Abstract
In heart, a robust regulatory mechanism is required to counteract the regenerative Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of ryanodine receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca2+ release by using a technique of imaging local Ca2+ release, or “Ca2+ spikes”, at subcellular sites; and we tracked the kinetics of Ca2+ release triggered by L-type Ca2+ channels. At 0 mV, Ca2+ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca2+ channels with the Ca2+ channel agonist, FPL64176, did not prolong or trigger secondary Ca2+ spikes, even though two-thirds of the sarcoplasmic reticulum Ca2+ remained available for release. Latency of Ca2+ spikes coincided with the first openings but not with the reopenings of L-type Ca2+ channels. After an initial maximal release, even a multi-fold increase in unitary Ca2+ current induced by a hyperpolarization to −120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca2+ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca2+ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.
In cardiac myocytes, Ca2+ release from sarcoplasmic reticulum (SR) is activated by the Ca2+-induced Ca2+ release (CICR) mechanism (1–3). Recent evidence suggests that sarcolemmal L-type Ca2+ channels are closely associated with a cluster of sarcoplasmic Ca2+ release channels, called ryanodine receptors (RyRs), in the diadic junctions forming discrete release units (4–10). According to the “cluster bomb” model (11), Ca2+ influx via Ca2+ channels serves as the local Ca2+ signal to activate the coupled RyR(s), causing further increase in local [Ca2+] and cross-activation of other RyRs within the release unit. These local release events have been visualized directly as “Ca2+ sparks” (12), and the recruitment of these events, as a function of Ca2+ channel activation and amplitude of unitary Ca2+ current (iCa), underlies the whole-cell Ca2+ transient induced by Ca2+ current (ICa) (13–15).
Despite improved understanding of the activation process, how Ca2+ release is terminated remains unclear. CICR, with its inherent positive feedback, is expected to operate in an “all-or-none” fashion. Regenerative activation of multiple RyRs within the release units should result in long-lasting Ca2+ sparks and unstable global Ca2+ oscillations. However, Ca2+ sparks of usually short duration (half-time of decay ≈22 ms) (12–14), and Ca2+ transients of graded amplitude and robust global stability occur in intact myocytes (16, 17), indicate that a regulatory mechanism(s) must exist to interrupt regenerative Ca2+ release. Several mechanisms have been proposed for the termination of Ca2+ release. (i) Ca2+-induced inactivation of Ca2+ release (2, 18): Binding of released Ca2+ to an inactivation site of RyRs shifts the channels to an inactivated state and shuts off Ca2+ release. This was originally put forward by Fabiato (2), based on studies in skinned myocytes, as the mechanism to counteract CICR. However, this process has not been demonstrated in intact myocytes (19). (ii) Adaptation of RyRs (20–22): Spontaneous decline in open probability of RyR channels in lipid bilayers after activation by a step increase in [Ca2+]; contrary to inactivation, the adapted channels can be reactivated by subsequent steps to higher [Ca2+]. (iii) Stochastic attrition (11): Simultaneous stochastic closing of RyRs in an active release unit of a few RyRs, reducing local [Ca2+] to a sub-threshold level and thereby extinguishing Ca2+ release. In addition, depletion or reduction of SR Ca2+ also may terminate Ca2+ release due to the lack of releasable Ca2+ or reduction in the gain of CICR (23, 24). To date, no conclusive supporting evidence has been provided for these putative mechanisms.
To elucidate the mechanistic nature of Ca2+ release termination, we applied a confocal-imaging technique (25), which enables both spatial and temporal resolution of local SR Ca2+ release fluxes, or “Ca2+ spikes”, at the t-tubular-SR junctional regions during strong depolarizations, to track the kinetics of local Ca2+ release; and we made use of the Ca2+ channel agonist FPL64176 (FPL) and specific voltage-clamp protocols to manipulate the properties of single Ca2+ channel currents for triggering Ca2+ release. We tested specifically (i) whether increasing open-duration and enhancing reopenings of Ca2+ channels prolongs Ca2+ release and triggers secondary Ca2+ release, respectively, (ii) whether the SR Ca2+ is depleted after a maximal Ca2+ release induced by ICa, and (iii) whether RyRs can be reactivated by a stronger Ca2+ stimulus immediately subsequent to a prior activation. Our results provide direct evidence indicating that Ca2+ release is terminated mainly by a use-dependent inactivation of RyRs, whereas stochastic closing or adaptation of RyRs, or depletion of SR Ca2+ is not the primary cause of release termination in intact ventricular myocytes.
