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(Circulation Research. 2007;101:590.)
© 2007 American Heart Association, Inc.

Cellular Biology

Voltage Dependence of Cardiac Excitation–Contraction Coupling

Unitary Ca²⁺ Current Amplitude and Open Channel Probability

Julio Altamirano, Donald M. Bers

From the Department of Physiology, Loyola University Chicago, Maywood, Ill. Present address for J.A.: Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore.

Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Chicago, Stritch School of Medicine, 2160 S First Ave, Maywood, IL 60153. E-mail dbers{at}lumc.edu

	Abstract

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Excitation–contraction coupling in cardiac myocytes occurs by Ca²⁺-induced Ca²⁺ release, where L-type Ca²⁺ current evokes a larger sarcoplasmic reticulum (SR) Ca²⁺ release. The Ca²⁺-induced Ca²⁺ release amplification factor or gain (SR Ca²⁺ release/I_Ca) is usually assessed by the V_m dependence of current and Ca²⁺ transients. Gain rises at negative V_m, as does single channel I_Ca (i_Ca), which has led to the suggestion that the increases of i_Ca amplitude enhances gain at more negative V_m. However, I_Ca=NP_oxi_Ca (where NP_o is the number of open channels), and NP_o and i_Ca both depend on V_m. To assess how i_Ca and NP_o separately influence Ca²⁺-induced Ca²⁺ release, we measured I_Ca and junctional SR Ca²⁺ release in voltage-clamped rat ventricular myocytes using "Ca²⁺ spikes" (confocal microscopy). To vary i_Ca alone, we changed [Ca²⁺]_o rapidly at constant test V_m (0 mV) or abruptly repolarized from +120 mV to different V_m (at constant [Ca²⁺]_o). To vary NP_o alone, we altered Ca²⁺ channel availability by varying holding V_m (at constant test V_m). Reducing either i_Ca or NP_o alone increased excitation–contraction coupling gain. Thus, increasing i_Ca does not increase gain at progressively negative test V_m. Such enhanced gain depends on lower NP_o and reduced redundant Ca²⁺ channel openings (per junction) and a consequently smaller denominator in the gain equation. Furthermore, modest i_Ca (at V_m=0 mV) may still effectively trigger SR Ca²⁺ release, whereas at positive V_m (and smaller i_Ca), high and well-synchronized channel openings are required for efficient excitation–contraction coupling. At very positive V_m, reduced i_Ca must explain reduced SR Ca²⁺ release.

Key Words: calcium-induced calcium release • excitation–contraction coupling

	Introduction

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Cardiac myocyte excitation–contraction coupling (ECC) occurs by Ca²⁺-induced Ca²⁺ release (CICR),^1–3 where a small Ca²⁺ current (I_Ca) through L-type Ca²⁺ channels (LCCs) locally controls a larger Ca²⁺ release from the sarcoplasmic reticulum (SR) via a closely apposed cluster of ryanodine receptors (RyRs).^4,5 Whole-cell SR Ca²⁺ release magnitude is finely graded by the amplitude of I_Ca, and both variables have similar, but not identical, bell-shaped dependence on membrane voltage (V_m).⁶ However, these events have underlying unitary components with different V_m dependences. There are 30 000 spatially discrete junctions or dyads per myocyte, each of which contains an average of 12 LCCs and 100 RyRs,^4,7 although the precise number of channels has variance that may be functionally important.⁸ Whole-cell Ca²⁺ transients result from the temporal and spatial summation of many independent Ca²⁺ release events known as Ca²⁺ sparks that are synchronized by I_Ca^9–13 and the characteristics of which are V_m independent.^11–14

I_Ca is the ensemble of single channel currents (i_Ca) through thousands of individual open channels (NP_o; I_Ca=i_CaxNP_o), and i_Ca amplitude and NP_o both depend on V_m (Figure 1d).¹⁵ At each dyad, CICR efficacy could be governed by either i_Ca amplitude or by local NP_o (which should parallel overall NP_o). Earlier voltage-clamp studies measuring global SR Ca²⁺ release¹⁰ or Ca²⁺ sparks^11–13 in voltage steps V_Test from constant holding potential (V_Hold) showed that the V_m dependence of ECC gain was similar to that predicted for i_Ca. They inferred that higher i_Ca amplitude at negative V_m, increases "coupling fidelity" (ie, the probability that i_Ca trough an open channel triggers a Ca²⁺ spark), causing the increase in CICR gain. However, because NP_o also depends on V_m (over the same range), NP_o changes might also be important, and no prior study separated the impact of i_Ca versus NP_o on CICR gain. That is our aim here.

