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(Circulation Research. 2003;93:40.)
© 2003 American Heart Association, Inc.

Cellular Biology

Ca²⁺ Scraps

Local Depletions of Free [Ca²⁺] in Cardiac Sarcoplasmic Reticulum During Contractions Leave Substantial Ca²⁺ Reserve

Thomas R. Shannon, Tao Guo, Donald M. Bers

From the Department of Physiology (T.R.S., T.G., D.M.B.), Loyola University Chicago, Maywood, Ill; Department of Molecular Biophysics and Physiology (T.R.S.), Rush University, Chicago, Ill.

Correspondence to Thomas R. Shannon, DVM, PhD, Department of Molecular Biophysics and Physiology, Rush University, 1750 W Harrison, Chicago, IL 60612. E-mail tshannon{at}rush.edu

	Abstract

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Free [Ca²⁺] inside the sarcoplasmic reticulum ([Ca²⁺]_SR) is difficult to measure yet critically important in controlling many cellular systems. In cardiac myocytes, [Ca²⁺]_SR regulates cardiac contractility. We directly measure [Ca²⁺]_SR in intact cardiac myocytes dynamically and quantitatively during beats, with high spatial resolution. Diastolic [Ca²⁺]_SR (1 to 1.5 mmol/L) is only partially depleted (24% to 63%) during contraction. There is little temporal delay in the decline in [Ca²⁺]_SR at release junctions and between junctions, indicating rapid internal diffusion. The incomplete local Ca²⁺ release shows that the inherently positive feedback of Ca²⁺-induced Ca²⁺ release terminates, despite a large residual driving force. These findings place stringent novel constraints on how excitation-contraction coupling works in heart and also reveal a Ca²⁺ store reserve that could in principle be a therapeutic target to enhance cardiac function in heart failure.

Key Words: calcium homeostasis • sarcoplasmic reticulum • ryanodine receptors • confocal imaging • membrane transport

	Introduction

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Measurement of cytosolic free [Ca²⁺] ([Ca²⁺]_i) is routine in most cell types and central to understanding the critical and ubiquitous roles of [Ca²⁺]_i in cellular signaling. In most cells, Ca²⁺ is stored in intracellular compartments, the endoplasmic or sarcoplasmic reticulum (ER or SR). The rapid release of this stored Ca²⁺ via inositol trisphosphate or ryanodine receptor (RyR) channels is the triggering event for many cellular signaling cascades, including muscle contraction. The total amount of Ca²⁺ stored in the SR ([Ca²⁺]_SRT) is critical to this Ca²⁺ signaling, by directly varying the amount available for release. In addition, in cardiac muscle, increasing [Ca²⁺]_SRT also increases fractional SR Ca²⁺ release for a given release trigger.^1–4 This is probably due to an effect of luminal Ca²⁺ on RyR gating.^5,6 The [Ca²⁺]_SRT dependence of release is nonlinear and extremely steep in the normal range of SR Ca²⁺ loads. Thus, variation in [Ca²⁺]_SRT may play an important role in regulating SR Ca²⁺ release.^1–4

Although [Ca²⁺]_SRT is important, SR Ca²⁺ is heavily buffered, and it is free intra-SR [Ca²⁺] ([Ca²⁺]_SR) that centrally determines (1) [Ca²⁺]_SRT, (2) the effect of intra-SR Ca²⁺ on the RyR, (3) the driving force for SR Ca²⁺ release, and (4) the maximal thermodynamic [Ca²⁺] gradient that the SR Ca²⁺-ATPase can establish. Knowledge about [Ca²⁺]_SR is increasingly important in understanding cardiac excitation-contraction coupling (ECC) and numerous processes in virtually all cells. [Ca²⁺]_SRT can be measured by releasing SR Ca²⁺ by activation of RyR in intact cells (eg, by caffeine).⁷ This gives quantitative data about [Ca²⁺]_SRT but not [Ca²⁺]_SR. Intra-SR–trapped fluorescent Ca²⁺ indicators and Ca²⁺-sensitive proteins targeted to organelles^8–16 can assess [Ca²⁺]_SR, but truly quantitative data have been challenging to obtain, especially in cardiac muscle.

