INTRODUCTION

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IN THE HEART, release of Ca²⁺ from the sarcoplasmic reticulum (SR) by the mechanism of Ca²⁺-induced Ca²⁺ release (CICR), triggered by depolarization-induced Ca²⁺ entry through surface membrane L-type Ca²⁺ channels, is the crucial cellular event underlying excitation-contraction (E-C) coupling and force development. Relaxation of cardiac cells is critically dependent on mechanisms lowering intracellular Ca²⁺ ([Ca²⁺]_i) through reuptake into the SR and/or transport across the sarcolemma. Reuptake of Ca²⁺ provides the necessary filling of the SR to allow release of a sufficient amount of Ca²⁺ for the next activation or, on the level of the intact heart, for the next heartbeat. Reuptake of Ca²⁺ occurs in an energy-consuming process by the SR Ca²⁺-ATPase. The SR Ca²⁺ pump is regulated by various cytoplasmic protein kinases. It has been shown that the 52-amino acid protein phospholamban is closely associated with the Ca²⁺ pump and is a key site of regulatory protein phosphorylation. Unphosphorylated phospholamban is inhibitory for the Ca²⁺ pump, whereas phosphorylation of phospholamban increases SR Ca²⁺ pump activity. This mechanism has been shown to be a major component of the positive inotropic effect of -adrenergic stimulation (for review of regulatory effect of phospholamban on myocardial contractility, see Ref. 20).

The study of the role of phospholamban in the regulation of cardiac function has recently been facilitated through the development of a phospholamban-deficient mouse model. The phospholamban-deficient mice, generated by gene-targeting methodology in embryonic stem cells (27), displayed hyperdynamic cardiac function with enhanced inotropic characteristics and accelerated relaxation (9, 20). Several recent studies using cells isolated from these hearts have investigated cellular and subcellular aspects of Ca²⁺ regulation and E-C coupling. These studies involved characterization of diastolic and systolic [Ca²⁺]_i changes and contractile parameters (38), loading of the SR (22), effect on Ca²⁺ sparks (33), L-type Ca²⁺ current (28, 33), and protein phosphorylation (19). Specifically, with regard to intracellular Ca²⁺ handling, systolic [Ca²⁺]_i is higher (22, 38), the SR Ca²⁺ load is increased (22, 33), and [Ca²⁺]_i transients decline faster (22, 33, 38) in phospholamban-deficient myocytes.

The goal of the present study was to investigate, with spatial information on the subcellular level, the role of phospholamban in spontaneous and electrically evoked Ca²⁺ signals in mouse ventricular myocytes. By use of confocal laser scanning microscopy, the specific spatial and temporal characteristics of electrically evoked [Ca²⁺]_i transients as well as spontaneous Ca²⁺ sparks and [Ca²⁺]_i waves were compared between wild-type (WT) and phospholamban-deficient (PLB-KO) mice. Our results show that triggered and spontaneous [Ca²⁺]_i transients declined faster and that spontaneous Ca²⁺ sparks and [Ca²⁺]_i waves occurred more frequently in PLB-KO myocytes. The enhanced reuptake of Ca²⁺ (and consequent increased local Ca²⁺ buffering) and an apparent reduction of the refractory time for SR Ca²⁺ release profoundly increased inhomogeneities of [Ca²⁺]_i distribution encountered during triggered transients as well as for spontaneous release events (sparks and waves). Overall, increased activity of the SR Ca²⁺ pump led to enhanced local Ca²⁺ gradients and inhomogeneous Ca²⁺ handling during E-C coupling and to an enhancement of spontaneous activity of cardiac ventricular myocytes. An initial account of this work was presented in abstract form (16).