View larger version (38K): [in this window] [in a new window]   Figure 1. Voltage dependence of ICa and SR Ca2+ release in OG-5N/EGTA-loaded myocytes. a, Two-dimensional image and longitudinal scan-line position. Normalized (F/F0) line-scan image during depolarization from VHold=–60 to 0 mV (right); ordinate is distance and abscissa is time. Trace below is spatially averaged F/F0 (bar=0.1 F/F0).The inset enlargement shows local Ca2+ release events. b, Peak ICa (filled) and whole-cell SR Ca2+ release flux (open) (F/F0) vs VTest (n=7 to 8). c, CICR gain (peak F/F0)/(peak ICa) vs VTest. d, Voltage dependence of unitary ICa determinants: activation curve (open circles) (NPo=ICa/[VTest–VCa], where VCa is ICa reversal potential). Solid curve is least-squares Boltzmann fit to mean data: V50=–20 mV, slope=4. The broken line indicates Vm dependence of iCa amplitude from Goldman–Hodgkin–Katz equation (iCa=FkVm [4PCa([Ca2+]o–[Ca2+]i exp[–2kVm])/(1–exp[2kVm])], where F=9.648x10–4 Coulombs/mol; PCa=4x10–5 cm/sec; k=1/25.7 mV–1; [Ca2+]o=1 mmol/L; [Ca2+]i=150 nmol/L). The ICa (filled circles) data are as in b.

Measuring local SR Ca²⁺ release events during ECC is limited by spatial overlap of signals from neighboring junctions. Spatial separation of release events requires either drastic reduction of the number of active LCCs (by negative V_Test or channel blockers)^11,13 or trapping released Ca²⁺ by mM EGTA in combination with a fast local Ca²⁺ indicator (Ca²⁺ spikes).^16–19 Here we measure Ca²⁺ spikes during voltage clamp to separate the role of i_Ca and NP_o in controlling ECC efficacy. This approach also ensures constant cytosolic and SR [Ca²⁺] at each pulse. Surprisingly, we found that increasing either i_Ca or NP_o alone decreases ECC gain. The results also suggest that the small i_Ca at a V_Test of 0 mV may be a highly effective trigger of SR Ca²⁺ release and also that redundancy of Ca²⁺ channel opening at individual junctions is critical in how local control of SR Ca²⁺ release occurs.