In the present study, we measure [Ca²⁺]_SR directly in a spatially resolved, dynamic manner in intact ventricular myocytes. Because the data are spatial as well as quantitative, we also assess whether appreciable diffusional delays exist between [Ca²⁺]_SR near release sites and sites far away.

Cardiac ECC works by local Ca²⁺-induced SR Ca²⁺ release, where Ca²⁺ current is the trigger.³ This inherently positive feedback would be expected to empty the SR, although indirect evidence suggests that SR Ca²⁺ release is incomplete during a normal heartbeat.^1–4 However, this is controversial and our understanding of cardiac ECC is limited by lack of knowledge of spatially resolved [Ca²⁺]_SR. Data presented here are critical to understanding how [Ca²⁺]_SR is involved in regulating the release process.

	Materials and Methods

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Myocyte Isolation and Indicator Loading
Animal protocols were approved by the Loyola University Animal Studies Committee. Ventricular myocytes were isolated from New Zealand White rabbits (Myrtle’s Rabbitry, Thompson Station, Tenn) as previously described² and were loaded with Fluo-5N AM (Molecular Probes) for 2 hours, and then 1.5 hours was allowed for deesterification and outward leak of cytosolic indicator, all at 37°C.¹⁷ All experiments were performed at 23°C. Fluorescence was measured both on confocal and epifluorescence microscopes at excitation=488 nm, emission >500 nm for Fluo-5N. Cells exposed to Di-8-ANNEPS to identify transverse tubules were imaged with excitation=488 nm and fluorescence emission at >600 nm. The image in Figure 2C was deconvolved as in Gonzalez et al.¹⁸

View larger version (49K): [in this window] [in a new window]   Figure 2. Cellular anatomy of the SR. A and B, Images of Fluo-5N–loaded myocytes before and after Di-8-ANNEPS addition to visualize transverse tubules. Di-8-ANNEPS colocalizes with Fluo-5N signal. C, Zoom of region in B, illustrating transverse tubules (T-T) with junctional and free SR regions in between (JSR and FSR).

All reagents and chemicals were purchased from Sigma Chemical Company except as indicated. Cell superfusate contained (in mmol/L) CaCl₂ 2, NaCl 140, KCl 4, MgCl₂ 1, HEPES 5, and glucose 10 (pH 7.4). Statistical significance was tested with two-way ANOVA. A value of P<0.05 was considered significant.

Fluo-5N Calibration
In vitro calibration was performed in intracellular solutions (control, in mmol/L, KCl 140, HEPES 40 [pH 7.2]) with the [Ca²⁺] indicated. Solutions were suspended within a fluorometer, and Fluo-5N fluorescence was measured under the indicated conditions. In vivo, F_Max was determined in an intact myocyte by adding 1 µmol/L isoproterenol (ISO), then subsequently adding 0.5 mmol/L tetracaine to block SR Ca²⁺ leak, and finally [Na⁺]_o was removed to raise both [Ca²⁺]_i and [Ca²⁺]_SR. In vivo, K_d was estimated in permeabilized cells (50 µg/mL saponin) with 10 mmol/L caffeine to allow [Ca²⁺] equilibration across the SR. Intracellular solution, as previously described,¹⁹ included (in mmol/L) Cs-glutamate 200, HEPES 10 (pH 7.2), Mg-ATP 5, phosphocreatine ditris 5, MgCl₂ 0.5, glutathione 10 (reduced form), 5 U/mL creatine phosphokinase, 8% dextran (MW 40 000), with variable free [Ca²⁺], 1 µmol/L FCCP, 1 µmol/L ruthenium red, 2 µmol/L oligomycin, and 8 µmol/L cyclosporine to limit mitochondrial Ca²⁺ uptake.