MATERIALS AND METHODS
Single Channel Recordings.
Ventricular myocytes were enzymatically isolated from adult male Wistar rats (200–250 g). Cell-attached, patch-clamp recordings of L-type Ca2+ channels were obtained by using Sylgard (Dow Corning Midland, MI)-coated, thick-walled borosilicate glass pipettes (5–7 MΩ). Pipettes were filled with solution contained (in mM): 0.01 FPL, 10 CaCl2, 130 tetraethylammonium chloride (TEA-Cl), and 10 Hepes (pH 7.4). Cells were bathed in high [K] solution, contained (in mM): 110 potassium aspartate, 30 KCl, 1 MgCl2, 5 Hepes, 5 EGTA, and 3 Na2ATP, pH 7.4, (free [Ca2+] = 100 nM) to approximately zero the membrane potential and enabled estimation of transpatch potentials. Unitary currents were recorded by using a cooled capacitor-feedback headstage (CV203B) and Axopatch 200B amplifier (Axon Instruments, Foster City, CA), low pass-filtered at 1 kHz, and digitized at 10 kHz. Data were collected and analyzed by using the pCLAMP software (Axon Instruments).
Simultaneous Measurement of ICa and SR Ca2+ Release Fluxes.
Myocytes were whole-cell voltage clamped with patch pipettes with tip resistance of 1.5–2.5 MΩ, and superfused with Tyrode’s solution containing (in mM): 137 NaCl, 2 CaCl2, 5.4 KCl, 1 MgCl2, 10 glucose, and 10 Hepes at pH 7.4, with 20 μM tetrodotoxin to block the sodium current. Membrane currents were measured with an Axopatch 200B patch-clamp amplifier. SR Ca2+ release fluxes were detected simultaneously with a novel laser confocal-imaging technique (25), by using the low affinity Ca2+ sensitive dye, Oregon Green 488 BAPTA-5N (OG-5N, Molecular Probes) in conjunction with high [EGTA], to minimize the resident time of free-released Ca2+ in the cytoplasm and to optimize the detection of localized high [Ca2+] in the release sites. The pipette solution contained (in mM): 105 CsCl, 10 NaCl, 5 MgATP, 10 Hepes, 20 TEA-Cl, 4 EGTA, 2 CaCl2, and 1 OG-5N at pH 7.2, to eliminate K+ currents and buffer-free [Ca2+] at 150 nM for adequate Ca2+ loading of SR. EGTA (4 mM) has no significant effect on the local Ca2+ signaling between L-type Ca2+ channels and RyRs (9). Confocal images were acquired by using a Zeiss LSM-410 inverted confocal microscope with a Zeiss Plan-Neofluor 40x oil immersion objective (NA = 1.3), and the confocal pinhole was set to render spatial resolutions of 0.4 μm in the x–y axis and 0.9 μm in the z-axis. OG-5N was excited by the 488 nm line of an argon laser, and fluorescence was measured at >515 nm. Images were taken in the line-scan mode, with 512 pixels/line (0.104 μm/pixel) scanned at 2.09 ms intervals, and processed by using idl software (Research System, Boulder, CO). Conventional whole-cell Ca2+ transients were performed in some myocytes with methods described previously (9). All external solutions bathing the myocytes were exchanged rapidly (≈200 ms) with a concentration-clamp system to avoid changes in SR loading, and caffeine was rapidly applied by using a pico-spritzer. All experiments were performed at room temperature (20–22°C).