	Materials and Methods

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Voltage Clamp
Enzymatically isolated rat ventricular myocytes were voltage clamped in a ruptured patch whole-cell configuration, allowing intracellular dialysis with a solution containing (in mmol/L): 115 CsCl, 10 NaCl, 10 tetraethylammonium chloride, 1 MgCl₂, 5 MgATP, 0.3 NaGTP, 20 Hepes, 1 CaCl₂ (free [Ca²⁺]150 nmol/L), 2 EGTA (K_d150 nmol/L), and 1 Oregon green 488 BAPTA-5N (OG-5N) (K_d31.4 µmol/L),¹⁶ pH 7.2 with CsOH. Patch pipettes filled with this solution had resistance of 1.5 to 2.5 M. Control bath solution contained (in mmol/L): 137 NaCl, 6 CsCl, 1 MgCl₂, 1 CaCl₂, 10 glucose, 10 Hepes, and 0.02 tetrodotoxin, pH 7.4 with NaOH. Cells were dialyzed for 10 minutes before data recording. Experiments were conducted at 23°C. Voltage control and I_Ca recording were achieved with an Axopatch 200 and pClamp 8 software (Axon Instruments), which also controlled bath solution exchange and confocal-scanning synchronization. In control conditions, I_Ca and Ca²⁺ release were activated by a series of 200-ms depolarizing steps (V_Test) to –40 to +30 mV in increments of 10 mV from V_Hold of –55 to –60 mV. Stability of SR Ca²⁺ load was confirmed with caffeine-induced Ca²⁺ transients. To vary NP_o, we altered V_Hold between –60 and –25 mV during 17 seconds. I_Ca was evoked by 200-ms depolarizing steps to 0 mV. To vary i_Ca amplitude over a wide range, we used 2 complementary protocols. (1) Rapid (100 ms) exchange of the bath solution [Ca²⁺] ([Ca²⁺]_o; 0.25 to 10 mmol/L) from the standard 1 mmol/L for 2 seconds. After 1.5 seconds of complete switch of external solution (sufficient time for T-tubular equilibration of [Ca²⁺]_o in preliminary I_Ca tests and prior studies²⁰ where =200 ms), cells were depolarized to 0 mV for 200 ms from V_Hold of –60 mV. (2) Cells were predepolarized to +120 mV (15 ms; V_Hold=–55 mV) to activate LCCs without Ca²⁺ influx and then repolarized to V_Test between 0 to +60 mV ("tail currents"). [Ca²⁺]_o (1 mmol/L) was constant throughout this protocol. Because tail currents can be contaminated easily, we repeated protocols in the same cell with CdCl₂ (1 mmol/L) present, and used the Cd²⁺-sensitive tail currents as I_Ca.²¹

Confocal Imaging of Ca²⁺ Release Flux
An inverted microscope (Eclipse, TE-2000-U, Nikon) was interfaced with a confocal scan head (Radiance 2100, controlled by Lasersharp 2000 software; Bio-Rad) using a Plan Fluor x40, 1.3 NA oil immersion objective lens. OG-5N was excited at 488 nm (argon laser at 3% to 6% of maximum), with emission collected at >500 nm. Confocal data were acquired in line-scan mode at 500 Hz (with a pixel size of 120 nm and pinhole optimized for resolution of 0.4 µm in the focal plane and <1 µm in the z-axis. Image processing used an algorithm written in IDL (Research Systems) provided by Dr H. Cheng (NIH, Baltimore, Md).¹⁶ Fluorescent images are normalized as F/F₀, where F is fluorescence intensity and F₀ is average fluorescence at rest. The fraction of active junctions during voltage-clamp pulses (200 ms) was determined using a spike threshold of F/F₀ of 0.2. For comparing SR Ca²⁺ release among different protocols, data were normalized to values for a depolarization from V_Hold of –60 to 0 mV with [Ca²⁺]_o=1 mmol/L. For the tail I_Ca protocol, values were normalized to those on repolarization to 0 mV.

Statistics
The data are presented as means±SEM. Paired Student’s t test or ANOVA, followed by all pairwise multiple comparison, were used when appropriate. P<0.05 was considered significant.

	Results

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Ca²⁺-Induced Ca²⁺ Release Gain, Membrane Potential, and Fractional Release
We measured whole-cell I_Ca and individual local SR Ca²⁺ release (Ca²⁺ spikes) with confocal microscopy (Figure 1a). Ca²⁺ spikes are discernable and proportional to release rate, as Ca²⁺ transiently binds to OG-5N before being absorbed by EGTA.¹⁶ Electrically (and caffeine) evoked Ca²⁺ spikes occur at regular intervals (2 µm) along the length of the cell, corresponding to the location of transverse tubule–SR junctions at the z-line of each sarcomere.¹⁶ These represent junctional SR Ca²⁺ release flux underlying ECC. The peak of the spatially averaged fluorescence (F/F₀) is an index of overall SR Ca²⁺ release flux.¹⁶ Figure 1b displays the typical bell-shaped V_m dependence of I_Ca and the whole-cell SR Ca²⁺ release flux (F/F₀). The classic V_m dependence of ECC gain computed from these data is in Figure 1c. Because i_Ca and NP_o are V_m sensitive (and change in opposite directions; Figure 1d), the rising phase of the gain curve, in principle, could be attributable to either increasing i_Ca, decreasing NP_o or both. Therefore, to assess the independent roles of NP_o and i_Ca on CICR gain, we devised voltage-clamp protocols to isolate their impact, independent of V_m at constant SR Ca²⁺ load.

