease of [Ca²⁺]_SR. During diastole, [Ca²⁺]_SR was 1 to 1.5 mmol/L and fell to 0.3 to 0.6 mmol/L during systole. The authors point out this significant amount of Ca²⁺ remaining within the SR even after release and discuss the implications this will have for the termination of systolic Ca²⁺ release. Importantly, they achieved subcellular spatial resolution. Although most of the signal came from sites spaced at sarcomere intervals assumed to be junctional SR, there was, however, some signal from other sites. Interestingly, the time course of the depletion signal was the same as the one at the putative junctional sites, indicating rapid diffusion of Ca²⁺ within the SR.

We expect that the ability to measure free SR Ca²⁺ concentration will have profound consequences for the study of SR and cell Ca²⁺ handling. As with other new methods, refinements will be needed. The indicator used (fluo-5N) has a K_d of 400 µmol/L. Given that Ca²⁺ is reported to be as high as 1.5 mmol/L in the present study, a lower-affinity indicator might be useful for some purposes. Another advance would be to show that the method works in species other than the rabbit. The authors (D.M. Bers, oral communication, June 2003) found that the technique worked much less well in rat cells, a result consistent with previous work using NMR indicators where an SR signal could be observed in rabbit but not rat hearts.⁷ However, even as it stands, this method should allow further important advances to be made. Two such possibilities are briefly described below.

Is SR Ca²⁺ Spatially Uniform Throughout the Cell?

As mentioned above, Shannon et al⁸ found little diffusion delay between junctional and other regions of the SR. It would also be useful to see whether there exist regions within the cell with a greater [Ca²⁺]_SR than others. For example, under Ca²⁺-overloaded conditions, it would be interesting to know whether Ca²⁺ waves start at regions with greater SR content than others.

What Happens to Free SR Ca²⁺ in Heart Failure?

Much evidence suggests that the contribution of the SR to the systolic Ca²⁺ transient decreases in heart failure, possibly as a result of a decrease of SR Ca²⁺ content. Various explanations have been provided for this including decreased SERCA activity and/or increased diastolic Ca²⁺ leak through the RyR.^1,2 As far as the decreased SERCA activity hypothesis is concerned, there are two distinct possibilities as shown in the Figure. Either there is a uniform decrease in SERCA in all parts of the SR (Figure, panel B) or the density of SR decreases but the remaining SR has a normal expression of SERCA (Figure, panel C). This could occur, for example, if the increase of cell volume that occurs in hypertrophy and failure is not accompanied by any increase of SR volume. As shown in the Figure, both hypotheses predict a decrease of SR Ca²⁺ as measured per cell volume but have very different predictions with regard to free SR Ca²⁺. The uniform decrease of SERCA predicts a decrease of [Ca²⁺]_SR (Figure, panel B). However, if the SR volume decreases (as a fraction of that of the cell), then there is no reason to expect any change of [Ca²⁺]_SR (Figure, panel C). Much previous work has shown that the fraction of the SR that is released during systole depends on the SR Ca²⁺ content.^5,9 Here, it is presumably the free Ca²⁺ that is relevant. On the first model then, the fraction of released Ca²⁺ should decrease in failure, whereas on the second, no such effect would be expected and the decrease of total Ca²⁺ release is simply due to a decrease of SR volume. There are also implications for arrhythmogenic diastolic Ca²⁺ release. If we assume that this occurs when SR Ca²⁺ reaches a certain threshold value,¹⁰ then this is less likely to occur if [Ca²⁺]_SR is decreased (Figure, panel B). However, if the free SR Ca²⁺ is not decreased in failure (Figure, panel C) then arrhythmias may be more likely to occur. Previous work has also pointed out that arrhythmogenic Ca²⁺ release occurs despite paradoxically decreased SR Ca²⁺ content.¹¹ That study suggested that sympathetic stimulation might elevate SR content to levels at which spontaneous Ca²⁺ release occurs. We speculate that the measured decrease of total SR Ca²⁺ (referred to the cell volume) need not be accompanied by a decrease of [Ca²⁺]_SR. It will be fascinating in future work to measure both free and total SR Ca²⁺. Finally, the use of low-affinity fluorescent indicators potentially provides a map of the distribution of SR within the cell. In principle, it should be possible to measure the volume of the cell with more than a threshold level of fluorescence and hence measure the SR volume to see whether it changes in failure.

View larger version (17K): [in this window] [in a new window]   Schematic diagram of the effects on SR Ca2+ handling of different ways of decreasing SERCA expression. The diagrams at the top show schematics of the cell (rectangle) with SR denoted as ovals and SERCA as filled circles. The shading gives an indication of SR free Ca2+ during diastole. The traces below show [Ca2+]i (top) and [Ca2+]SR (bottom). A, Control. B, Effect of removing 50% of SERCA uniformly from the SR. C, Effect of removing half the SR but leaving the SERCA concentration in the remaining SR the same as control. Note that in B and C the cytoplasmic Ca2+ transient is slowed. However, depending on whether the decrease of SERCA occurs with no change (B) or a proportional decrease (C) of total SR volume, then the resting (diastolic) level of [Ca2+]SR is respectively decreased or unaffected. Note that the modeling is very simplified and takes no account of the effects of changes of SR Ca2+ on the fraction of Ca2+ released.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

References

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