Data Analysis.
Single Ca2+ channel records were leak and capacitive current eliminated by subtracting the original traces with blank sweeps. Open events were idealized by half-height criteria, and single channel patches were verified by the absence of stacked openings in entire data sets (>900 sweeps). Amplitudes of iCa were estimated from the Gaussian distributions of single channel currents, and open-time distribution was fitted with a bi-exponential probability distribution function. Latency distributions and averaged currents of the first openings and reopenings of Ca2+ channels were constructed from idealized events. Ca2+ release-induced inactivation of ICa was quantified as the fraction of peak ICa at 25 ms of clamp pulses (I25 ms/Ipeak) and compared before and after complete depletion of SR Ca2+ with 10 mM caffeine. SR Ca2+ release fluxes were determined from line-scan confocal images as described by Song et al. (25). Briefly, spatially averaged OG-5N fluorescent signals from confocal images were normalized with basal fluorescence and expressed as F/F0. The change in the OG-5N signal (ΔF/F0), in the presence of high EGTA concentration, is the sum of two components, a prominent spike component directly related to SR Ca2+ release fluxes (fr), and a small pedestal component (fs) representing the weighted running integral of the release fluxes, thus ΔF/F0 = fr + fs, where fs = α∫frdt. fs and α were determined experimentally by fitting fs to the pedestal level of ΔF/F0 after repolarization, at which Ca2+ release was expected to be zero. fr was then generated by subtracting fs from the ΔF/F0 trace. Numerical simulation by using realistic buffer kinetics and concentrations showed that Ca2+ spikes reproduced well the waveform of Ca2+ fluxes, with an “on” and “off” response time of <1 ms, and the amplitude of the spike was linearly related to Ca2+ release flux over a wide range (25). All data were expressed as mean ± SEM and were compared by using paired t tests. P values <0.05 were considered statistically significant.
RESULTS AND DISCUSSIONS
Stochastic Closing of RyRs and Termination of SR Ca2+ Release.
Previous numerical analysis of whole-cell Ca2+ transients (26, 27) and our recent direct measurement of SR Ca2+ release fluxes (25) revealed that Ca2+ release occurs and terminates shortly after the onset of a depolarizing pulses. Because L-type Ca2+ channel openings are brief (28–30), and reopenings of Ca2+ channels are inhibited to a large extent by the inactivation induced by Ca2+ release from SR (8–10) and Ca2+ influx via Ca2+ channels (29, 30), the transient nature of SR Ca2+ release may simply reflect the random stochastic closing of RyRs in the absence of a sustained trigger (11). In this scenario, an increase in the open duration of Ca2+ channels should provide a more prolonged stimulus to sustain the regenerative activation of RyRs; and reopenings of Ca2+ channels after release termination should provide new stimuli to reactivate the extinguished RyRs. To test this paradigm, the Ca2+ channel agonist FPL64176 (FPL) (31, 32) was used to prolong the open duration and enhance the reopenings of Ca2+ channels. Fig. 1 shows single Ca2+ channel currents recorded under cell-attached mode with Ca2+ (10 mM) as the charge carrier in the presence of FPL (10 μM). After depolarization to 0 mV, the probability of an active sweep was 0.60 ± 0.05, and open probability (Po) of the active sweeps was 0.28 ± 0.04 (n = 3). The high Po was mainly due to a prolonged open lifetime (mean open time = 15.9 ± 2.9 ms, n = 3 patches) (Fig. 1 A and B), which was ≈2 orders of magnitude longer than those recorded without FPL (mean open-time = 0.27 ms) (28). Latency distributions show that the first openings dominated the first 10 ms, and reopenings of Ca2+ channels constituted virtually all of the events after 30 ms of the clamp pulse, giving rise to the prominent maintained phase of ICa (Fig. 1 C and D). Additionally, FPL abolished the inactivation of Ca2+ channel induced by Ca2+ release. Under control conditions, whole-cell ICa elicited at 0 mV inactivated rapidly (I25 ms/Ipeak = 0.13 ± 0.01, n = 13); the rate of inactivation was significantly reduced when SR Ca2+ release was abolished by 10 mM caffeine (I25 ms/Ipeak = 0.28 ± 0.02, P < 0.05). In the presence of FPL, inactivation of ICa was prolonged significantly (I25 ms/Ipeak = 0.71 ± 0.03, n = 13); inhibition of Ca2+ release with caffeine had minimal effect on the maintained phase and the rate of inactivation of ICa (I25 ms/Ipeak = 0.73 ± 0.03, n = 13) (Fig. 1 E and F). Reopenings of Ca2+ channels, thus, were not prevented by Ca2+ release triggered by their own first openings in the presence of FPL and provided multiple stimuli to the coupled RyRs during a depolarizing pulse.
