From the Pharmacology Group, School of Pharmacy,
University of Lausanne, 1015 Lausanne and § Division of
Clinical Biochemistry, Department of Internal Medicine, University
Medical Center, 1211 Geneva 4, Switzerland
Received for publication, July 13, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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Mitochondrial Ca2+
concentration ([Ca2+]m) was monitored in C2C12
skeletal muscle cells stably expressing the Ca2+-sensitive
photoprotein aequorin targeted to mitochondria. In myotubes,
KCl-induced depolarization caused a peak of 3.03 ± 0.14 µM [Ca2+]m followed by an
oscillatory second phase (5.1 ± 0.1 per min). Chelation of
extracellular Ca2+ or blockade of the voltage-operated
Ca2+ channel attenuated both phases of the KCl response.
The inhibitor of the sarcoplasmic reticulum Ca2+-ATPase,
cyclopiazonic acid, reduced the amplitude of the KCl-induced [Ca2+]m peak and prevented the oscillations,
suggesting that these were generated intracellularly. No such
[Ca2+]m oscillations occurred with the nicotinic
agonist carbachol, cyclopiazonic acid alone, or the purinergic agonist
ATP. In contrast, caffeine produced an oscillatory behavior, indicating
a role of ryanodine receptors as mediators of the oscillations. The
[Ca2+]m response was desensitized when cells were
exposed to two consecutive challenges with KCl separated by a 5-min
wash, whereas a second pulse of carbachol potentiated
[Ca2+]m, indicating differences in intracellular
Ca2+ redistribution. Cross-desensitization between KCl and
carbachol and cross-potentiation between carbachol and KCl were
observed. These results suggest that close contacts between
mitochondria and sarcoplasmic reticulum exist permitting
Ca2+ exchanges during KCl depolarization. These newly
demonstrated dynamic changes in [Ca2+]m in
stimulated skeletal muscle cells might contribute to the understanding
of physiological and pathological processes in muscular disorders.
Duchenne muscular dystrophy is one of the most severe myopathies.
Its primary defect is the lack of dystrophin, a cytoskeletal protein
linking the extracellular matrix with the cytoskeleton at the
sarcolemmal membrane. The pathogenesis of Duchenne muscular dystrophy
is not well characterized; however, a dysregulation of Ca2+
handling is proposed to play an important role (for review, see Ref.
1). An abnormal increase in cytosolic Ca2+ in dystrophic
muscle could activate Ca2+-dependent proteases
and disturb the metabolic regulation of cells leading to muscle
degeneration (2, 3). There is some controversy about this
Ca2+ elevation (1, 4). Some groups (5, 6) showed that basal
cytosolic Ca2+ concentration
([Ca2+]c)1
was higher in dystrophic skeletal muscle cells relative to controls, whereas other laboratories (7, 8) found that
[Ca2+]c was only increased under stress. To
elucidate this contention, the redistribution of Ca2+ into
intracellular organelles should be examined. In this context, the
mitochondrion is a good candidate, since it is 1) able to buffer part
of [Ca2+]c (9, 10); 2) able to answer to the
energy demand of cells by tuning the creatine/phosphocreatine ratio
(11, 12); and 3) able to produce ATP via the increase of mitochondrial
Ca2+ concentration ([Ca2+]m) (13) and
the activation of key enzymes of oxidative phosphorylation (14). The
control of [Ca2+]m homeostasis is not only
important for the regulation of metabolism but also for the propagation
of the Ca2+ signal throughout cells and for
Ca2+-regulated cell functions (15, 16). This is illustrated
by the correlation of mitochondrial Ca2+ uptake with
insulin secretion on the one hand (17) and catecholamine secretion on
the other hand (18).