Fast caffeine-induced Ca²⁺ release (Figure 2a) was used to verify the stability of SR Ca²⁺ load. It also allowed us to identify all release sites along a scan line (100% active junctions) and to infer the overall fractional release of Ca²⁺ from the SR (using the pedestal of [Ca²⁺]_i elevation after the peak release sensed by OG-5N equilibrated with EGTA; vertical blue arrows). Whereas I_Ca could often activate 80% of junctions,¹⁷ the integrated SR Ca²⁺ release was only 50% of that released by caffeine (Figure 2b).

View larger version (49K): [in this window] [in a new window]   Figure 2. Caffeine-induced Ca2+ transients, fraction of active junctions, and fractional SR Ca2+ release. a, Line-scan images of Ca2+ release during depolarization from –60 to 0 mV (top) and rapid caffeine application without depolarization (bottom). Traces are spatially averaged fluorescence (mean of 8 cells). b, Pooled data of fraction of active junctions (left) in response to ICa (black) and caffeine (red), steady-state fluorescence pedestal (F/F0) (middle) and fractional SR Ca2+ release (right).

Altering the NP_o at Constant i_Ca
The effect of altered NP_o alone on CICR gain was assessed by varying Ca²⁺ channel availability by changing V_Hold, with Ca²⁺ release evoked by depolarizations to the same V_Test (such that i_Ca was always identical; Figure 3a).²² Indeed, changes of V_Hold over this range do not prolong single channel open time^23,24 and did not significantly alter normalized I_Ca time course when SR Ca²⁺ release was blocked (data not shown). Thus, changes in I_Ca in Figure 3 reflect changes in NP_o. This allowed variation of NP_o over a wide range (>10-fold; n=23 to 24). The confocal line-scan images show that the number of Ca²⁺ spikes (active junctions) increase at negative V_Hold (higher NP_o; Figure 3c), and this was also evident as increased global Ca²⁺ spike amplitude and integrated SR Ca²⁺ release (see Figure I in the online data supplement at http://circres.ahajournals.org). Indeed, gradation of global SR Ca²⁺ release flux with NP_o is largely caused by the number of junctions firing (see linear correlation in Figure 3b). There was also a minor change in mean Ca²⁺ spike amplitude (20% to 25%), which correlates with and may be secondary to the larger fraction of active junctions (see the online data supplement). This may be attributable to recruitment of multiple junctions within the confocal-assessed volume.¹⁷ ECC gain (fraction of active junctions/peak I_Ca) decreased monotonically as a function of V_Hold (with increasing NP_o), indicating that CICR is more efficient when fewer LCCs open (Figure 3d). Thus reduced NP_o could explain the increasing ECC gain at negative V_m in Figure 1c. However, can increasing i_Ca also explain it?

View larger version (43K): [in this window] [in a new window]   Figure 3. NPo and SR Ca2+ release flux. a, Voltage-clamp protocol to vary NPo (at constant iCa) and ICa records (top), simultaneous line-scan confocal images and spatially averaged OG-5N/EGTA signals (bottom). b, Pooled data for fraction of active junctions (local Ca2+ spikes) plotted vs SR Ca2+ release (peak F/F0; n=23 cells). c, Pooled ICa amplitude (filled circles) and fraction of active junctions (open circles) vs VHold. Black curve (ICa) is a least-squares Boltzmann fit (V50=–34.1 mV, slope=5). d, CICR gain as fraction of active junctions/ICa (normalized to value at VHold=–60 mV) vs trigger ICa. Curve is least-squares single-exponential fit (k=1.2).