	Results

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The rabbit ventricular myocyte shown in Figure 1A is loaded with Fluo-5N, a low-affinity Ca²⁺ indicator that has extremely low fluorescence when Ca²⁺-free.¹⁷ While there is surely some indicator in cytosol and mitochondria, [Ca²⁺] is submicromolar in these compartments, such that the fluorescence is primarily from the SR (where expected [Ca²⁺]_SR is 1 mmol/L). This SR localization is supported by 3 observations. First, the fluorescence pattern is localized to Z lines and transverse tubules (stained by the lipophilic fluorophore Di-8-ANNEPS), exactly as expected for cardiac junctional SR (JSR, Figures 2A and 2B). That is, there is higher fluorescence near transverse tubules (site of capacious JSR) and weaker fluorescence strands through the sarcomere (site of more wispy, less dense longitudinal or free SR, FSR). Moreover, [Ca²⁺]_SR is expected to be the same throughout the resting SR, so brightness may reflect the expected ultrastructural SR organization. The periodic bright fluorescence regions (1.9-µm spacing; Figure 1B) correspond to sarcomeric spacing. Second, this distinct pattern is abolished by rapid application of 10 mmol/L caffeine, which causes SR Ca²⁺ release to the cytosol (Figure 1B). The remaining sporadic bright spots and perinuclear rings that are little affected by caffeine (and avoided in Figure 1B) probably reflect Fluo-5N compartmentalization in non-SR regions with high [Ca²⁺]. Third, permeabilization of the sarcolemma with saponin does not alter the fluorescence pattern appreciably (not shown). Thus, the image at high zoom in Figure 2C illustrates the anatomy of the SR as it wraps around the myofilaments (dark regions within the sarcomere between wispy areas of FSR) with the junctional SR located at the Z lines.

View larger version (67K): [in this window] [in a new window]   Figure 1. Fluorescence signals from intra-SR Fluo-5N. A, Fluorescence is abolished by 10 mmol/L caffeine application at striations but not in non-SR regions. B, Enlargement of part of A (with intensity profile). Striations are periodic every 1.9 µm, consistent with intrasarcomeric spacing and are abolished by caffeine.

Figure 3A shows whole-cell fluorescence changes that reflect transient [Ca²⁺]_SR depletions during twitches and also caffeine-induced [Ca²⁺]_SR depletions. After caffeine removal, [Ca²⁺]_SR only partially recovers unless pacing is resumed. The caffeine-sensitive fluorescence (attributed to the SR) at 0.5-Hz stimulation is 53.2±1.8% (n=14, Figure 3A) of the total. All subsequent data refer only to this caffeine-sensitive component. During the twitch, fluorescence declines to a minimum of 75% at 100 ms after [Ca²⁺]_SR starts to decline and recovers with a time constant () of 100 ms (Figure 3B). Figure 3C shows that the [Ca²⁺]_SR depletion measured at a single SR junction in confocal microscopy is quite similar to the global [Ca²⁺]_SR signal. We refer to these local [Ca²⁺]_SR depletions as "Ca²⁺ scraps" (since they are the intra-SR correlate of evoked Ca²⁺ sparks).²⁰ Moreover, when Ca²⁺ scraps at junctional and free SR regions (as in Figure 2C) are compared, there is little kinetic difference (Figure 3D). This indicates that longitudinal diffusion within the SR is faster than we could readily detect (assuming SR Ca²⁺ release occurs at JSR).²¹ Thus, Ca²⁺ diffusion from free to junctional SR does not appreciably limit SR Ca²⁺ availability for release, even at a single twitch.

View larger version (26K): [in this window] [in a new window]   Figure 3. [Ca2+]SR measurements. A, Whole-cell [Ca2+]SR during twitches evoked at 1 Hz (black) or 0.5 Hz (red), interrupted during application of 10 mmol/L caffeine (used to deplete SR Ca2+). Pacing was resumed only for 0.5 Hz. B, Single-twitch [Ca2+]SR signal (1 Hz, average of 4) as fraction of caffeine-sensitive diastolic fluorescence at 1 Hz (Fd1). Exponential curve fit (=100 ms) of [Ca2+]SR recovery in red. C, Measurement at a single junction (as identified in Figure 1D), showing similar characteristics (with 20 mmol/L butanedione monoxime, 4 ms/line). D, Separate signals analyzed from JSR and FSR regions. Traces are average from 93 JSR and 29 FSR regions, with amplitude normalized.