Confocal images of SR Ca2+ release fluxes showed that depolarizations to 0 mV activated spatially discrete, localized Ca2+ spikes, which occurred and terminated within 40 ms after the onset of clamp pulse (Fig. 2A). They usually occurred only once at each release site (≈1.8 μm apart), without reactivation during the later part of depolarization. Estimations based on anatomical data (5, 33, 34) suggest that a resolvable volume (0.144 μm3) of confocal images encompasses multiple diadic junctions; hence, a Ca2+ spikes represents the ensemble Ca2+ release fluxes originated from multiple release units within the same site. Rapid application of FPL (10 μM, 10 s before a clamp-pulse) caused an immediate enhancement of ICa (Ipeak = 0.92 ± 0.11 nA in control and 3.64 ± 0.34 nA in FPL, n = 10, P < 0.05), a slowing of its inactivation, and an increase in the spatially averaged release transient (ΔF/F0 = 0.18 ± 0.02 in control, and 0.23 ± 0.02 in FPL, n = 10, P < 0.05). Surprisingly, the time course of Ca2+ release was unaltered (time to peak = 17.2 ± 1.2 ms in control and 15.2 ± 0.9 ms in FPL; time to 75% relaxation = 35.9 ± 2.1 ms in control and 34.2 ± 2.6 ms in FPL, n = 10); and no major secondary Ca2+ release was observed despite the presence of a prominent maintained ICa. The disparity between the kinetics of ICa and Ca2+ release is illustrated by superimposing the peak normalized ICa and spatially averaged Ca2+ release transients (Fig. 2A Right Bottom). These results were confirmed by using the first derivative of “conventional” Indo-1 Ca2+ transient (d[Ca2+]/dt), as an empirical indicator of Ca2+ release (data not shown). Latency analysis shows that the occurrence of Ca2+ spikes, mostly within 2–10 ms of depolarization, coincided with the first latency of Ca2+ channels, but was completely dissociated from Ca2+ channel reopenings (Fig. 2B). These results argue against the stochastic attrition (11) as the primary mechanism for terminating Ca2+ release, because it predicts an increase in open duration of L-type Ca2+ channel would prolong Ca2+ release, and multiple Ca2+ channel reopenings would simply give rise to multiple release events. Moreover, the finding that Ca2+ release at 0 mV was activated exclusively by the first openings of Ca2+ channels (35) is consistent with the previous finding that Ca2+ release is gated by the initial phase of ICa (7, 14, 26, 27, 31, 36), but is in contrast to the observation that the latency distribution of Ca2+ sparks in the presence of Ca2+ channel blocker resembles the time course of ICa (13, 15). In the latter case, however, only a few Ca2+ release units were triggered at the onset, hence leaving plenty of unfired RyRs for activation in the later part of depolarizing pulses.
Recovery of Ca2+ release also was examined by applying a second depolarizing pulse at different intervals (50–1200 ms) after a maximal initial release at 0 mV. An apparent absolute refractory period of ≈150 ms was observed, followed by a second phase of recovery of Ca2+ release with a half-time of ≈500 ms (data not shown), similar to the previous observation on the interactions of evoked Ca2+ release with Ca2+ waves (38). The refractory period after a maximal Ca2+ release was, hence, significantly longer than that following a spontaneous spark (≈30 ms) (39) generated by only a single/few RyRs.