Altering i_Ca at Constant NP_o
In the limiting case, where i_Ca is nearly 0, it may not activate SR Ca²⁺ release, but the intrinsic i_Ca dependence of gain has not been previously measured. Here we altered i_Ca amplitude (at constant NP_o) using 2 different, but complementary, protocols. First, i_Ca was varied by abruptly changing [Ca²⁺]_o just before (and only during) the depolarization to a constant V_Test (ensuring constant NP_o). Figure 4a shows I_Ca, confocal line scans, and spatially averaged Ca²⁺ release in a representative cell. The fraction of active junctions decreased as [Ca²⁺]_o and i_Ca were lowered, especially at [Ca²⁺]_o1 mmol/L. These results show 2 key points. First, most of the drop in the fraction of junctions firing (Figure 4b) occurs only at very low i_Ca ([Ca²⁺]_o < 0.5 mmol/L) and is not increased as i_Ca is elevated by raising [Ca²⁺]_o from 1 to 10 mmol/L. This implies that the small i_Ca at 0 mV has a high coupling fidelity and can effectively trigger Ca²⁺ release and that raising i_Ca does not evoke additional release.²⁵ Second, increasing i_Ca over a broad range causes a monotonic decline in ECC gain (Figure 4c). Both of these points indicate that increasing i_Ca (eg, at more negative V_m) neither enhances coupling fidelity nor increases ECC gain (in contrast with earlier interpretations).

View larger version (39K): [in this window] [in a new window]   Figure 4. iCa vs SR Ca2+ release flux. a, Protocol and ICa with rapidly switched [Ca2+]o. Small effects of [Ca2+]o on NPo via surface potential effects are minimal at Vm=0 mV, because NPo is near maximum. Simultaneous line-scan images and spatially averaged fluorescence (bottom). b, Pooled data for peak ICa (closed circles) and fraction of active junctions (open circles) vs [Ca2+]o, (n=10 to 26 cells). Curves are least-squares exponential fits to mean data. c, CICR gain assessed as in Figure 3, plotted vs trigger ICa. Curve indicates a single exponential fit (k=0.1).

The second method to manipulate i_Ca at constant NP_o extended this analysis to lower i_Ca and also simulated better physiological action potentials (APs), where real ECC occurred. Here (Figure 5a), we first fully activated channels (high NP_o) by depolarization to +120 mV (which prevents Ca²⁺ entry). After 15 ms, the cell was repolarized to different V_Test to increase Ca²⁺ driving force and i_Ca (tail current), but with the same initial NP_o at each pulse.²¹ The magnitude of Ca²⁺ release (and fraction of active junctions) was largest with a V_Test near 0 mV (largest tail I_Ca) and decreased at more positive V_Test (Figure 5a and 5b). Because LCCs were preactivated during the pulse to +120 mV, the mean Ca²⁺ spike latency was short and unaffected by V_Test (supplemental Figure II). Figure 5c shows that ECC gain increases as i_Ca is reduced, even at the lowest i_Ca. This is surprising, because i_Ca amplitude ought to be limiting at some level. However, these data suggest that under relatively physiological conditions, at positive V_m, the intrinsic low coupling fidelity of small i_Ca may be counteracted by the synchronous activation of multiple LCCs.

View larger version (39K): [in this window] [in a new window]   Figure 5. iCa vs SR Ca2+ release flux evoked by repolarization. a, Voltage-clamp protocol and Cd2+-sensitive tail ICa (constant [Ca2+]o= 1 mmol/L). At VTest+50 mV, no Ca2+ was released until repolarization to VHold. b, Pooled peak ICa data (closed circles) and fraction of active junctions (open circles) as a function of VTest (n=8). Curve indicates single exponential fit to mean ICa (k=0.04). c, CICR gain plotted vs trigger ICa.