	METHODS

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Cardiac cell preparation. Ventricular myocytes were isolated from species-matched WT and PLB-KO mice (35-45 g) hearts by an enzymatic Langendorff perfusion procedure as described previously (14, 22). Animals were anesthetized by intraperitoneal injection of pentobarbital sodium (70 mg/kg). Myocytes were used 1-6 h after isolation. For experimentation, the cells were placed in an experimental chamber (~0.5 ml) that allowed for continuous superfusion with Tyrode solution and electrical field stimulation with 2-ms voltage pulses of suprathreshold amplitude applied through parallel platinum wires. All experiments were performed at room temperature (20-22°C).

Ca²⁺ measurements. Cardiac myocytes were loaded with Ca²⁺ indicator by exposure to 5 µM fluo 3-AM (Molecular Probes, Eugene, OR) for 20-25 min at 20°C. The cells were subsequently washed for 20 min in extracellular solution to allow sufficient time for deesterification. For fluorescence measurements, the coverslip with the cells was mounted on the stage of an inverted microscope (Axiovert 100, Zeiss, Germany) equipped with an ×40 objective (Plan-Neofluor, oil, NA 1.3, Zeiss). The microscope was attached to a confocal laser scanning unit (LSM 410, Zeiss). Fluo 3 fluorescence was excited with the 488-nm line of an argon ion laser. Emitted fluo 3 fluorescence was measured at a wavelength of >515 nm. [Ca²⁺]_i images were calculated according to the formula (8): [Ca²⁺]_i = K_DR/(K_D/[Ca²⁺]_rest  R + 1), where R is the normalized fluorescence (F/F₀), F₀ is the fluo 3 fluorescence at rest, and K_D is the dissociation constant for the Ca²⁺-fluo 3 complex. A value of 1.1 µM was assumed for K_D. This value represents a minimal estimate of the K_D of fluo 3 in the cytoplasmic environment (12, 15). Resting [Ca²⁺]_i ([Ca²⁺]_rest) was taken as 100 nM for both WT and PLB-KO cells, since it has been shown that [Ca²⁺]_rest was not different in the two preparations (22, 38). Line plots of local [Ca²⁺]_i transients were spatially averaged over a distance of 1.5 µm unless noted otherwise. This corresponds to a spatial average of 4-6 pixels dependent on the magnification (zoom factor) that was chosen for the recording of the confocal images.

Solutions. The cells were superfused continuously with a physiological salt solution (standard Tyrode solution) composed of (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and 10 HEPES, titrated to pH 7.3 with NaOH.

