Alteration in Calcium Handling at the Subcellular Level in
mdx Myotubes*
Valerie
Robert
,
Maria Lina
Massimino,
Valeria
Tosello,
Robert
Marsault,
Marcello
Cantini,
Vicenzo
Sorrentino§¶, and
Tullio
Pozzan
From the Department of Biomedical Sciences, CNR Center of
Biomembranes, University of Padova, 35131 Padova,
§ DIBIT, San Raffaele Scientific Institute, 20132 Milano,
and ¶ Department of Neurosciences, Section of Molecular
Medicine, University of Siena, 53100 Siena, Italy
Received for publication, July 17, 2000, and in revised form, August 29, 2000
 |
ABSTRACT |
In this study, we have tested the hypothesis that
augmented [Ca2+] in subcellular regions or
organelles, which are known to play a key role in cell survival, is the
missing link between Ca2+ homeostasis alterations and
muscular degeneration associated with muscular dystrophy. To this end,
different targeted chimeras of the Ca2+-sensitive
photoprotein aequorin have been transiently expressed in subcellular
compartments of skeletal myotubes of mdx mice, the animal
model of Duchenne muscular dystrophy. Direct measurements of the
[Ca2+] in the sarcoplasmic reticulum,
[Ca2+]sr, show a higher steady state level at
rest and a larger drop after KCl-induced depolarization in
mdx compared with control myotubes. The peaks in
[Ca2+] occurring in the mitochondrial matrix of
mdx myotubes are significantly larger than in controls upon
KCl-induced depolarization or caffeine application. The augmented
response of mitochondria precedes the alterations in the
Ca2+ responses of the cytosol and of the cytoplasmic region
beneath the membrane, which become significant only at a later stage of myotube differentiation. Taking into account the key role played by mitochondria Ca2+ handling in the control of cell death,
our data suggest that mitochondria are potential targets of impaired
Ca2+ homeostasis in muscular dystrophy.
 |
INTRODUCTION |
Although it is well established that the lack of dystrophin
expression is the primary genetic defect in Duchenne's muscular dystrophy (DMD),1 the
mechanism leading to progressive muscle damage is still largely unknown
(1). It has been suggested that an elevation of cytosolic Ca2+ concentration ([Ca2+]c), under
resting conditions, and a concurrent activation of
Ca2+-dependent proteases may represent the
mechanistic link between the genetic defect and the DMD phenotype (2).
The differences in [Ca2+]c between normal and
dystrophic muscles have been found also in myotubes and in the
classical animal model of the disease, the mdx mouse.
Several groups (3, 4), however, have been unable to confirm these data,
and the question remains controversial.
Evidence has been accumulated over the last few years indicating that a
key aspect of the Ca2+ signaling pathway is represented by
its spatial and temporal complexity. Localized changes in the cytosol,
much larger than those occurring in the bulk cytosol, are known to
occur close to the mouth of Ca2+ channels, and these
localized events are pivotal in triggering important cellular events
such as secretion, gene expression, and metabolic activation. In this
respect, mitochondria represent a privileged sensor of local
[Ca2+] increases. Not only their Ca2+
accumulation depends on microdomains of high Ca2+ generated
in their vicinity, but their capacity to take up Ca2+ is
essential to shape the kinetics of cytoplasmic Ca2+ changes
(5). Last, but not least, mitochondrial Ca2+ accumulation
results in activation of ATP production under physiological conditions
(6) but leads to initiation of apoptotic signaling when excess
Ca2+ is taken up by the organelles (7). Taking into
account the key role played by mitochondria Ca2+ handling
in the control of cell death, our data suggest that mitochondria are
potential targets of impaired Ca2+ homeostasis in muscular dystrophy.