Composite Results
Figure 6a illustrates the relative efficacy of I_Ca, with different underlying unitary properties, in recruiting local SR Ca²⁺ release events. At an I_Ca of 8 pA/pF, the experimental conditions are the same. However, as one lowers I_Ca by reducing NP_o (black filled), the drop off in ECC efficacy is more severe than when i_Ca is reduced (red open and blue open, for the same I_Ca value). Thus a larger number of Ca²⁺ release events are recruited when i_Ca is small (but NP_o is high) than when NP_o is low (but i_Ca large). This underscores the importance of NP_o in regulating CICR efficacy. Figure 6b shows CICR gain as a function of I_Ca and emphasizes that reducing either i_Ca or NP_o alone increases CICR gain. Returning to the classic observation in Figure 1c, where ECC gain increases sharply at more negative V_Test, it can now be appreciated that this effect must be mediated by the decrease in NP_o (consistent with Figures 1d and 3d). In contrast, the increase in i_Ca at negative test V_m (attributable to enhanced driving force) would be expected to decrease gain (as demonstrated in Figures 4c and 5c).

View larger version (14K): [in this window] [in a new window]   Figure 6. Composite results from varying NPo and iCa. a, Relative efficacy of NPo and iCa to activate local Ca2+ release events. Fraction of active junctions plotted vs trigger ICa with variable NPo (black closed circles) or iCa (open circles). b, CICR gain as a function of trigger ICa.

	Discussion

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Cardiac SR Ca²⁺ release is graded by I_Ca, but which unitary component (i_Ca versus NP_o) controls CICR gain at different V_m is controversial. Because gain has similar V_m dependence as the expected i_Ca (Figure 1c and 1d), this was taken to indicate that the increase in i_Ca amplitude enhances its capability to trigger release (higher coupling fidelity) and dictates high gain at negative V_m. However, the explicit i_Ca dependence of gain or coupling fidelity is unknown. NP_o is also influenced by V_m over the same range as i_Ca (Figure 1d), and no previous work has separated the influence of NP_o and i_Ca, but rather both components change simultaneously as V_m varies.^10–13

Our aims were to separately measure NP_o and i_Ca dependence of CICR and to better explain the V_m dependence of ECC gain. Our data show that reducing either i_Ca or NP_o alone increased CICR gain and that a decrease in NP_o has a stronger effect on the recruitment of Ca²⁺ release sites than does a decrease in i_Ca. We suggest that the amplitude of i_Ca at 0 mV (with 1 mmol/L [Ca²⁺]_o) may be sufficient to effectively trigger uniform SR Ca²⁺ release. Hence, additional I_Ca (either as i_Ca or NP_o) will be redundant or wasted, because it will not trigger additional Ca²⁺ sparks (and thus decreases ECC gain). This disproves the idea that increasing i_Ca at negative V_m can increase gain. However, at positive V_m, where i_Ca is rather small, effective SR Ca²⁺ release might require synchronous activation of a group of LCCs. Moreover, the decline in SR Ca²⁺ release that occurs at very positive square V_m pulses must be mainly attributable to the progressively decline in i_Ca, because NP_o is expected to remain maximal.

CICR Gain and Fractional Release
The OG-5N/EGTA fluorescence signals provide an accurate estimate of global and junctional SR Ca²⁺ release.¹⁶ Here we counted active junctional Ca²⁺ release sites (Ca²⁺ spikes) to assess CICR gradation. Notably, similar results were obtained when the whole-cell fluorescence peak was used to assess SR Ca²⁺ release (see linear correlation in Figure 3b).

Because altered SR Ca²⁺ content can dramatically affect ECC gain,²⁶ presumably because of an effect of intra-SR [Ca²⁺] on RyR gating,²⁷ SR Ca²⁺ content must be controlled in studies of CICR gain. Here we accomplished this by dialysis of the cell with strongly buffered [Ca²⁺]_i and long times between pulses (17 seconds). We demonstrated that despite the high concentration of these Ca²⁺chelators, total SR Ca²⁺ release was consistent. Fractional SR Ca²⁺ release on strong depolarization (V_Test=0 mV) was 50% of that evoked by caffeine, consistent with previous estimates without Ca²⁺ chelators.²⁶ Thus, this method of trapping released Ca²⁺ does not greatly modify the release process itself. This is convenient but may also mean that the shut-off of SR Ca²⁺ release does not depend entirely on [Ca²⁺] outside the SR (which should be limited somewhat by 1 mmol/L of the fast Ca²⁺ buffer OG-5N). It would be consistent with some contribution of decreasing intra-SR Ca²⁺ in the termination of SR Ca²⁺ release.^28,29

Figure 1b shows that, with these Ca²⁺ chelators, the classic V_m dependence of CICR^10–13,17 is recapitulated. Previous reports with different experimental details all came to the same reasonable conclusion that increasing i_Ca could explain why ECC gain rises at increasingly negative test V_m. However, none of them separated the impact of i_Ca versus NP_o as we have done to further test this conclusion.