In recent years, chimeras of the Ca2+-sensitive
photoprotein aequorin with unique cellular targets contributed to the
understanding of the cross-talk between intracellular compartments
(19). In particular, contacts between endoplasmic reticulum and
mitochondria were demonstrated in the HeLa cell line (20). Furthermore,
it appears that release of Ca2+ from endoplasmic reticulum
located near mitochondria leads to the formation of microdomains of
high [Ca2+], thus permitting mitochondrial
Ca2+ uptake via a low affinity uniporter (10, 21).
To investigate compartmental Ca2+ distribution in skeletal
muscle, we used aequorin targeted to mitochondria (mtAeq). The C2C12 murine skeletal muscle cell line was studied as a model to analyze cellular responses to various Ca2+ raising agents (Fig.
1). These include depolarizing agents
such as KCl at high concentration, which causes direct depolarization of the plasma membrane activating the voltage-operated Ca2+
channel and releasing Ca2+ from the sarcoplasmic reticulum
(SR) via the ryanodine receptor (RyR) (22); carbachol (CCh), which
induces indirect depolarization by the activation of the nicotinic
acetylcholine receptor and a subsequent Na+ entry;
extracellular ATP, which activates P2U-purinergic receptors leading to the formation of inositol 1,4,5-trisphosphate
(IP3) and Ca2+ release from the SR through the
IP3 receptor (23); cyclopiazonic acid (CPA), which blocks
the sarcoplasmic reticulum Ca2+-ATPase (SERCA); and
caffeine (Caf) as a RyR activator (22). [Ca2+]c
increases are transmitted to mitochondria via an electrogenically driven uniporter leading to transient increases in
[Ca2+]m.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Scheme of the presumed sites of action
of the agents affecting [Ca2+]m tested in
skeletal muscle cells. In myotubes, Ca2+ can be
released from the SR by cell depolarization or by purinergic
stimulation. KCl and CCh, a nicotinic acetylcholine receptor
(nAChR) agonist, induce cell membrane depolarization
(+++). The voltage-sensitive Ca2+ channel that
functions as the dihydropyridine receptor (DHPR) then
interacts with the RyR, and Ca2+ is released from the SR.
Caf itself can activate the RyR directly. Ca2+-induced
Ca2+ release could reinforce RyR stimulation (+). ATP
activates the purinergic receptor (P2UR)
leading, via phospholipase C (PLC) activation, to the
formation of IP3 which in turn releases Ca2+
from the SR via the IP3 receptor
(IP3R). The elevated
[Ca2+]c can be redistributed to mitochondria via
the Ca2+ uniporter (U) or to the SR via the
SERCA that can be inhibited by cyclopiazonic acid (CPA).
Further abbreviations used are: -Bgt,
-bungarotoxin; Nif, nifedipine.
The present study shows that KCl-induced depolarization and caffeine
caused a transient peak in [Ca2+]m followed by an
oscillatory second phase in C2C12 myotubes. These
[Ca2+]m oscillations were not observed with ATP,
CPA, or the other depolarizing agent CCh. These results suggest
privileged appositions of SR and mitochondria as described for other
cell types (20, 24, 25).
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EXPERIMENTAL PROCEDURES |
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Materials--
Coelenterazine, Fura-2/AM, and Mitotracker were
from Molecular Probes (Eugene, OR). Carbachol and carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP) were from Fluka
(Buchs, Switzerland). Atropine, -bungarotoxin, cyclopiazonic acid,
and caffeine were from Sigma. Digitonin was from Merck. G418 was from
Calbiochem. Mouse monoclonal anti-HA-probe (F-7) IgG2 was
from Santa Cruz Biotechnology (Santa Cruz, CA), and fluorescein-labeled
goat anti-mouse IgG from Pierce. CGP 37157 was from Tocris (Bristol, UK).
