CA2Ⴙ SCRAPS LOCAL DEPLETIONS OF FREE CA2Ⴙ IN CARDIAC SARCOPLASMIC RETICULUM DURING CONTRACTIONS LEAVE SUBSTANTIAL CA2Ⴙ RESERVE
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Ca2ⴙ Scraps
Local Depletions of Free [Ca2ⴙ] in Cardiac Sarcoplasmic Reticulum
During Contractions Leave Substantial Ca2ⴙ Reserve
Thomas R. Shannon, Tao Guo, Donald M. Bers
Abstract—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. (Circ Res. 2003;93:40-45.)
Key Words: calcium homeostasis 䡲 sarcoplasmic reticulum 䡲 ryanodine receptors 䡲 confocal imaging
䡲 membrane transport
easurement of cytosolic free [Ca2⫹] ([Ca2⫹]i) is routine This gives quantitative data about [Ca2⫹]SRT but not [Ca2⫹]SR.
M in most cell types and central to understanding the
critical and ubiquitous roles of [Ca2⫹]i in cellular signaling. In
Intra-SR–trapped fluorescent Ca2⫹ indicators and Ca2⫹-
sensitive proteins targeted to organelles8 –16 can assess
most cells, Ca2⫹ is stored in intracellular compartments, the [Ca2⫹]SR, but truly quantitative data have been challenging to
endoplasmic or sarcoplasmic reticulum (ER or SR). The rapid obtain, especially in cardiac muscle.
release of this stored Ca2⫹ via inositol trisphosphate or In the present study, we measure [Ca2⫹]SR directly in a
ryanodine receptor (RyR) channels is the triggering event for spatially resolved, dynamic manner in intact ventricular
many cellular signaling cascades, including muscle contrac- myocytes. Because the data are spatial as well as quantitative,
tion. The total amount of Ca2⫹ stored in the SR ([Ca2⫹]SRT) is we also assess whether appreciable diffusional delays exist
critical to this Ca2⫹ signaling, by directly varying the amount between [Ca2⫹]SR near release sites and sites far away.
available for release. In addition, in cardiac muscle, increas- Cardiac ECC works by local Ca2⫹-induced SR Ca2⫹ re-
ing [Ca2⫹]SRT also increases fractional SR Ca2⫹ release for a lease, where Ca2⫹ current is the trigger.3 This inherently
given release trigger.1– 4 This is probably due to an effect of positive feedback would be expected to empty the SR,
luminal Ca2⫹ on RyR gating.5,6 The [Ca2⫹]SRT dependence of although indirect evidence suggests that SR Ca2⫹ release is
release is nonlinear and extremely steep in the normal range incomplete during a normal heartbeat.1– 4 However, this is
of SR Ca2⫹ loads. Thus, variation in [Ca2⫹]SRT may play an controversial and our understanding of cardiac ECC is limited
important role in regulating SR Ca2⫹ release.1– 4 by lack of knowledge of spatially resolved [Ca2⫹]SR. Data
Although [Ca2⫹]SRT is important, SR Ca2⫹ is heavily buff- presented here are critical to understanding how [Ca2⫹]SR is
ered, and it is free intra-SR [Ca2⫹] ([Ca2⫹]SR) that centrally involved in regulating the release process.
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 Materials and Methods
maximal thermodynamic [Ca2⫹] gradient that the SR Ca2⫹-
ATPase can establish. Knowledge about [Ca2⫹]SR is increas- Myocyte Isolation and Indicator Loading
ingly important in understanding cardiac excitation- Animal protocols were approved by the Loyola University Animal
Studies Committee. Ventricular myocytes were isolated from New
contraction coupling (ECC) and numerous processes in Zealand White rabbits (Myrtle’s Rabbitry, Thompson Station, Tenn)
virtually all cells. [Ca2⫹]SRT can be measured by releasing SR as previously described2 and were loaded with Fluo-5N AM (Mo-
Ca2⫹ by activation of RyR in intact cells (eg, by caffeine).7 lecular Probes) for 2 hours, and then 1.5 hours was allowed for
Original received April 23, 2003; revision received May 22, 2003; accepted May 22, 2003.