In this study, we tested the hypothesis that the differences in
cytoplasmic [Ca2+] in muscles lacking dystrophin might be
amplified in specific cellular regions, in particular within the
mitochondrial matrix. We addressed this issue directly by using the
strategy of targeted aequorin that we previously developed and used to
analyze Ca2+ handling in different types of cells ranging
from cell lines to primary cultures of neurons or rat skeletal myotubes
(8-11). To evaluate the importance of mechanical stress as a key
factor in the development of muscle degeneration (12, 13), analysis of
[Ca2+] homeostasis at the subcellular levels was carried
out both at day 7 of culture (when spontaneous contractions are
minimal) and in older cultures (day 11), i.e. in cells
harboring a more mature contractile machinery that undergo frequent
contractions. The observation that upon stimulation the
Ca2+ response of mitochondria is augmented in
mdx cells compared with controls, even before significant
alterations of the [Ca2+]c, suggests that
derangement in Ca2+ handling by these organelles may
play a pivotal role in degeneration of dystrophic fibers.
 |
MATERIALS AND METHODS |
Cell Culture--
Myotubes were prepared from hind leg muscles
of 1-3-day-old normal (C57BL101) and mdx mice. The muscle
was minced and submitted to three successive treatments with 0.125%
trypsin. Cells were resuspended in DMEM supplemented with 10% fetal
calf serum and were seeded at a density of 3 × 105
cells onto 13-mm coverslips coated with collagen for aequorin measurements or at a density of 106 cells onto 24-mm
collagen-coated coverslips for GFP detection.
Chimeras and Transfection--
The different constructs have
been described in detail previously (8-10). Transfection was carried
out on the 2nd day using the calcium-phosphate method. After 12 h
of incubation with the calcium-phosphate precipitate, cells were
washed, and the growth medium was replaced with DMEM + 2% horse serum
to induce fusion of myoblasts. All the experiments were then performed
at days 7 and 11 of culture.
[Ca2+] Measurements with Aequorin--
For the
cell transfected with the cytAEQ, the mitAEQ, and the pmAEQ constructs,
reconstitution of the functional aequorin occurs in DMEM supplemented
with 1% serum and 5 µM coelenterazine for 1 h at
37 °C. For the srAEQ, we have shown previously that to obtain an
efficient reconstitution, it is necessary to reduce drastically the
[Ca2+] in the lumen of the store. This is accomplished by
incubating the cells for 1 h at 4 °C in a Krebs-Ringer buffer
(125 mM NaCl, 5 mM KCl, 1 mM
Na3PO4, 1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES, pH 7.4)
containing 5 µM coelenterazine, 30 µM N,tert-butylhydroquinone, and 1 mM EGTA (11). After the reconstitution step, the cells were
placed in a perfused, thermostated chamber in close proximity to a low
noise photomultiplier, with a built-in amplifier-discriminator. The
output of the discriminator was captured by a Thorn-EMI photon counting
board and stored in an IBM-compatible computer for further analyses.
Aequorin photon emission was calibrated off line into [Ca2+] values using a computer algorithm based on the
Ca2+ response curve of wild-type and mutant aequorins, as
described previously (14).
Confocal Examination of the pmGFP--
Transfected cells were
observed with a Nikon RCM8000 real time confocal microscope.
Statistical Analysis--
All data are reported as means ± S.D. Statistical differences between control and mdx cells
were evaluated by a one-tailed Student's t test, a
p value <0.05 was considered statistically significant.
 |
RESULTS |
The Stimulated [Ca2+] Peak in the Mitochondria Is
Elevated in mdx Myotubes--
Fig. 1
shows that perfusion of myotubes, transfected with cytosolic and
mitochondria targeted aequorins, with high KCl (125 mM) to
depolarize the plasma membrane results in rapid increases of
[Ca2+] in both compartments. In absolute terms, the
[Ca2+] increases in the mitochondrial matrix are about
10-fold higher than in the cytosol, as previously reported for rat
myotubes (10). On average, the rises in [Ca2+]c
were indistinguishable in control and mdx myotubes (1.37 ± 0.06 µM, n = 9,, versus 1.38 ± 0.09 µM n = 10, respectively), whereas the peak rises in the mitochondrial
matrix, [Ca2+]m, were statistically higher in the
cells from affected animals (16.9 ± 1.1 µM,
n = 11, in mdx versus 12.2 ± 1.1 µM, n = 12, in controls
p < 0.01). The pre-stimulatory levels of
[Ca2+] were indistinguishable in the two myotube
populations for either compartment (around 100 nM). This
conclusion, however, should be taken with caution since the calibration
of the aequorin signal, accurate for concentrations above 300-400
nM, becomes subject to major uncertainties at lower levels.