Cell Culture-- C2C12 murine skeletal muscle cells were grown in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with essential amino acids, vitamins, 10 µg/ml ciproxine, 10% fetal calf serum (FCS, Life Technologies, Switzerland), and 500 µg/ml G418 for continuous selection of C2C12 myoblasts expressing the transgenes. Cultures were incubated at 37 °C in a water-saturated atmosphere of 5% CO2 in air and were passaged twice a week. For experiments, cells were seeded in 4- or 6-well plates at 104 cells per cm2 in DMEM, 10% FCS. After 3-4 days in culture, confluence was reached, and differentiation of C2C12 myoblasts into post-mitotic myotubes was induced by reducing the FCS concentration in the culture medium from 10 to 2%.
C2C12 Cell Line Stably Expressing Aequorin Targeted to Mitochondria-- C2C12 cells were stably transfected with a pcDNAI expression vector containing a cDNA encoding aequorin targeted to the mitochondria (26). After 1 day in culture, two 80-cm2 flasks of C2C12 myoblasts (~125'000 cells/flask) were transfected by calcium phosphate precipitation as follows. Culture medium was replaced by fresh DMEM, 10% FCS 1 h before transfection. mtAeq/pcDNAI (20 µg) and plasmid RSVNeo (6 µg) were added to 750 µl of 250 mM CaCl2. This solution was slowly added to 750 µl of phosphate containing solution (in mM: NaCl 280, NaH2PO4 750, Na2HPO4 750, Hepes 50, pH 7) and incubated for 40 min at room temperature. The calcium phosphate/DNA precipitate (750 µl) was added to each flask, and cells were incubated overnight. Medium was removed, and cells were exposed to 15% glycerol in DMEM, 10% FCS for 2 min. They were incubated overnight in DMEM, 10% FCS and were split at a 1:6 ratio as described (27). Selection for transfected cells was done with 1.5 mg/ml G418 for 18 days. Colonies were isolated with glass rings, and two stable cell lines, C2C12 mtAeq 8 and C2C12 mtAeq 11, were established. The mitochondrial localization of the Ca2+-sensitive photoprotein aequorin was verified by specific immunostaining (17). Photon quantification after digitonin (100 µM) permeabilization showed that both clones exhibited sufficient mtAeq expression for quantitative [Ca2+]m analysis. The capacity of myoblasts to differentiate into myotubes was compared and prompted the choice of clone 8 which was used up to passage 16.
Mitochondrial [Ca2+] Measurement-- C2C12 myoblasts were seeded on gelatin-coated ThermanoxTM coverslips of 13-mm diameter (Nunc, Life Technologies, Inc.). After 8-11 days in culture, [Ca2+]m was measured in a population of myotubes (about 60,000 cells/coverslip) as follows. The mtAeq was reconstituted with coelenterazine (5 µM) in DMEM for 2-4 h before the experiment. The coverslip in a 0.5-ml chamber thermostated at 37 °C was placed 5 mm from the photon detector. Cells were superfused at a rate of 1 ml/min with physiological salt solution (PSS, in mM: NaCl 145, KCl 5, MgCl2 1, Hepes 5, glucose 10, and CaCl2 1.2, pH 7.4). Stimuli were usually applied for 5 min (unless otherwise stated) in PSS except for high KCl solution which contained (in mM) NaCl 95, KCl 55, MgCl2 1, Hepes 5, glucose 10, and CaCl2 0.12, pH 7.4. Emitted luminescence was detected by a photomultiplier apparatus (EMI 9789, Thorn-EMI, UK) and recorded every second using a computer photon-counting board (EMI C660) as described previously (17). As published (28-31), the relationship between recorded counts and [Ca2+] is shown in Equation 1,
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(Eq. 1) |
Cytosolic [Ca2+] Measurement-- C2C12 cells were seeded on gelatin-coated glass coverslips of 22 mm diameter. After 45 min loading in 5 µM Fura-2/AM in the dark at room temperature, cells were washed twice with PSS. The coverslip was placed in a thermostated chamber at 37 °C on the stage of a fluorescence microscope (Nikon Diaphot, Küsnacht, Switzerland). After 3 min of stabilization in PSS, myotubes were excited at alternative wavelengths of 340 and 380 nm, and emission was recorded at 510 nm. The PhoCal software (Life Science Resources Ltd., Cambridge, UK) was used to analyze the collected data. The ratio R between the emitted light at 340 and 380 nm permitted calculation of [Ca2+]c according to the equation formulated by Grynkiewicz et al. (32). [Ca2+]c determinations were done independently of the [Ca2+]m measurements on separate coverslips.