Figure 4A shows [Ca²⁺]_SR and contractions at different pacing frequencies. As frequency increases, diastolic [Ca²⁺]_SR increases, as does the extent of [Ca²⁺]_SR depletion and the contraction amplitude. With ß-adrenergic activation by ISO, there was a further increase in diastolic [Ca²⁺]_SR and extent of [Ca²⁺]_SR depletion and faster [Ca²⁺]_SR recovery ( decreased from 155 to 70 ms), consistent with the expected acceleration of SR Ca²⁺-ATPase by ISO. Figures 4B through 4D show mean diastolic F (F_d versus that at 1 Hz, F_d1), peak fractional depletion (F/F_d=25±2.6% for 1 Hz), and the minimum systolic value of [Ca²⁺]_SR. All three parameters increase with frequency and ISO. This is consistent with a [Ca²⁺]_SR-dependent increase in fractional release and also clearly demonstrates that [Ca²⁺]_SR depletion is incomplete during a twitch.^1,2 The of refilling (Figure 4E) speeds up with frequency, consistent with frequency-dependent acceleration of relaxation and [Ca²⁺]_i decline.³

View larger version (36K): [in this window] [in a new window]   Figure 4. Pacing frequency and ISO enhance [Ca2+]SR and depletion. A, Cell contraction (arbitrary units) and Fluo-5N fluorescence for steady-state twitches. B, Mean diastolic fluorescence, where subscripts refer to frequency or ISO at 1 Hz (n=14 cells; normalized to Fd1). C, Fractional decline in [Ca2+]SR during steady-state twitches (normalized to diastolic F, Fd; n=14). D, Minimum caffeine-sensitive F during systole, where Fd-ISO=518±68. E, Time constant for recovery of [Ca2+]SR during twitches. *P<0.05.

Because F may not be linearly related to [Ca²⁺]_SR, calibrations are needed for greater quantitative evaluation. In vitro, Fluo-5N Ca²⁺ affinity (K_d=135 µmol/L) in solution measured in a fluorometer is not altered by Mg or tetracaine (used below), although tetracaine partially quenches fluorescence (Figure 5A). The presence of protein decreases both maximal fluorescence as well as apparent affinity of Fluo-5N for Ca²⁺ (Figure 5A). At cellular protein concentrations (50 to 100 mg/mL), Fluo-5N affinity is reduced 3-fold (typical for fluorescent Ca²⁺ indicators in cells).^22,23

View larger version (25K): [in this window] [in a new window]   Figure 5. Fluo-5N calibration and [Ca2+]SR. A, In vitro calibration in intracellular solutions with the [Ca2+] indicated ±1 mmol/L MgCl2 or 0.5 mmol/L tetracaine and different bovine serum albumin (BSA) concentrations. B, In vivo FMax determination in an intact myocyte with 1 µmol/L ISO, then 0.5 mmol/L tetracaine is added to block SR Ca2+ leak and [Na]o is removed to drive [Ca2+]i and [Ca2+]SR up (mean FMax=1.37±0.09xFd1). C, Permeabilized cell (50 µg/mL saponin) with 10 mmol/L caffeine to allow [Ca2+] equilibration across the SR.

Maximal fluorescence (F_Max) in myocytes was defined by stimulating SR Ca²⁺-ATPase by ISO and blocking SR Ca²⁺ release by tetracaine (which dramatically increases SR Ca²⁺ content).²⁴ Note that F did not rise much on tetracaine addition (even when we accounted for the modest quench by tetracaine). To further raise [Ca²⁺]_SR, extracellular Na⁺ was abruptly removed (causing Ca²⁺ entry via Na⁺-Ca²⁺ exchange), which caused spontaneous contractions and corresponding Ca²⁺ depletions (Figure 5B). Under these conditions (with the RyR inhibited), the SR Ca²⁺ pump should approach a limiting [Ca²⁺]_SR/[Ca²⁺]_i gradient,²⁵ and [Ca²⁺]_SR should rise by the same factor as [Ca²⁺]_i (and the elevation of average [Ca²⁺]_i is indicated by the cellular contracture in Figure 5B). Since F still did not increase appreciably, despite the substantial rise in [Ca²⁺]_SRT expected with this protocol, F_Max represents saturation of intra-SR Fluo-5N. F_Min is taken as F_Caf.