NP_o and CICR Gain
The actual number of LCCs at a single dyad varies,⁸ but an average dyad probably contains 12 LCCs.^1,4,7 Only a fraction of these open on any given pulse, and the average number, NP_o in a single dyad, should reflect global NP_o. We found that reducing I_Ca by lowering NP_o increases gain while reducing the number of active junctions (Figure 3c and 3d), presumably by limiting the number of Ca²⁺ channel openings at a given junction and the junctions where any openings happen. This may explain why, at more negative V_m (where NP_o is low, but i_Ca is large), ECC gain is high because there is almost no redundant I_Ca. That is, a single Ca²⁺ channel opening can trigger a Ca²⁺ spark at 1 dyad,^12,13,19 and because P_o is very low, there are almost no dyads where more than 1 Ca²⁺ channel opens (which would be redundant or wasted openings). As V_m becomes less negative and P_o increases, multiple channels will open at some dyads; however, if the first opening suffices to trigger Ca²⁺ release, then subsequent openings are redundant for ECC. This will be true as long as the coupling fidelity of i_Ca at 0 mV is sufficient, and our data that increasing i_Ca always decreases gain suggest that this may be the case.

The latency of Ca²⁺ spike recruitment should be related to Ca²⁺ channel first opening latency.^12,19,30,31 For high NP_o (and adequate i_Ca), a first opening will occur earlier on average (even with opening being random).¹⁵ Accordingly, we found that the average latency of Ca²⁺ spikes was dramatically increased (2 fold) at low NP_o (supplemental Figure II). Although 1 open LCC may suffice to trigger CICR at each site,^12,13,19 a cluster of functionally available LCCs might ensure that at least 1 will open on depolarization and will help trigger relatively synchronized Ca²⁺ release.^19,30 Although multiple openings create a safety margin to assure high fidelity synchronized signaling,¹⁹ this also creates redundant (or wasted) Ca²⁺ entry and reduction in gain at higher I_Ca (high NP_o).

The precise number of active LCCs necessary to trigger a Ca²⁺ spark at all V_m is not known, but the following simple numbers may provide quantitative perspective. An I_Ca of 8 pA/pF during the first 20 ms in a 30-pL myocyte would require 90 000 single channel openings (for i_Ca of 0.33 pA for 0.5 ms) or 25% of the Ca²⁺ channels. If there are 30 000 junctions per myocyte, this implies an average of 3 LCC openings per junction. It is thus not surprising that reducing NP_o rapidly reduces the fraction of active junctions from the relatively large value at V_m=0 mV (Figure 6a). We did not study the 30% of LCCs on the surface sarcolemma,³² but these likely also colocalize with RyRs at surface junctions.⁵ Although Brette et al^32,33 measured slower I_Ca inactivation for surface versus transverse tubular I_Ca, they reported similar ECC gain for both.

Single L-Type Ca²⁺ Channel Current Amplitude and CICR Gain
We tested whether increasing i_Ca amplitude increases gain and coupling fidelity (using rapid [Ca²⁺]_o changes at constant NP_o). SR Ca²⁺ release and percentage of active junctions were nearly maximum at [Ca²⁺]_o1 mmol/L, with little increase at 10 mmol/L (despite nearly double the i_Ca; Figure 4b). This indicates that the coupling fidelity for i_Ca at [Ca²⁺]_o=1 mmol/L (at 0 mV) is near optimal for CICR. Extra Ca²⁺ influx then increases the denominator in the gain equation without commensurate SR Ca²⁺ release enhancement, such that gain decreases at higher i_Ca. Similar decreases in ECC gain are seen with long channel openings induced by Bay K 8644,³⁴ where presumably the initial i_Ca is sufficient to trigger release (and the rest is redundant).