Statistical Analysis--
Where applicable, values are expressed
as means ± S.E., and significance of difference was calculated by
one-way analysis of variance and two-tailed Student's t
test for unpaired data. Traces are representative of at least 3 experiments performed at least in duplicate.
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RESULTS |
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Effect of KCl on [Ca2+]m and Its Modulation
by Mitochondrial Agents--
Exposure of C2C12 myotubes to a
depolarizing solution of KCl (55 mM) for 5 min led to an
increase in [Ca2+]m, from a base line of
0.20 ± 0.01 to 3.03 ± 0.14 µM (n = 38, Fig.
2A). A delay of 14.8 ± 1.0 s was observed between the addition of the stimulus and the
onset of the [Ca2+]m elevation, and the time from
the onset to the maximum was 23.4 ± 1.3 s. The peak was
immediately followed by [Ca2+]m oscillations,
with a frequency of 5.1 ± 0.1 per min. The duration of KCl
exposure affected these [Ca2+]m oscillations. A
brief (20 s) KCl pulse elicited a [Ca2+]m peak
without oscillations (2.22 ± 0.02 µM, n = 4; Fig. 2A, inset), whereas oscillations were
observed with longer (1 min) KCl pulses. KCl induced an elevation in
[Ca2+]m 6 to 7 times higher than the one of
[Ca2+]c (Fig. 2A). No oscillations of
cytosolic Ca2+ were detected under KCl stimulation even if
the Fura-2/AM concentration was reduced from 5 to 2 µM to
attenuate its Ca2+ buffering capacity (data not shown).
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In further experiments, the mitochondrial membrane potential was blunted with the uncoupler FCCP (1 µM) which dissipates the proton gradient. After this pretreatment, the KCl-induced [Ca2+]m increase was completely blocked (Fig. 2B). This result confirms the correct localization of aequorin in mitochondria. As KCl induced a transient [Ca2+]m increase, the extrusion pathways from mitochondria were studied. The inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP 37157 (10 µM) did not affect the KCl-induced [Ca2+]m response (Fig. 2C). Preincubation of the myotubes with cyclosporin A (1 µM), a blocker of the mitochondrial permeability transition pore (33), also failed to modify KCl-induced [Ca2+]m transient or oscillations (data not shown).
Modulation of KCl-induced [Ca2+]m Increase by
Agents Not Acting on Mitochondria--
Extracellular Ca2+
depletion by 3 mM EGTA reduced the 55 mM
KCl-induced [Ca2+]m transient by 56 ± 17%
to 1.32 ± 0.34 µM at peak (n = 5),
maintaining the oscillations (Fig.
3A). When nifedipine (1 µM), a blocker of the voltage-operated L-type
Ca2+ channel, was added 20 min before and during KCl
stimulation, a similar [Ca2+]m attenuation as
Ca2+ depletion with EGTA was observed (Fig. 3B).
Interestingly, when SR Ca2+ refilling was blocked by CPA
(100 µM) 5 min before and during KCl stimulation, the
amplitude of the KCl-induced [Ca2+]m peak was
reduced and the oscillations were totally prevented (Fig.
3C).
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Other [Ca2+]m Raising Agents--
The
occurrence of [Ca2+]m oscillations following
stimuli other than KCl was analyzed in C2C12 myotubes. The cholinergic agonist CCh, known to induce similar levels of depolarization as KCl
(23), elicited a [Ca2+]m transient similar to KCl
(3.24 ± 0.23 µM, n = 21, Fig.