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@rush.edu
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000079967.11815.19
40
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Shannon et al Local SR Depletions 41
Figure 1. Fluorescence signals from
intra-SR Fluo-5N. A, Fluorescence is
abolished by 10 mmol/L caffeine applica-
tion at striations but not in non-SR
regions. B, Enlargement of part of A
(with intensity profile). Striations are peri-
odic every 1.9 m, consistent with
intrasarcomeric spacing and are abol-
ished by caffeine.
deesterification and outward leak of cytosolic indicator, all at 37°C.17 cence is primarily from the SR (where expected [Ca2⫹]SR is
All experiments were performed at 23°C. Fluorescence was mea- ⬇1 mmol/L). This SR localization is supported by 3 obser-
sured both on confocal and epifluorescence microscopes at excita-
vations. First, the fluorescence pattern is localized to Z lines
tion⫽488 nm, emission ⬎500 nm for Fluo-5N. Cells exposed to
Di-8-ANNEPS to identify transverse tubules were imaged with and transverse tubules (stained by the lipophilic fluorophore
excitation⫽488 nm and fluorescence emission at ⬎600 nm. The Di-8-ANNEPS), exactly as expected for cardiac junctional
image in Figure 2C was deconvolved as in Gonzalez et al.18 SR (JSR, Figures 2A and 2B). That is, there is higher
All reagents and chemicals were purchased from Sigma Chemical fluorescence near transverse tubules (site of capacious JSR)
Company except as indicated. Cell superfusate contained
and weaker fluorescence strands through the sarcomere (site
(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 of more wispy, less dense longitudinal or free SR, FSR).
ANOVA. A value of P⬍0.05 was considered significant. Moreover, [Ca2⫹]SR is expected to be the same throughout the
resting SR, so brightness may reflect the expected ultrastruc-
Fluo-5N Calibration tural SR organization. The periodic bright fluorescence re-
In vitro calibration was performed in intracellular solutions (control, gions (1.9-m spacing; Figure 1B) correspond to sarcomeric
in mmol/L, KCl 140, HEPES 40 [pH 7.2]) with the [Ca2⫹] indicated.
spacing. Second, this distinct pattern is abolished by rapid
Solutions were suspended within a fluorometer, and Fluo-5N fluo-
rescence was measured under the indicated conditions. In vivo, FMax application of 10 mmol/L caffeine, which causes SR Ca2⫹
was determined in an intact myocyte by adding 1 mol/L isoproter- release to the cytosol (Figure 1B). The remaining sporadic
enol (ISO), then subsequently adding 0.5 mmol/L tetracaine to block bright spots and perinuclear rings that are little affected by
SR Ca2⫹ leak, and finally [Na⫹]o was removed to raise both [Ca2⫹]i caffeine (and avoided in Figure 1B) probably reflect Fluo-5N
and [Ca2⫹]SR. In vivo, Kd was estimated in permeabilized cells (50
g/mL saponin) with 10 mmol/L caffeine to allow [Ca2⫹] equilibra-
compartmentalization in non-SR regions with high [Ca2⫹].
tion across the SR. Intracellular solution, as previously described,19 Third, permeabilization of the sarcolemma with saponin does
included (in mmol/L) Cs-glutamate 200, HEPES 10 (pH 7.2), not alter the fluorescence pattern appreciably (not shown).
Mg-ATP 5, phosphocreatine ditris 5, MgCl2 0.5, glutathione 10 Thus, the image at high zoom in Figure 2C illustrates the
(reduced form), 5 U/mL creatine phosphokinase, 8% dextran (MW anatomy of the SR as it wraps around the myofilaments (dark
40 000), with variable free [Ca2⫹], 1 mol/L FCCP, 1 mol/L
ruthenium red, 2 mol/L oligomycin, and 8 mol/L cyclosporine to
regions within the sarcomere between wispy areas of FSR)
limit mitochondrial Ca2⫹ uptake. with the junctional SR located at the Z lines.
Figure 3A shows whole-cell fluorescence changes that reflect
Results transient [Ca2⫹]SR depletions during twitches and also caffeine-
The rabbit ventricular myocyte shown in Figure 1A is loaded induced [Ca2⫹]SR depletions. After caffeine removal, [Ca2⫹]SR
with Fluo-5N, a low-affinity Ca2⫹ indicator that has ex- only partially recovers unless pacing is resumed. The caffeine-
tremely low fluorescence when Ca2⫹-free.17 While there is sensitive fluorescence (attributed to the SR) at 0.5-Hz stimula-
surely some indicator in cytosol and mitochondria, [Ca2⫹] is tion is 53.2⫾1.8% (n⫽14, Figure 3A) of the total. All subse-
submicromolar in these compartments, such that the fluores- quent data refer only to this caffeine-sensitive component.
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42 Circulation Research July 11, 2003
Figure 3. [Ca2⫹]SR measurements. A, Whole-cell [Ca2⫹]SR during
twitches evoked at 1 Hz (black) or 0.5 Hz (red), interrupted dur-
ing 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 dia-
stolic 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 characteris-
tics (with 20 mmol/L butanedione monoxime, 4 ms/line). D, Sep-
arate signals analyzed from JSR and FSR regions. Traces are
average from 93 JSR and 29 FSR regions, with amplitude
normalized.