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Fig. 1.
Cytoplasmic and mitochondrial
[Ca2+] increases caused by plasma membrane
depolarization. Skeletal myotubes were transfected with chimeras
that encode for the aequorin expressed either in the cytosol or in the
mitochondria. [Ca2+] measurements were done at day 7 of
culture after a step of reconstitution in DMEM supplemented with 1%
fetal calf serum in which 5 µM coelenterazine was added.
The coverslip was then placed in the thermostated chamber of the
luminometer and perfused with Krebs-Ringer buffer containing 1 mM Ca2+. Where indicated cells were stimulated
with 125 mM KCl (iso-osmotic substitution of NaCl) which
results in a rapid Ca2+ spike in the cytosol and the
mitochondria. Typical traces for myotubes transfected with the cytAEQ
(A) or mitAEQ (C) and corresponding mean steady
state levels of the Ca2+ spike amplitude (B and
D, respectively) are shown for mdx (black
line) and control (Cont) cells (hatched
line). *, p < 0.01 mdx
versus control cells.
|
|
Microdomains of High Ca2+ Involved in the Altered
Mitochondrial Responses--
Given that the
[Ca2+]m rises are secondary to
[Ca2+]c increases, the above results may appear
contradictory. However, some evidence now indicates that mitochondrial
Ca2+ uptake depends on microdomains of high
[Ca2+] generated in the proximity of the organelles in
close contact to Ca2+ channels of the endo-sarcoplasmic
reticulum or of the plasma membrane, rather than on the bulk increase
in [Ca2+]c (8). The larger increase of
[Ca2+]m observed in mdx myotubes could
thus depend on larger increases of [Ca2+] in such
microdomains. To investigate this issue directly, in a first set of
experiments, myotubes were transfected with an aequorin chimera (pmAEQ)
whose expression in HeLa or A5r7 cells was shown to be restricted to
the inner surface of the plasma membrane (9). We employed the low
Ca2+ affinity mutant of pmAEQ, given that it has been
previously demonstrated that only this construct correctly measures the
high [Ca2+] occurring in this region. To verify the
correct targeting of the expressed protein in living cells, we took
advantage of the observation that the subcellular localization of
aequorin is entirely dependent on the targeting strategy (in the case
of pmAEQ, the fusion with SNAP25). Accordingly, a construct where the
fluorescent protein GFP is fused at the C terminus of SNAP25 has the
same subcellular localization of a construct containing aequorin (data not shown). Fig. 2A shows that
the distribution of the pmGFP in skeletal myotubes is largely
restricted to the inner surface of the plasma membrane, although a
small diffuse intracellular signal is also observed. As shown in Fig.
2B, upon depolarization with high KCl, the peak level of
Ca2+ increase measured with the pmAEQ is over 1 order of
magnitude higher than in the bulk cytosol, but no significant
difference was observed between the two myotube populations (14.1 ± 1.2 µM, n = 6, for mdx
versus 16.3 ± 3.7 µM, n = 6, for control cells). A much lower Ca2+ increase (around
1 µM) was observed with this aequorin construct when
cells were exposed to KCl in the absence of Ca2+ or in the
presence of extracellular Ca2+ to caffeine, confirming that
this probe is selectively suited to monitor the [Ca2+]
changes occurring underneath the plasma membrane upon opening of
Ca2+ channels located there.

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Fig. 2.
Sub-plasmalemmal [Ca2+]
changes. A, confocal image of mdx myotubes
transiently expressing pmGFP. B, [Ca2+]
measurements of myotubes transfected with pmAEQ. All other conditions
are as described in Fig. 1. The columns represent the mean
level of the Ca2+ spike induced by KCl stimulation in cells
transfected with the pmAEQ (C). Bar, 15 µm.
Cont, control.
|
|
An alternative explanation to account for the larger
[Ca2+]m increases, despite minor differences in
the bulk cytoplasm or underneath the plasma membrane, is that more
Ca2+ is released from the SR. In fact, we and others (8,
10) have previously demonstrated that effective Ca2+ uptake
by mitochondria depends on the strategic location of the latter
organelles close to the Ca2+ release channels.