4D). However, as compared with
KCl, no oscillations followed (Fig. 4A). The nicotinic
blocker -bungarotoxin (100 nM) almost completely
attenuated the CCh-induced response, whereas the muscarinic antagonist
atropine (10 µM) had no effect (Fig.
5, A and B),
highlighting that C2C12 myotubes are exclusively sensitive to nicotinic
stimulation and confirming the observations by other groups (23, 24). Activation of nicotinic receptors causes cell depolarization by Na+ entry and, to a much lesser extent, Ca2+
entry via the associated channel. A dependence on extracellular Ca2+ was shown by the fact that EGTA reduced the
CCh-induced [Ca2+]m peak (Fig.
5C).
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The purinergic agonist ATP (1 mM) induced a biphasic
[Ca2+]m response initiated by a peak of 1.37 ± 0.12 µM (n = 14, Fig. 4, B
and D), followed by an oscillatory phase. However, these oscillations were not as regular and frequent as the KCl-induced oscillations. Moreover, ATP concentrations of less than 1 mM were unable to elicit an oscillatory phase (data not
shown). In Ca2+-free medium (3 mM EGTA), 1 mM ATP elicited two different [Ca2+]m
patterns; in 8 of 15 preparations, it caused a small [Ca2+]m peak without subsequent oscillations
(Fig. 6A), and in 7 of 15 preparations, it induced a smaller biphasic
[Ca2+]m transient (Fig. 6B).
Pretreatment of cells with the SERCA inhibitor CPA slightly delayed the
ATP response without affecting the second phase (Fig. 6C).
Thus, it appears that the [Ca2+]m oscillations
caused by ATP are not of the same origin as those induced by KCl.
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CPA alone (100 µM) raised [Ca2+]m to 1.51 ± 0.31 µM (n = 9) before a slow return to basal level occurred (Fig. 4, C and D). As with CCh, this second phase did not exhibit [Ca2+]m oscillations.
The RyR activator caffeine elicited a [Ca2+]m peak followed by oscillations with a pattern similar to the one observed with KCl stimulation (Fig. 4, E and F). At 5 mM Caf, [Ca2+]m was increased to 1.35 ± 0.05 µM (n = 3), and [Ca2+]m oscillations appeared during the decay phase. This was also observed at 3.5 and 7 mM, whereas 20 mM Caf caused a [Ca2+]m peak (4.51 ± 0.19 µM, n = 3) without associated oscillations.
Desensitization of [Ca2+]m Caused by Repeated
Stimulations--
C2C12 myotubes were stimulated twice with the same
[Ca2+]m raising agent, and both pulses were
separated by a 5-min wash. KCl (55 mM) caused a
[Ca2+]m desensitization, i.e. the
second peak was significantly smaller than the first peak induced by
KCl (Fig. 7, A and
F). On the other hand, 45Ca2+ influx
into cells caused by the second KCl pulse was essentially the same as
the one caused by the first pulse, i.e. 206 ± 19 and 206 ± 22% of basal influx, respectively (n = 5, performed at least in
triplicate).2 This suggests
that the voltage-operated Ca2+ channel is not desensitized
by repeated KCl stimulations. ATP (1 mM) or CPA (100 µM) also induced [Ca2+]m
desensitization (Fig. 7, B, C, and F). In
contrast to KCl desensitization, a second pulse of CCh (100 µM) elicited a higher [Ca2+]m peak
than the first one, indicating a potentiation (Fig. 7, D and
F). However, if the wash time was reduced from 5 to 2 min, a
double stimulation with CCh tended to a [Ca2+]m
desensitization (Fig. 7E).