We also used a voltage-clamp protocol more like an action potential (Figure 5), where the range of i_Ca analysis was extended to smaller values than in Figure 4. This protocol with a brief prepulse to +120 mV mimics the AP upstroke in rapidly activating a large number of Ca²⁺ channels but without Ca²⁺ influx (constant NP_o). Then sudden repolarization increases the Ca²⁺ electrochemical gradient, causing a rapid influx through open channels, as occurs during early AP repolarization.¹⁸ Still ECC gain increased at even smaller i_Ca (Figure 5c). This is surprising because, at some very low i_Ca, one should see ineffective triggering of Ca²⁺ release (low coupling fidelity) and hence decreased gain. Notably, the results for pseudo-AP pulses (Figure 5b) differ from usual square pulses to positive V_m (Figure 1b). With the positive prepulse SR, Ca²⁺ release declines less dramatically with decreasing I_Ca than for the traditional square pulse. That is, it takes a 6-fold decrease in i_Ca in the +prepulse experiment (Figure 5b) to reduce SR Ca²⁺ release by 50%, whereas only a 3-fold i_Ca reduction in the classic square pulse experiment (Figure 1b) produces a comparable decrease in SR Ca²⁺ release. We pose 2 explanations.

First, high NP_o with strong depolarization (or AP peak) may synchronize tail i_Ca (or peak I_Ca during early AP repolarization) even when i_Ca amplitude is small, thereby enhancing coupling fidelity (eg, simultaneous subthreshold i_Ca summate to assure triggering). Square pulses to lower V_m may activate many channels, but less synchronously, so SR Ca²⁺ release may be less reliably triggered. This is consistent with Sah et al,¹⁸ who showed that Ca²⁺ spark frequency during an AP depends strongly on early repolarization rate, and we found shorter latency of Ca²⁺ spikes during the tail I_Ca protocol, regardless of V_Test (supplemental Figure II). Hence, during APs, the expected decrease in i_Ca coupling fidelity may be counteracted by the well-synchronized high number of open channels.

Second, the strong positive prepulse or AP peak may drive Ca²⁺ influx via Na⁺/Ca²⁺ exchange (before Ca²⁺ entry occurs via LCCs).^12,35–40 Whereas some data with action potential and voltage-clamp–induced Ca²⁺ transients^41,42 suggest that Ca²⁺ entry via Na⁺/Ca²⁺ exchange is not critical for physiological ECC, our present results leave open the following possibility. This early Ca²⁺ entry may not trigger Ca²⁺ release by itself but could elevate local [Ca²⁺]_i such that a smaller, and possibly well-synchronized, local i_Ca exhibits higher coupling fidelity (again, versus the simple square pulse).

In conclusion, our results suggest that the relatively small i_Ca at 0 mV (with 1 mmol/L [Ca²⁺]_o) is sufficient for triggering Ca²⁺ sparks, such that a single channel opening may trigger SR Ca²⁺ release (even at 0 mV). At more negative V_m, the gain is increased because there is less redundancy of LCC openings per junction (lower NP_o), and this effect more than offsets the increasing i_Ca (which by itself decreases gain). At positive V_m (with square depolarizations), where i_Ca becomes much smaller, the decline in SR Ca²⁺ release and ECC gain may be related to a decrease in i_Ca coupling fidelity, despite multiple channel openings. Finally, more efficient SR Ca²⁺ release during an AP may be achieved by synchronized opening of LCCs during the AP upstroke or bolstering of local [Ca²⁺]_i.

	Acknowledgments

 
We thank Dr K. S. Ginsburg for discussions and J. Acevedo and B. French for technical assistance.

Sources of Funding

Supported by NIH grants HL30077 and HL64724.

Disclosures

None.

	Footnotes

 
Original received September 26, 2005; resubmission received March 19, 2007; revised resubmission received June 19, 2007; accepted July 11, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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