Ca2+ handling by the SR was thus investigated directly by
transfecting myotubes with a selectively localized aequorin, srAEQ. In
this case, reconstitution of an active aequorin with coelenterazine requires prior depletion of Ca2+ from the organelle,
followed by refilling (see "Materials and Methods"). Fig.
3 shows that upon re-addition of
Ca2+ to the medium, the [Ca2+] within the SR
rapidly increases up to a steady state level of about 400 µM (429 ± 14 µM, n = 9), a value close to that found in rat myotubes (11). Under the same
conditions, the steady state level in the SR of mdx myotubes
is significantly higher, 596 ± 68 µM
(n = 9). The difference in the intralumenal level of
[Ca2+] between the SR of myotubes prepared from controls
and mdx mice concerns not only the steady state level but
also the changes caused by KCl that are larger in mdx
myotubes. To confirm further that in mdx myotubes the
increased Ca2+ response in the mitochondria is due to a
larger release of Ca2+ from the SR, we have examined the
effect of caffeine. This drug directly causes the opening of ryanodine
receptors in the SR terminal cysternae without directly affecting
plasma membrane channels. The [Ca2+]m peak upon
addition of 50 mM caffeine is higher in mdx than
in control cells (10.7 ± 0.8 µM, n = 6, for mdx versus 7.26 ± 0.33 µM,
n = 6, for control cells, p < 0.01)
(Fig. 4, A and B).
However the difference in caffeine response between mdx and
control cells is lost when the drug is applied at a lower concentration
(Fig. 4, C and D). For 10 mM
caffeine, mdx and control myotubes revealed a clear
difference in the shape and amplitude of the Ca2+
responses. In mdx myotubes, the rising phase was slower and
less ample but was followed by a sustained elevation of the signal. This lower sensitivity of mdx cells to caffeine was evident
also for the [Ca2+] changes occurring in the cytoplasm.
We thus tested the possibility that a qualitative or quantitative
difference in ryanodine receptor isoform expression could be
responsible for the different caffeine sensitivity of the two myotube
preparations. The level of ryanodine receptor was thus quantitatively
assessed by Western blotting using antibodies selectively recognizing
types 1-3. No difference between mdx or control cultures in
the level of expression of any of these channels was observed, however
(data not shown).

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Fig. 3.
[Ca2+] changes within the
SR. Kinetics of the [Ca2+] changes in myotubes
transfected with the srAEQ construct (A). B, the
columns represent the mean steady state level of the
[Ca2+]sr. C shows the effect of KCl on
[Ca2+]sr. *, p < 0.01 mdx versus control (Cont) cells,
n = 9 for each cell population.
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Fig. 4.
[Ca2+] changes in the
mitochondria as induced by caffeine. Typical traces of
[Ca2+] measurements on myotubes transfected with the
mitAEQ construct and treated with 50 (A), 30 (C),
and 10 mM caffeine (D). B, the
columns represent the mean levels of the [Ca2+] induced
by 50 mM caffeine (B). *, p < 0.01 mdx versus control (Cont) cells,
n = 6 for each cell population.
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Evolution of [Ca2+] Alterations with Myotube
Maturation--
Taken together the data reported above demonstrate
that, despite no obvious difference in Ca2+ handling in the
bulk cytosol and at the plasma membrane level, the mitochondria are
strategically located to reveal the alterations in Ca2+
homeostasis occurring in the SR of mdx cells. It has been
suggested that the defect in Ca2+ homeostasis is
exacerbated in mdx cells upon application of mechanical stress (12, 13). A simple and physiological way to test this hypothesis
is to prolong the time in culture. The maturation of the myotubes, in
fact, correlates with the development of the cytoskeletal architecture
and with the intensification of spontaneous contractile events. In the
experiments shown in Fig. 5, the mean peak rise in cytosol, mitochondria, and sub-plasma membrane region of
control and mdx myotubes was compared at day 11 of culture. Unlike the results obtained in 7-day-old cultures, the peak increases caused by KCl were significantly higher in mdx compared with
control myotubes, not only in the mitochondria but also in the other
two compartments. We also tried to measure the [Ca2+] in
the sarcoplasmic reticulum at this stage of culture. However, the
protocol used to deplete the SR during the reconstitution step induced
cell hypercontracture and detachment from the plate. This effect was
particularly evident in mdx myotubes.