To test whether K_d=400 µmol/L is appropriate for intra-SR Fluo-5N, we used permeabilized myocytes (with RyRs opened by caffeine). When [Ca²⁺] was stepped from 50 nmol/L (F_Min) to 400 µmol/L (F₄₀₀) to 10 mmol/L (F_Max; Figure 5C), we found that F₄₀₀ was, on average, 47.5% of F_Max-F_Min. This confirms that 400 µmol/L is an appropriate K_d in situ.

Using these calibrations, we found that diastolic [Ca²⁺]_SR increased from 0.48 to 0.92 to 1.65 mmol/L from 0.1 to 1 Hz (Figures 6A and 6B). These values are similar to time-averaged whole-heart NMR estimates of [Ca²⁺]_SR (1.5 mmol/L)¹² and our estimates (1 mmol/L), based on [Ca²⁺]_SR and [Ca²⁺]_SRT in SR vesicles plus cellular [Ca²⁺]_SRT.²⁵ However, both of those results lacked kinetic or spatial information.

View larger version (30K): [in this window] [in a new window]   Figure 6. Dynamic [Ca2+]SR profiles. A, Calibrated [Ca2+]SR signals. B, Mean diastolic [Ca2+]SR and fractional twitch depletion at different frequencies.

The extent of [Ca²⁺]_SR depletion increased from 24% to 63% over this range of frequencies. Diastolic ISO data were not calibrated because F_d was too near F_Max. Minimum [Ca²⁺]_SR attained during a twitch varied from 0.36 to 0.61 mmol/L (even with ISO). These [Ca²⁺]_SR measurements support previous, less direct [Ca²⁺]_SRT measurements that suggested incomplete SR Ca²⁺ depletion.^1,2,4 Importantly, Figures 3B and 3C also indicate that JSR in individual junctions depletes only partially. These data are not consistent with recent models of ECC where local [Ca²⁺]_SR was projected to be 95% depleted early during the twitch, thereby limiting further SR Ca²⁺ release.²⁶

	Discussion

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We report the first direct measurements of [Ca²⁺]_SR during individual contractions and with subsarcomeric spatial resolution. These measurements and method provide valuable new quantitative information that is at the very heart of cardiac ECC. It has also been argued that SR Ca²⁺ depletion does not participate in the shutoff of SR Ca²⁺ release,²¹ and indeed we show that SR Ca²⁺ release stops at local [Ca²⁺]_SR 0.4 mmol/L when there is still a large driving force for SR Ca²⁺ release. Cardiac ECC models have assumed that there is a major time delay (up to seconds) between recovery of [Ca²⁺]_SR near Ca²⁺ uptake sites (FSR) and at release sites (JSR).²⁷ Such purported major time lags between JSR and FSR do not seem to occur during release (Figure 3C), and the [Ca²⁺]_SR is restored rapidly during the twitch (even in the JSR). The apparent delay or restitution of SR Ca²⁺ release (eg, at premature heartbeats) is probably due mainly to recovery of RyR (and/or L-type Ca²⁺ channel) availability, rather than the amount of releasable SR Ca²⁺.

We propose that [Ca²⁺]_SR depletion is dynamically involved in terminating SR Ca²⁺ release, due to direct effects on RyR gating (not by exhausting available SR Ca²⁺). Indeed, the SR retains a Ca²⁺ reserve, which is pharmacologically accessible, as indicated by caffeine-induced Ca²⁺ transients, which completely deplete [Ca²⁺]_SR (Figure 1B). When [Ca²⁺]_SR is below 40% to 50% of its control value, resting SR Ca²⁺ leak (Ca²⁺ spark frequency) is very small and a normal Ca²⁺ current trigger fails to release appreciable SR Ca²⁺.^1–4,20,28 This demonstrates that luminal [Ca²⁺]_SR dynamically modulates SR Ca²⁺ release (and leak) during both diastole and ECC.