This is consistent with a [Ca2⫹]SR-dependent increase in frac-
Figure 2. Cellular anatomy of the SR. A and B, Images of Fluo-5N– tional release and also clearly demonstrates that [Ca2⫹]SR deple-
loaded myocytes before and after Di-8-ANNEPS addition to visual-
ize transverse tubules. Di-8-ANNEPS colocalizes with Fluo-5N sig-
tion is incomplete during a twitch.1,2 The of refilling (Figure
nal. C, Zoom of region in B, illustrating transverse tubules (T-T) 4E) speeds up with frequency, consistent with frequency-
with junctional and free SR regions in between (JSR and FSR). dependent acceleration of relaxation and [Ca2⫹]i decline.3
Because F may not be linearly related to [Ca2⫹]SR, calibrations
During the twitch, fluorescence declines to a minimum of ⬇75% are needed for greater quantitative evaluation. In vitro, Fluo-5N
at ⬇100 ms after [Ca2⫹]SR starts to decline and recovers with a Ca2⫹ affinity (Kd⫽135 mol/L) in solution measured in a
time constant () of ⬇100 ms (Figure 3B). Figure 3C shows that fluorometer is not altered by Mg or tetracaine (used below),
the [Ca2⫹]SR depletion measured at a single SR junction in although tetracaine partially quenches fluorescence (Figure 5A).
confocal microscopy is quite similar to the global [Ca2⫹]SR The presence of protein decreases both maximal fluorescence as
signal. We refer to these local [Ca2⫹]SR depletions as “Ca2⫹ well as apparent affinity of Fluo-5N for Ca2⫹ (Figure 5A). At
scraps” (since they are the intra-SR correlate of evoked Ca2⫹ cellular protein concentrations (50 to 100 mg/mL), Fluo-5N
sparks).20 Moreover, when Ca2⫹ scraps at junctional and free SR affinity is reduced ⬇3-fold (typical for fluorescent Ca2⫹ indica-
regions (as in Figure 2C) are compared, there is little kinetic tors in cells).22,23
difference (Figure 3D). This indicates that longitudinal diffusion Maximal fluorescence (FMax) in myocytes was defined by
within the SR is faster than we could readily detect (assuming stimulating SR Ca2⫹-ATPase by ISO and blocking SR Ca2⫹
SR Ca2⫹ release occurs at JSR).21 Thus, Ca2⫹ diffusion from free release by tetracaine (which dramatically increases SR Ca2⫹
to junctional SR does not appreciably limit SR Ca2⫹ availability content).24 Note that F did not rise much on tetracaine addition
for release, even at a single twitch. (even when we accounted for the modest quench by tetracaine).
Figure 4A shows [Ca2⫹]SR and contractions at different pacing To further raise [Ca2⫹]SR, extracellular Na⫹ was abruptly re-
frequencies. As frequency increases, diastolic [Ca2⫹]SR increases, moved (causing Ca2⫹ entry via Na⫹-Ca2⫹ exchange), which
as does the extent of [Ca2⫹]SR depletion and the contraction caused spontaneous contractions and corresponding Ca2⫹ deple-
amplitude. With -adrenergic activation by ISO, there was a tions (Figure 5B). Under these conditions (with the RyR inhib-
further increase in diastolic [Ca2⫹]SR and extent of [Ca2⫹]SR ited), the SR Ca2⫹ pump should approach a limiting [Ca2⫹]SR/
depletion and faster [Ca2⫹]SR recovery ( decreased from 155 to [Ca2⫹]i gradient,25 and [Ca2⫹]SR should rise by the same factor as
70 ms), consistent with the expected acceleration of SR Ca2⫹- [Ca2⫹]i (and the elevation of average [Ca2⫹]i is indicated by the
ATPase by ISO. Figures 4B through 4D show mean diastolic F cellular contracture in Figure 5B). Since F still did not increase
(Fd versus that at 1 Hz, Fd1), peak fractional depletion (⌬F/ appreciably, despite the substantial rise in [Ca2⫹]SRT expected
Fd⫽25⫾2.6% for 1 Hz), and the minimum systolic value of with this protocol, FMax represents saturation of intra-SR Fluo-
[Ca2⫹]SR. All three parameters increase with frequency and ISO. 5N. FMin is taken as FCaf.
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Shannon et al Local SR Depletions 43
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.