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Fig. 5.
[Ca2+] measurements on myotubes
at day 11 of culture. Typical traces of [Ca2+]
responses generated by KCl stimulation and corresponding mean levels of
the Ca2+ spike amplitude for myotubes transfected with
cytAEQ (A and B), mitAEQ (C and
D), and pmAEQ (E and F).
*p < 0.01 mdx versus control
(Cont) cells, n = 6 for each construct and
cell population.
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|
 |
DISCUSSION |
The hypothesis that in DMD the missing link between the absence of
dystrophin and muscle degeneration is represented by a derangement in Ca2+ homeostasis has been proposed
several years ago, but no firm conclusion has yet been reached. In
fact, whereas different groups (2, 13, 15) reported that the resting
levels of cytosolic [Ca2+] are higher in mdx
or DMD compared with control cells and that this increase is a
determinant factor leading to protease activation and subsequent
muscular degeneration, other investigators (3, 4) could not confirm
this difference. Controversial data have also been reported for the
peak levels of [Ca2+]c upon stimulation, some
groups finding larger rises in mdx (15), others similar to
controls (16), and some even reporting reductions (17). We have
re-addressed this issue by using a different methodological and
conceptual approach. We reasoned that if the resting and stimulated
[Ca2+] is indeed slightly modified in
dystrophin-deficient cells, it could be predicted that by directly
investigating Ca2+ handling in regions and organelles,
where the cytosolic signal is amplified, it should be possible to
verify more stringently the hypothesis.
Under resting conditions, a consistent difference in
[Ca2+] between mdx and controls was observed
in the SR lumen. This finding, however, is consistent with the
hypothesis of Steinhardt's group given that the SR accumulates
Ca2+ up to a concentration that is over 3000-fold that of
the cytosol. Furthermore, considering that (i) the
Kd for Ca2+ of the SERCAs is close to
the resting cytosolic level and (ii) that the steady state value of
Ca2+ within the SR depends on the kinetic balance between
Ca2+ influx and efflux, it was predicted that a small
increase in the mean [Ca2+] of the cytosol would result
in a much larger accumulation of the cation in the lumen of the store.
Our findings are therefore consistent with the proposal that in
mdx myotubes there is a small increase of resting cytosolic
Ca2+. We here show that this small difference is then
amplified in the SR.
During stimulation, as a consequence of the higher steady state
[Ca2+] in the SR of mdx cells, more
Ca2+ is released from the SR of dystrophin-deficient cells
compared with controls. This results in an amplified response within
mitochondria, whereas the increase in the bulk cytosolic
[Ca2+] is unaffected. The explanation for this apparent
discrepancy lies in the capacity of mitochondria located close to the
ryanodine receptors to buffer effectively the Ca2+ coming
out from the SR, minimizing the differences in bulk cytosolic Ca2+ changes between the two myotube populations. It should
be also stressed that the values of [Ca2+]m
reported here are the mean of the whole organelle populations, and
probably higher values are reached in the mitochondria closer to the
release sites. The conclusion that the primary difference between
mdx and control cells depends on the larger release from the
SR is further supported by the observation that (i) a higher Ca2+ accumulation in the mitochondria is induced by 50 mM caffeine, which only causes Ca2+ release
from the SR, and that (ii) no modification of the Ca2+ peak
in the sub-plasmalemmal space is observed between the mdx and control cells upon KCl depolarization. It should be stressed that
this is the first direct measurement in a muscle cell of the levels of
[Ca2+] in this latter subcellular compartment.
Our results also demonstrate that quantitative or phenotypic
modifications of ryanodine receptors could not account for the altered
response of mdx myotubes, given that the expression level of
the three ryanodine receptor isoforms is unaffected. We can only
speculate, at the moment, on the reason for this reduced sensitivity to
caffeine. These results may also help explain the controversies between
different groups concerning the peak values of
[Ca2+]c measured upon stimulation. In fact, the
differences between mdx and control cells may vary depending
also on the type and concentration of stimulus used.