	RESULTS

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Spatiotemporal characteristics of electrically evoked [Ca²⁺]_i transients in WT and PLB-KO myocytes. We first investigated the kinetics of electrically evoked macroscopic [Ca²⁺]_i transients recorded from single isolated ventricular myocytes from WT and PLB-KO mice. Figure 1 compares a series of [Ca²⁺]_i transients recorded after a period of rest of ~30 s. In both WT and PLB-KO mouse preparations, postrest potentiation and a negative staircase of the [Ca²⁺]_i transients were observed. Closer inspection of the time course of the negative staircase (Fig. 1) revealed that the time-dependent decrease in the amplitude of the [Ca²⁺]_i transients tended to be slower in the PLB-KO cells compared with WT myocytes. Because the negative staircase from the potentiated state is often interpreted as a successive cellular (SR) Ca²⁺ loss due to competition between SR uptake and plasma membrane Ca²⁺ extrusion, the slowing of the stimulation-dependent SR Ca²⁺ loss in the PLB-KO mouse is in agreement with the enhanced SR Ca²⁺ uptake in these cells. Some part of this negative staircase might also be attributed to partial refractoriness of E-C coupling (1, 2). This refractoriness appeared to be reduced in PLB-KO myocytes (see below), so that the effect may be less important in PLB-KO myocytes. The relative contributions of these two factors, however, cannot be unequivocally distinguished in these experiments. On the level of individual [Ca²⁺]_i transients, no significant difference was observed in the rate of rise of [Ca²⁺]_i; however, the decline of [Ca²⁺]_i was markedly accelerated in the PLB-KO preparation (Fig. 1C). [Ca²⁺]_i decayed with a time constant  (monoexponential fit to rate of decay of [Ca²⁺]_i) of 75 ms, whereas, in the WT preparation, [Ca²⁺]_i declined, with a  of 190 ms. This increased rate of decay of [Ca²⁺]_i was again indicative of the accelerated reuptake of Ca²⁺ into the SR.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Postrest potentiation, negative staircase, and kinetics of intracellular Ca2+ ([Ca2+]i) transients in phospholamban-deficient (PLB-KO) myocytes. Both wild-type (WT; A) and PLB-KO (B) myocytes revealed postrest potentiation and a negative staircase of electrically evoked [Ca2+]i transients. Negative staircase was more pronounced in WT myocytes. C: comparison of kinetics of normalized steady-state [Ca2+]i transients from WT and PLB-KO myocytes.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Spatial organization of electrically evoked [Ca2+]i transients in WT and PLB-KO myocytes. A: line-scan image and localized [Ca2+]i profiles of electrically evoked [Ca2+]i transients revealed spatially homogeneous elevations of [Ca2+]i in WT cells. Line was positioned along the longitudinal axis of the cell. B: electrically evoked [Ca2+]i transients in PLB-KO myocytes showed pronounced spatial inhomogeneities of [Ca2+]i and localized failures of SR Ca2+ release of variable subcellular locations ( in [Ca2+]i profiles). C: spontaneous [Ca2+]i wave during electrically evoked [Ca2+]i transients in PLB-KO myocytes. [Ca2+]i wave propagated through cellular regions where previous or subsequent electrical stimulation failed to trigger release of Ca2+ from SR, indicating functional Ca2+ release throughout entire scanned region of cell. Scale bars = 20 µm. Arrows indicate time of electrical stimulation with extracellular electrodes.