SR Ca2+ Depletion and Termination of Ca2+ Release.
The refractoriness of SR after a maximal Ca2+ release could be due to global or local depletion of SR Ca2+. To explore this possibility, high concentration of caffeine was applied to myocytes, which was exposed to FPL for 10 s, via a pico-spritzer at 40 ms after depolarization to cause complete release of Ca2+ from the SR. The rapid injection of caffeine caused a large release of Ca2+ during the otherwise silent later period of the clamp pulse (Fig. 3A), with Ca2+ spikes occurring at sites activated by ICa before the caffeine application. The amount of Ca2+ released by ICa compared with caffeine, quantified by integrating their respective release transients, had a ratio of 1:2.2 (Fig. 3B). Assuming the total releasable SR Ca2+ equals the sum of Ca2+ released by ICa and caffeine, the fractional release of Ca2+ induced by ICa was 33.1 ± 3.4% (n = 6), consistent with previous estimations (24, 40). This indicates that SR Ca2+ was not depleted after the initial release, and a substantial amount of Ca2+ was immediately available for release when RyRs were allowed to reopen. Moreover, the reduction of SR Ca2+ by one-third was unlikely the primary mechanism for the refractoriness of SR because ICa is able to trigger Ca2+ release after a similar reduction of Ca2+ loading (40), and Ca2+ release can be elicited within 2–3 depolarizing pulses immediately after complete depletion of SR by caffeine(9, 17, 40), suggesting that SR is capable of releasing Ca2+ with an even lower Ca2+ content (less than two-thirds of normal load). Moreover, spontaneous Ca2+ sparks were observed after 64% reduction in SR Ca2+ loading (41). However, the data do not exclude the possibility that the partial SR Ca2+ depletion may play a contributing role in terminating Ca2+ release by altering RyR activities (42, 43), and reducing the gain of CICR (24).
Inactivation vs. Adaptation of RyRs in Terminating SR Ca2+ Release.
The refractoriness of SR to reopenings of L-type Ca2+ channels could be due to RyR adaptation or inactivation. To distinguish between these two possibilities, we devised a voltage-clamp protocol to test whether the once fired RyRs could be reactivated by a stronger Ca2+ stimulus, as would be predicted by the adaptation, but not by the inactivation hypothesis. Depolarizing pulses to either −30, 0, +30, or 60 mV were applied for 50 ms, followed by a hyperpolarization to −120 mV. Single channel recordings in the presence of FPL showed that iCa at −30, 0, and +30 mV were −0.42, −0.21, and −0.06 pA, respectively. Hyperpolarizing steps to −120 mV caused an instantaneous jump of iCa to −1.00 pA (Fig. 4A) due to an increase in the electrochemical gradient for Ca2+ influx. According to the adaptation hypothesis, the multi-fold increase in iCa during the hyperpolarizing steps should provide a local [Ca2+] sufficient to reactivate the adapted RyRs (20), even though these RyRs no longer respond to the smaller iCa of Ca2+ channel reopenings during depolarization. However, when this protocol was applied to intact myocytes, hyperpolarizing steps subsequent to the maximal Ca2+ releases elicited by depolarizations to −30 and 0 mV failed to trigger, or only activated a minimal Ca2+ release, despite the activation of a larger tail than initial ICa (Fig. 4 B and C). This result argues against the adaptation of RyRs but supports the hypothesis of strong inactivation of RyRs after their initial activation.