Considering the sarcolemmal localization of dystrophin, most of the
studies exploring the mechanism of [Ca2+]c
modifications in dystrophic muscles have been focused on altered
Ca2+ influx. Changes of Ca2+ channel activities
or appearance of novel forms of Ca2+ leak channels, notably
of stretch-activated Ca2+ channels, have been reported (18,
19). In addition, tetrodotoxin-sensitive spontaneous contractile
activity of myotubes in culture exacerbates the stimulated
Ca2+ increases (12, 13). We confirmed these observations
since in more mature cultures (day 11) characterized by frequent
spontaneous contractions, the peak increases caused by KCl was found to
be significantly higher, both in the cytosol and in the
sub-plasmalemmal space. Hence, our data strengthen and extend previous
observations demonstrating that alterations of Ca2+
handling progressively amplify as mechanical work augments.
How does the alteration in Ca2+ handling relate to the
muscle fiber degeneration typical of this disease? Our data clearly do not exclude that activation of proteolysis may be involved, but they
suggest the possibility that other pathways may be as or even more
relevant. In fact, several groups have reported the presence of
apoptotic fibers in both mdx mice or DMD patients before
muscular necrosis becomes evident (20, 21), and evidence is
accumulating in favor of a causal relation between elevations in
[Ca2+] in the mitochondria and the activation of
apoptosis (5). In turn, very recent evidence has been published
relating the [Ca2+] within the ER-SR and the activation
of the apoptotic process. In particular overexpression of Bcl-2, the
well known anti-apoptotic protein, results in a reduction of steady
state [Ca2+] in the ER (and reduced Ca2+
peaks in the mitochondrial matrix upon stimulation) (22), whereas overexpression of SERCA, and a higher Ca2+ accumulation in
the ER, accelerates spontaneous cell death (23). These and other data
thus suggest the existence of a causal link between the level of
Ca2+ within the stores and the activation-inhibition of
apoptosis (24). As to the mitochondrial pathways that could be affected by derangements in Ca2+ homeostasis, obvious
candidates appear at the opening of the permeability transition pore
and the ensuing release of pro-apoptotic factors or/and ATP synthesis
(24). Of interest, early changes in mitochondrial functions have been
demonstrated in muscle of mdx mice with a reduction of 50%
of the activity of the respiratory chain (25).
 |
ACKNOWLEDGEMENTS |
We thank Dr. A. Conti for carrying out
Western blot analysis of ryanodine receptors, L. Pasti for confocal
microscopy, S. Jouaville for helpful discussions, and G. Ronconi and M. Santato for expert technical assistance.
 |
FOOTNOTES |
*
This work was supported by Telethon Grants 845 and 1226, by
the Italian Association for Cancer Research Grants ACC990092 and 98130471, Union European Programs for Biotechnology Grant
BIO4CT960382, Training and Mobility for Researchers Grant TMR 980236, CNR Biotechnology Project 9900445PF49, by the Italian Minister of
University and Scientific Research (MURST), 1998-1999, and by the
Armenise Harvard Foundation (to T. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biomedical
Sciences, University of Padova, Via G. Colombo 3, 35121, Padova, Italy.
Tel.: 0039-049-8276067; Fax: 0039-049-8276049; E-mail: pozzan@civ.
bio.unipd.it.
Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M006337200
 |
ABBREVIATIONS |
The abbreviations used are:
DMD, Duchenne
muscular dystrophy;
[Ca2+]c, cytosolic
Ca2+ concentration;
[Ca2+]m, mitochondrial Ca2+ concentration;
[Ca2+]sr, Ca2+ concentration in the
sarcoplasmic reticulum;
GFP, green fluorescent protein;
pmGFP, sub-plasmalemmal green fluorescent protein;
cytAEQ, cytosolic aequorin,
mitAEQ, mitochondrial aequorin;
pmAEQ, sub-plasmalemmal aequorin;
srAEQ, aequorin expressed in the sarcoplasmic reticulum. SERCA,
sarco-endoreticulum Ca2+ ATPase;
SR, sarcoplasmic
reticulum;
ER, endoplasmic reticulum;
DMEM, Dulbecco's modified
Eagle's medium.
 |
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