Ca²⁺ sparks in WT and PLB-KO ventricular myocytes. It is generally accepted that the whole cell [Ca²⁺]_i transients in cardiac cells reflect the temporal and spatial summation of individual elementary Ca²⁺ release events, referred to as Ca²⁺ sparks (8, 15, 24, 25). Ca²⁺ sparks originate at morphologically discrete sites of the SR by the concerted opening of a few release channels organized in clusters (3). Amplitude and kinetics of Ca²⁺ sparks critically depend on the SR Ca²⁺ load and the rate of Ca²⁺ uptake (11). Both parameters are known to be larger and faster in PLB-KO cells. Furthermore, PLB-KO myocytes have been shown (27) to contain fewer ryanodine receptors and/or Ca²⁺ release channels, possibly as a compensatory mechanism for the enhanced SR Ca²⁺ pumping rate. To investigate the effects of phospholamban ablation on elementary Ca²⁺ release events, we analyzed the spatiotemporal characteristics of spontaneous Ca²⁺ sparks in WT and PLB-KO myocytes. Spontaneous Ca²⁺ sparks were much more frequent in PLB-KO cells, probably due to the higher SR Ca²⁺ load. In PLB-KO myocytes, spark frequencies of up to 82 sparks/s along a 100-µm scan line could be observed. Based on a vertical resolution of ~1 µm in our confocal recording system, this corresponds to a spark frequency of ~3,000 sparks · pl¹ · s¹ (e.g., see Fig. 6A). As shown in Fig. 3, Ca²⁺ sparks in PLB-KO mice were typically larger in amplitude (Fig. 3B). The average spark amplitudes (expressed as F/F₀) was 2.06 ± 0.77 (n = 53; mean ± SD) in PLB-KO myocytes and 0.95 ± 0.27 (n = 49) WT cells, respectively. Furthermore, [Ca²⁺]_i declined faster in PLB-KO myocytes, and the overall spatial spread of sparks was narrower compared with sparks observed in WT myocytes (Fig. 3A). Figure 4 represents a gallery of local Ca²⁺ release events recorded from the same PLB-KO cell, illustrating the heterogeneity among individual sparks. Ca²⁺ sparks in PLB-KO cells showed large variations in amplitude (Fig. 4A). However, the kinetics of rise and decay of [Ca²⁺]_i were virtually identical between large and small sparks (Fig. 4, inset I; normalized transients marked by filled circle and filled square), suggesting that the two sparks originated from the same focal plane and the difference in amplitude was not the result of an out-of-focus event. In cells from PLB-KO mice, there were frequently individual sites of repetitive release (Fig. 4A, trace at bottom). Sparks could trigger additional sparks from immediately adjacent release sites (Fig. 4, B-D), sometimes giving rise to "mini"-[Ca²⁺]_i waves (Fig. 4B). Within the continuous diagonal band of elevated fluorescence of the propagating [Ca²⁺]_i wave, individual release sites could be identified as "hot spots" of high [Ca²⁺]_i. The distance between these hot spots corresponded approximately to the width of a sarcomere, and therefore the distribution of Ca²⁺ between two adjacent release sites reflects intrasarcomeric [Ca²⁺]_i gradients occurring during wave propagation. These intrasarcomeric [Ca²⁺]_i gradients during wave propagation were hardly resolvable in WT myocytes. They clearly support the notion that [Ca²⁺]_i wave propagation is a process of sequential recruitment of Ca²⁺ sparks at the SR-T tubule junctions (see Ref. 6). The presence of such gradients in PLB-KO cells also suggests that a significant amount of Ca²⁺ release was taken up locally into neighboring SR elements before it could trigger release from those SR units. This could explain, in part, the spatial inhomogeneities during twitch [Ca²⁺]_i transients (Fig. 2).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Comparison of spontaneous Ca2+ sparks in WT and PLB-KO myocytes. Line-scan images and [Ca2+]i profiles of sparks recorded from WT (A) and PLB-KO (B) myocytes. In PLB-KO cells, [Ca2+]i declined with time constant () of 45 ms, whereas, in WT cells, average  of 3 sparks shown was 101 ms. Scale bars = 10 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Variability of spontaneous Ca2+ sparks in PLB-KO myocytes. A: line-scan image and subcellular [Ca2+]i profiles, recorded from region marked by black lines. Two sites revealed marked differences in spark amplitude and frequency; however, kinetics of individual sparks was similar despite large differences in amplitude. Inset I: overlay of normalized Ca2+ sparks marked by  and . B: spontaneous Ca2+ sparks could trigger additional sparks in immediate vicinity or in close temporal succession from same site (). Inset II: comparison of normalized [Ca2+]i profiles of an individual ( in A) with "double spark" (). C: example of a double spark triggering an additional spark at a nearby release site (). Inset III: comparison of normalized [Ca2+]i profiles from an individual spark (×) and a double spark (). D: examples of sparks triggering additional sparks in immediate vicinity. Long duration of second localized [Ca2+]i transient is temporal summation of 2 or more sparks occurring at same location in rapid sequence. Scale bar = 10 µm.

[Ca²⁺]_i waves in PLB-KO myocytes. [Ca²⁺]_i waves have been shown to occur in cardiac cells as a result of Ca²⁺ overload of the SR and are caused by spatially propagating regenerative release (by CICR) and reuptake of Ca²⁺. The spontaneous [Ca²⁺]_i waves observed in PLB-KO cells revealed several distinct and unique features not observed in WT myocytes. Overall spontaneous [Ca²⁺]_i waves were more likely to occur in PLB-KO cells, consistent with the overall higher SR Ca²⁺ load in these cells (9, 22, 33). Figure 5 shows an example of multiple spontaneous [Ca²⁺]_i waves in PLB-KO myocytes. Typical features of [Ca²⁺]_i waves in PLB-KO cells are the multiplicity and short time intervals in which they can occur at the same cellular location (see also Fig. 6). Frequently the [Ca²⁺]_i waves appeared to be aborted; i.e., they failed to propagated throughout the entire cell. As shown in Fig. 5 (insets), abortion of a [Ca²⁺]_i wave did not occur abruptly. Typically, the continuous wave front, as visualized in the line-scan image, broke down into distinct localized release events before wave propagation came to a complete stop. The efficient and accelerated reuptake of released Ca²⁺ into the SR, together with narrow spatial spread of the wave front (see below), tended to interrupt the regenerative wave propagation mechanism of CICR and could explain the high frequency of abortive [Ca²⁺]_i waves. Such aborted waves were rare in WT myocytes.