Yasui et al. (22) showed that depolarization to +30 mV, in the presence of FPL, elicits a transient Ca2+ release that terminates despite continued ICa, yet additional Ca2+ release is triggered by the tail current after repolarization; this has been interpreted as the evidence for adaptation of RyRs in situ. When our myocytes were first depolarized to +30 (or +60 mV) to activate a submaximal Ca2+ release, the subsequent hyperpolarizing step indeed triggered a “tail” release transient (Fig. 4B Right). However, the total amount of Ca2+ released by the depolarizing pulse and the subsequent hyperpolarizing step was similar to the maximal Ca2+ release at −30 or 0 mV (Fig. 4D), indicating that the large tail iCa did not trigger additional Ca2+ release from RyRs that fired during depolarization; rather, the tail transients likely represent the activation of release units that were not opened by the small iCa during the submaximal initial release. Because a Ca2+ spike is the ensemble release fluxes from multiple release units within a junctional site, in the latter case, the tail currents should elicit larger Ca2+ spikes in junctional sites where only a few release units were activated by the preceding depolarizing pulse, and trigger smaller Ca2+ release in sites where most of the release units were fired during the initial activation. Indeed, such a complementary release pattern was apparent at individual junctional sites: large initial Ca2+ spikes were often followed by either no or small tail releases at the same sites (Fig. 5A, sites 2, 4, and 6), whereas small or no initial releases were usually associated with larger tail Ca2+ spikes (Fig. 5A, sites 1, 3, and 5). Quantitatively, the amplitude of the initial Ca2+ spikes, elicited in 34 different release sites, correlated negatively (r = −0.44, n = 170, P < 0.001) with the tail Ca2+ spikes (Fig. 5B). These results clearly indicate that the large tail Ca2+ spikes were not generated by the reactivation of adapted RyRs because they were originated from sites where the initial activation of RyR was absence or minimal. The tail current failed to elicit secondary Ca2+ spikes from sites of large initial release further supports the inactivation of once activated RyRs at subcellular release sites.
Concluding Remarks.
Using the imaging technique to directly measure SR Ca2+ release fluxes, in conjunction with the unique agonist, FPL64176, to manipulate the gating properties of L-type Ca2+ channels, we have provided compelling evidence that SR Ca2+ release during excitation–contraction coupling is terminated mainly by a local inactivation of RyRs in intact myocytes, whereas stochastic attrition, depletion of SR Ca2+, and the adaptation of RyRs observed in lipid-layers (20, 21) do not participate or only play a contributing role in terminating Ca2+ release in situ. This inactivation of RyRs may depend on the high local [Ca2+]consequential to their own Ca2+ release, as suggested previously in skinned fibers (2, 18), SR vesicles (44), and in single RyRs in lipid bilayers (45–47), as well as recently in intact myocytes (48), showing that the rate of Ca2+ spark termination is related to the magnitude of release flux. However, the possibility that the process is obligated to the activation of RyRs per se (49) cannot be excluded. Nevertheless, the inactivation process is highly localized, and use-dependent, and may be modulated, e.g., by cyclic adenosine monophosphate (21), Ca2+/calmodulin-dependent protein kinases (50, 51), or FK506-binding protein (52–54). Because of this use-dependent inactivation, RyRs once activated are precluded from reactivation during a cardiac cycle; therefore, it interrupts the positive feedback of CICR, providing both global and micro-stability in cardiac myocytes. Since the mobilization of Ca2+ from intracellular stores through Ca2+ release channels is pivotal for signal transduction in many cell types, our findings as well as our experimental approaches may have important applications to studies of Ca2+ signaling in general.
Acknowledgments
We thank Dr. D. T. Yue for the valuable comments and Dr. H. Spurgeon and B. Ziman for the technical supports. This work was supported by the National Institutes of Health Intramural Research Programs (to H.C., M.D.S., and E.G.L.), extramural grant (HL-52652, to J.S.K.S.), and an American Heart Association Established Investigator award (to J.S.K.S.).
ABBREVIATIONS
- RyRs
ryanodine receptors
- SR
sarcoplasmic reticulum
- CICR
Ca2+-induced Ca2+ release
- FPL
Ca2+ channel agonist, FPL64176
- OG-5N
Oregon Green 488 BAPTA-5N
- iCa
unitary Ca2+ current
- ICa
whole-cell Ca2+ current
Footnotes
This paper was submitted directly (Track II) to the Proceedings Office.
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