To test whether Kd⫽400 mol/L is appropriate for intra-SR ected to be ⬇95% depleted early during the twitch, thereby
Fluo-5N, we used permeabilized myocytes (with RyRs opened limiting further SR Ca2⫹ release.26
by caffeine). When [Ca2⫹] was stepped from 50 nmol/L (FMin) to
400 mol/L (F400) to 10 mmol/L (FMax; Figure 5C), we found that Discussion
F400 was, on average, 47.5% of FMax⫺FMin. This confirms that We report the first direct measurements of [Ca2⫹]SR during
400 mol/L is an appropriate Kd in situ.
individual contractions and with subsarcomeric spatial reso-
Using these calibrations, we found that diastolic [Ca2⫹]SR
lution. These measurements and method provide valuable
increased from 0.48 to 0.92 to 1.65 mmol/L from 0.1 to 1 Hz
new quantitative information that is at the very heart of
(Figures 6A and 6B). These values are similar to time-averaged
cardiac ECC. It has also been argued that SR Ca2⫹ depletion
whole-heart NMR estimates of [Ca2⫹]SR (1.5 mmol/L)12 and our
does not participate in the shutoff of SR Ca2⫹ release,21 and
estimates (⬇1 mmol/L), based on [Ca2⫹]SR and [Ca2⫹]SRT in SR
vesicles plus cellular [Ca2⫹]SRT.25 However, both of those results indeed we show that SR Ca2⫹ release stops at local [Ca2⫹]SR
lacked kinetic or spatial information. ⬇0.4 mmol/L when there is still a large driving force for SR
The extent of [Ca2⫹]SR depletion increased from 24% to Ca2⫹ release. Cardiac ECC models have assumed that there is
63% over this range of frequencies. Diastolic ISO data were a major time delay (up to seconds) between recovery of
not calibrated because Fd was too near FMax. Minimum [Ca2⫹]SR near Ca2⫹ uptake sites (FSR) and at release sites
[Ca2⫹]SR attained during a twitch varied from 0.36 to (JSR).27 Such purported major time lags between JSR and
0.61 mmol/L (even with ISO). These [Ca2⫹]SR measurements FSR do not seem to occur during release (Figure 3C), and the
support previous, less direct [Ca2⫹]SRT measurements that [Ca2⫹]SR is restored rapidly during the twitch (even in the
suggested incomplete SR Ca2⫹ depletion.1,2,4 Importantly, JSR). The apparent delay or restitution of SR Ca2⫹ release
Figures 3B and 3C also indicate that JSR in individual (eg, at premature heartbeats) is probably due mainly to
junctions depletes only partially. These data are not consistent recovery of RyR (and/or L-type Ca2⫹ channel) availability,
with recent models of ECC where local [Ca2⫹]SR was proj- rather than the amount of releasable SR Ca2⫹.
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44 Circulation Research July 11, 2003
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 mol/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⫾0.09⫻Fd1). C, Permeabilized cell (50 g/mL saponin) with 10 mmol/L caffeine to allow [Ca2⫹] equilibration across the SR.
We propose that [Ca2⫹]SR depletion is dynamically involved in to release appreciable SR Ca2⫹.1– 4,20,28 This demonstrates that
terminating SR Ca2⫹ release, due to direct effects on RyR gating luminal [Ca2⫹]SR dynamically modulates SR Ca2⫹ release (and
(not by exhausting available SR Ca2⫹). Indeed, the SR retains a leak) during both diastole and ECC.
Ca2⫹ reserve, which is pharmacologically accessible, as indi- The fact that SR Ca2⫹ release does not go to completion even
cated by caffeine-induced Ca2⫹ transients, which completely locally (as expected for positive feedback) rules out substrate
deplete [Ca2⫹]SR (Figure 1B). When [Ca2⫹]SR is below 40% to limitation as the cause of release termination but leaves two
50% of its control value, resting SR Ca2⫹ leak (Ca2⫹ spark potential types of inactivation.29,30 One mechanism, stochastic
frequency) is very small and a normal Ca2⫹ current trigger fails 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 inactiva-
tion (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 ICa) 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
Figure 6. Dynamic [Ca2⫹]SR profiles. A, Calibrated [Ca2⫹]SR sig-
nals. B, Mean diastolic [Ca2⫹]SR and fractional twitch depletion inactivating the channel faster and terminating release at a
at different frequencies. slightly higher [Ca2⫹]SR. However, our observation that release
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Shannon et al Local SR Depletions 45
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Acknowledgments heart. Biophys J. 1997;73:1524–1531.
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E. Ríos and L. Blatter for their help. Biophys J. 2002;83:59–78.
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Downloaded from http://circres.ahajournals.org/ by guest on September 28, 2015Ca2+ Scraps: Local Depletions of Free [Ca2+] in Cardiac Sarcoplasmic Reticulum During
Contractions Leave Substantial Ca2+ Reserve
Thomas R. Shannon, Tao Guo and Donald M. Bers
Circ Res. 2003;93:40-45; originally published online June 5, 2003;
doi: 10.1161/01.RES.0000079967.11815.19
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