View larger version (81K): [in this window] [in a new window]   Fig. 5.   Aborted [Ca2+]i waves in PLB-KO myocytes. A: line-scan image showing multiple spontaneous [Ca2+]i waves, some of which failed to propagate throughout entire cell. Inset: wave front of an aborted [Ca2+]i wave at higher magnification. Calibration bar = 10 µm. B: 3-dimensional representations ("surface plot") of line-scan image shown in A. Arrows indicate time dimension.

View larger version (77K): [in this window] [in a new window]   Fig. 6.   Enhanced Ca2+ spark and spontaneous [Ca2+]i wave activity triggered by action potentials. A: action potential (AP)-triggered [Ca2+]i transient was followed by a long-lasting period of enhanced Ca2+ spark activity. Bottom, [Ca2+]i profile averaged over entire width of line-scan image. Averaged [Ca2+]i remained above resting levels during period of increased spark occurrence. B: [Ca2+]i transient triggered by an electrically evoked AP was followed by an interval of enhanced spark activity that led to occurrence of multiple spontaneous [Ca2+]i waves. Bottom, cell-averaged [Ca2+]i profile revealing irregular changes of [Ca2+]i during period of enhanced spark and wave activity. Calibration bar = 20 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 7.   Spatiotemporal pattern of spontaneous [Ca2+]i waves in PLB-KO myocytes. A: series of 2-dimensional confocal fluorescence images of spontaneous [Ca2+]i waves recorded at time intervals of 186 ms. Circular [Ca2+]i waves originate at multiple cellular foci. B: spontaneous [Ca2+]i waves originate simultaneously at variable subcellular locations leading to inhomogeneous distribution of [Ca2+]i. C: spatial profiles of normalized [Ca2+]i wave front recorded from WT and PLB-KO myocytes.

View larger version (34K): [in this window] [in a new window]   Fig. 8.   Three-dimensional propagation pattern of [Ca2+]i waves in PLB-KO myocytes. A: line-scan image of a spontaneous [Ca2+]i wave apparently leaving and reentering focal plane. B: local [Ca2+]i transients recorded from equidistant sites separated by ~25 µm (black lines in A) revealed elevations of [Ca2+]i with constant delays consistent with constant propagation of [Ca2+]i wave (apparent propagation velocity = 63 µm/s). Line plots were spatially averaged over distances indicated by black bars to right of A. C: [Ca2+]i profiles recorded at sites indicated by arrows in A. Local [Ca2+]i transients revealed an increase of amplitude and rate of rise of [Ca2+]i during propagation of Ca2+ release, suggesting that [Ca2+]i wave was propagating into confocal plane.

View larger version (73K): [in this window] [in a new window]   Fig. 9.   Peculiarities of propagation patterns of spontaneous [Ca2+]i waves in PLB-KO myocytes. A: line-scan image of multiple spontaneous [Ca2+]i waves that apparently failed to annihilate each other. B: line-scan image of a [Ca2+]i wave reflected at cell border and apparently propagating backward into its own refractory space. For details, see text.

	DISCUSSION

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In this study, we presented new information about how removal of the physiological inhibition of the SR Ca²⁺ pump by phospholamban altered the spatiotemporal pattern of Ca²⁺ signaling during E-C coupling in mouse ventricular cells. We have investigated, with the use of confocal laser scanning microscopy and a genetically engineered mouse model, how the absence of phospholamban altered temporal and spatial properties of electrically evoked [Ca²⁺]_i transients, influenced the occurrence of spontaneous Ca²⁺ sparks, and affected characteristics of [Ca²⁺]_i waves.