The fact that SR Ca²⁺ release does not go to completion even locally (as expected for positive feedback) rules out substrate limitation as the cause of release termination but leaves two potential types of inactivation.^29,30 One mechanism, stochastic attrition, would be when a sufficient number of Ca²⁺ channels in a junction (L-type and RyR) close by chance to allow local [Ca²⁺]_i to fall and break the positive-feedback loop. This is unlikely to produce reliable termination of SR Ca²⁺ release, given the high number of channels at a junction (unless their gating is tightly coupled).^21,29–31 The second major class would be a time-dependent RyR inactivation, which could depend explicitly on [Ca²⁺]_i, [Ca²⁺]_SR, or both. There is evidence for [Ca²⁺]_i-dependent inactivation (or adaptation).²¹ DelPrincipe et al³² found that after a global cellular SR Ca²⁺ release, restitution required >1 second and suggested that SR Ca²⁺ depletion and slow functional repletion were the likely explanation (because discrete local Ca²⁺ releases showed much faster recovery). Our data indicate that the SR refills rather rapidly and suggests that this restitution depends more on recovery of RyR (or I_Ca) availability.

Interestingly, our own data demonstrate a trend upward in [Ca²⁺]_SR minimum during a twitch with increased release (Figure 4D). This would be consistent with a cytosolic Ca²⁺-dependent inactivation site on the RyR, which binds more Ca²⁺, thus inactivating the channel faster and terminating release at a slightly higher [Ca²⁺]_SR. However, our observation that release stops (and can only be very weakly activated) when [Ca²⁺]_SR <0.5 mmol/L also suggests an equally important regulatory role for [Ca²⁺]_SR (perhaps by influencing how RyR responds to [Ca²⁺]_i).^5,6 Thus, both [Ca²⁺]_SR and [Ca²⁺]_i may both contribute dynamically to the shutoff of SR Ca²⁺ release. A corollary is that both RyR availability and [Ca²⁺]_SR must recover to achieve restitution. So far, our results suggest that [Ca²⁺]_SR recovers somewhat faster (100 ms) than does the RyR (300 ms).^32,33

[Ca²⁺]_SR dictates the effect of SR Ca²⁺ on RyR gating, the driving force for Ca²⁺ release, but it is also limited thermodynamically. For diastolic [Ca²⁺]_i=100 to 150 nmol/L at 0.5 to 1 Hz (respectively), the [Ca²⁺]_SR/[Ca²⁺]_i gradient would be 9200 to 11 000. A gradient of 10 000 implies a high (78%) energetic efficiency for the SR Ca²⁺-ATPase (for 2 Ca²⁺/ATP) and a cytosolic G_ATP of 59 kJ/mol.^25,34

Our data provide the first direct quantitative examination of what is happening dynamically to local intra-SR free [Ca²⁺] during cardiac ECC. This is especially important because [Ca²⁺]_SR (rather than [Ca²⁺]_SRT) is the central thermodynamic parameter that governs buffering, allosteric regulation, driving gradient, and transport limits. Thus, in cardiac myocytes, [Ca²⁺]_SR is a major determinant of Ca²⁺ release and contraction. Low [Ca²⁺]_SR may also limit cardiac function in heart failure.^35,36 Intra-SR Ca²⁺ diffusion is rapid, and local [Ca²⁺]_SR never gets much less than 50% of its diastolic value, even with strong activation of ECC. The less than complete depletion of local [Ca²⁺]_SR during normal SR Ca²⁺ release implies a residual SR Ca²⁺ reserve that might be pharmacologically accessible for treatment of diseases such as heart failure. The experimental approach described here should be very useful in further studies of SR Ca²⁺ in cardiac myocytes and other cell types. This novel approach should allow new mechanistic and quantitative questions to be addressed.

	Acknowledgments

 
This work was funded by NIH grant HL30077 and HL64098 (D.M.B.) and AHA grant 0030381Z (T.R.S.). The authors thank Drs E. Ríos and L. Blatter for their help.

	Footnotes

 
Original received April 23, 2003; revision received May 22, 2003; accepted May 22, 2003.

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