Illuminating sarcoplasmic reticulum calcium.

The bulk of the Ca 2 that activates contraction in the heart comes from the sarcoplasmic reticulum (SR). Calcium is released by the process of Ca -induced Ca release (CICR) in which the entry of a small amount of Ca across the cell membrane triggers the release of much more from the SR. This mechanism depends on the fact that Ca entry from the extracellular fluid (via the L-type Ca current) increases the probability that the SR Ca release channel (ryanodine receptor, RyR) is open. The greater the open probability of the RyR, the greater the release of Ca from the SR and therefore the larger the contraction of the heart. A major factor determining the contractility of the heart is the Ca content of the SR. As one might expect, the more Ca 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 (Ca overload) results in Ca release even in the absence of a trigger. When this Ca release occurs in diastole, it can activate inward membrane currents and produce afterdepolarizations. In the field of heart failure, the decrease of the systolic Ca transient is generally associated with a decrease of SR Ca content, although the precise mechanisms responsible for this remain controversial.1,2 Given the importance of SR Ca 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 Ca into the cytoplasm (either by applying caffeine or rapid cooling3,4) and then measure the amplitude of the resulting contraction or increase of [Ca ]i. This method produces a qualitative measure of SR Ca content, although, if the Ca buffering properties of the cytoplasm are known, the total amount of Ca released from the SR can be calculated from the increase of [Ca ]i. If one assumes that all the SR Ca is released, then the calculated release of total Ca is equivalent to the amount originally stored in the SR. Another way to quantify the amount released depends on the fact that the Ca released from the SR is largely pumped out of the cell by the electrogenic Na -Ca 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 Ca originally stored in the SR.6 The above methods give a measure of the total amount of Ca 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 Ca 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) Ca concentrations have been measured by putting Ca -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 Ca indicator fluo-5N to measure free SR Ca ([Ca ]SR) continuously during stimulation. The data show that each systolic contraction is accompanied by the expected decrease of [Ca ]SR. During diastole, [Ca ]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 Ca remaining within the SR even after release and discuss the implications this will have for the termination of systolic Ca 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 Ca within the SR. We expect that the ability to measure free SR Ca concentration will have profound consequences for the study of SR and cell Ca handling. As with other new methods, refinements will be needed. The indicator used (fluo-5N) has a Kd of 400 mol/L. Given that Ca 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.