The PLB-KO mouse model has proved to be extremely useful for studying the role of SR pump regulation by physiological messengers in the intact cell. Several laboratories have contributed to the structural and functional characterization of this preparation. Specifically, effects on E-C coupling and Ca²⁺ regulation have been of interest. Alteration in contractile properties and [Ca²⁺]_i transients (22, 38), L-type Ca²⁺ current (28, 33), -adrenergic regulation (19, 38), and changes in intracellular protein levels (9) crucial for Ca²⁺ homeostasis and E-C coupling have been studied extensively in PLB-KO myocytes.

Spatial inhomogeneities of [Ca²⁺]_i during E-C coupling in PLB-KO ventricular myocytes. The "local control" model of E-C coupling in mammalian ventricular cells (36) suggests that Ca²⁺ influx through individual L-type Ca²⁺ channels triggers Ca²⁺ release from discrete SR Ca²⁺ release sites. These release sites are close associations of SR and surface or T-tubule membrane, forming diadic or triadic junctions that contain clusters of Ca²⁺ release channels (i.e., ryanodine receptors). The introduction of confocal microscopy to study the subcellular Ca²⁺ dynamics in cardiac cells has provided substantial experimental support for the local control model of Ca²⁺-induced Ca²⁺ release in the myocardium (for review, see Ref. 7). Specifically, the identification of Ca²⁺ sparks (8) as well as the voltage dependence of these subcellular release transients (24) suggested that the global whole cell [Ca²⁺]_i transient was the result of temporal and spatial summation of elementary release events (sparks) originating from discrete subcellular locations. Although in confocal line-scan images under control conditions electrically evoked [Ca²⁺]_i transients appear spatially quite uniform as the result of synchronized activation of release sites along the scanned line (Fig. 2C; see also Refs. 4, 6, 25), localized stochastic failures of E-C coupling have been recognized in rat myocytes (4). These failures became evident under conditions in which the SR Ca²⁺ load decreased during negative staircase or when reduction of Ca²⁺ influx by Ca²⁺-channel blockers (nifedipine or Cd²⁺) resulted in the breakdown of electrically evoked [Ca²⁺]_i transients into individual Ca²⁺ sparks (4, 25). As illustrated in Fig. 2, such distinct spatial inhomogeneities together with local failures of SR Ca²⁺ release could be observed in PLB-KO myocytes even under control conditions. In these cells, individual release sites became apparent as individual Ca²⁺ sparks, and occasionally some of these sites failed to release Ca²⁺ with electrical stimulation (Fig. 2B, filled circles). These failures did not appear to occur at fixed locations, since sites with failures of Ca²⁺ release could reveal release during the following stimulation. Furthermore, local depletion of the SR could be excluded as the cause of local failures, since, in the same cell, [Ca²⁺]_i waves occurred that propagated through these same sites, indicating local release of Ca²⁺. Because spontaneous [Ca²⁺]_i waves are the hallmark of SR Ca²⁺ overload (e.g., Ref. 37), local depletion phenomena are unlikely to produce the observed failures of triggered release.

Spontaneous Ca²⁺ signals in PLB-KO myocytes: increased occurrence of Ca²⁺ sparks and [Ca²⁺]_i waves. Differences between WT and PLB-KO mice regarding spontaneous Ca²⁺ signals include Ca²⁺ sparks of higher and larger variability in amplitude but narrower spatial definition and more rapid decline of [Ca²⁺]_i. The latter is in contrast to a previous study in which an accelerated decay of Ca²⁺ sparks in PLB-KO myocytes was only observed when the SR Ca²⁺ content was reduced to levels found in WT cells (33). Ca²⁺ sparks as well as spontaneous [Ca²⁺]_i waves, both of which occurred more frequently, could be a consequence of an increased SR Ca²⁺ load of PLB-KO myocytes (9, 22). The maximal spark frequencies observed in PLB-KO myocytes was at least an order of magnitude higher than typical for cardiac myocytes (15, 34). The frequency of spontaneous Ca²⁺ sparks was often increased following a train of electrically triggered action potentials (see Fig. 6A). Often this interval of enhanced spark activity led to a period of spontaneous [Ca²⁺]_i wave occurrence (Fig. 6B). The whole cell [Ca²⁺]_i transient (Fig. 6B, line profile) observed under these conditions was reminiscent of the Ca²⁺ signals underlying delayed afterdepolarization due to SR Ca²⁺ overload in cardiac myocytes, which have been shown to be spatially inhomogeneous and to propagate within myocytes as typical [Ca²⁺]_i waves (29).

Narrower wave profile and aborted [Ca²⁺]_i waves. The PLB-KO myocytes demonstrated a narrower spatial and temporal wave profile than the WT myocytes. This is entirely consistent with the faster SR Ca²⁺ pump rate in the PLB-KO hearts and the faster decline and narrower spatial spread of the Ca²⁺ sparks (Fig. 3). Thus, despite evidence for an increased SR Ca²⁺ load ([Ca²⁺]_i waves) in PLB-KO myocytes, the kinetics of spontaneous subcellular [Ca²⁺]_i transients (sparks) indicated that SR Ca²⁺ uptake was still operating at a very high rate and that the highly active SR Ca²⁺ pump was essentially buffering the local Ca²⁺ release from entering adjacent regions. This same characteristic could explain the observation of [Ca²⁺]_i gradients between adjacent release sites during wave propagation (see miniwave in Fig. 4B) and apparent abortion of [Ca²⁺]_i waves (e.g., Fig. 5A); i.e., the ability of a wave to trigger Ca²⁺ release in the next region may be limited by the buffering action of the SR Ca²⁺ pumps along the way. This suggests a remarkably strong negative-feedback action of the SR Ca²⁺ pump in the PLB-KO mouse, especially because it appears able to abort Ca²⁺ waves even at very high SR Ca²⁺ contents. The high SR Ca²⁺ content by itself would be expected to exert a positive feedback, actually enhancing wave activity and propagation. The same mechanism of local buffering through reuptake of Ca²⁺ could also explain why extremely high spark activity did not always lead to the occurrence of spontaneous [Ca²⁺]_i waves (Fig. 6, cf. A and B).

Reduced refractoriness of SR Ca²⁺ release in PLB-KO myocytes. The apparent reduced refractoriness of the SR Ca²⁺ release channels seen in the PLB-KO mouse was somewhat unexpected. This characteristic allowed the same site to produce repeated Ca²⁺ sparks with a very short delay (Fig. 4B) and Ca²⁺ sparks with double peaks (Fig. 4C), [Ca²⁺]_i waves following the same subcellular path in close temporal succession (Fig. 6C) and [Ca²⁺]_i waves to cross one another without annihilation (Fig. 9). Several possible mechanisms could be responsible for these observations. Satoh et al. (34) showed that there is an apparently intrinsic refractoriness of SR Ca²⁺ release after a twitch, demonstrable as a transient reduction in the frequency of Ca²⁺ sparks in rat and rabbit myocytes despite constant SR Ca²⁺ load. They also showed that this refractory period seemed to be overcome when the SR approached the Ca²⁺-overloaded state. A similar mechanism may be responsible for the shorter refractory period seen here in the PLB-KO myocytes, since SR Ca²⁺ load was consistently higher in PLB-KO cells compared with WT myocytes (9, 22, 33). The higher SR Ca²⁺ load might also limit any luminal Ca²⁺ depletion (26) from contributing to refractoriness. In addition to these possibilities, it has to be kept in mind that the kinetics of the indicator fluo 3 may limit its ability to discriminate some changes in free [Ca²⁺]_i (12), may obscure local gradients of [Ca²⁺]_i (3), and may contribute to the appearance of nonannihilating [Ca²⁺]_i waves.