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Cross-reactivity between KCl and Other
[Ca2+]m Raising Agents--
C2C12 myotubes were
challenged with KCl and another [Ca2+]m raising
agent, and both pulses were separated by a 5-min wash. When KCl
stimulation was followed by CCh, [Ca2+]m
desensitization occurred. The CCh-induced [Ca2+]m
peak was reduced by ~50% compared with a single CCh response (Fig.
8A). In contrast, a second
pulse of KCl after CCh stimulation increased
[Ca2+]m up to 3 µM as observed with
a single KCl stimulation. However, the subsequent oscillatory
[Ca2+]m phase was potentiated by the previous CCh
pulse (Fig. 8B). The frequency of the oscillations remained
unchanged, but the area under the curve of the KCl-induced
[Ca2+]m transient was ~3 times higher than the
area under the curve of a standard KCl response (364 ± 18 versus 125 ± 6 µM·s, n 4, p < 0.0001), whereas the time from the onset of
the KCl-induced [Ca2+]m increase to the return to
basal [Ca2+]m level was similar (386 ± 15 versus 357 ± 10 s, n
4, p = 3.5). There was no cross-reactivity between KCl and
the purinergic agonist ATP (1 mM, Fig. 8, C and
D), whereas a first pulse with CPA (100 µM)
attenuated the KCl-induced [Ca2+]m transient
(Fig. 8, E and F).
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DISCUSSION |
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The present study reports for the first time that C2C12 myotubes stably expressing aequorin targeted to mitochondria display [Ca2+]m oscillations. This oscillatory behavior was elicited by KCl depolarization, whereas nicotinic stimulation did not cause oscillations. The oscillations were prevented by SERCA inhibition but not by blockade of Ca2+ entry. They were also produced by caffeine. Thus, RyR are strong candidates as triggers for [Ca2+]m oscillations. The other important results concern the desensitization of the [Ca2+]m response by KCl and the potentiation of [Ca2+]m by CCh when these stimuli were applied twice.
The fact that the [Ca2+]m oscillations were detected in a cell population indicates that C2C12 myotubes are synchronized to some degree and respond as a single unit. Cell-to-cell communication can be expected to occur because myotubes result from the fusion of several myoblasts. An electrical coupling between cells is unlikely, as KCl clamped all cells to the same depolarized potential until KCl removal. Ca2+ itself or other second messengers such as IP3 could be involved in the oscillatory mechanism as has been shown in other cell types (34). As no spontaneous contractions occurred in the C2C12 myotubes used in this study, motion did not interfere with the analysis. [Ca2+]m oscillations were always observed following KCl-triggered depolarizations lasting longer than 1 min (Fig. 2A). When Ca2+ influx was prevented either by EGTA or by blockade of the voltage-operated Ca2+ channel with nifedipine, the whole [Ca2+]m response to KCl was diminished, but the oscillations were preserved (Fig. 3). Thus, Ca2+ entry into cells contributes to [Ca2+]m elevation but is not responsible for the oscillations. It thus appears that the "pacemaking" mechanisms for the oscillations are located intracellularly. When the SERCA was inhibited by CPA, KCl depolarization produced a [Ca2+]m peak, but no oscillations were observed (Fig. 3C). It is therefore likely that the SR communicates with mitochondria to generate [Ca2+]m oscillations. As opposed to KCl-stimulated [Ca2+]m oscillations, those produced by ATP were insensitive to CPA but depended on extracellular Ca2+. The latter is in line with findings on purinergic stimulation in C2C12 myotubes (23).
In contrast to a previous report (35), the [Ca2+]m oscillations observed in the present study were not synchronized with cytosolic Ca2+ oscillations. In MIN6 cells, Nakazaki et al. (35) proposed that repetitive Ca2+ microdomains are responsible for cytosolic and mitochondrial oscillations. In C2C12 myotubes, spatially restricted pulsatile Ca2+ hotspots could occur in the proximity of the SR. Colocalization of mitochondria and SR could permit Ca2+ exchanges between these organelles and subsequent oscillations. Contacts between mitochondria and SR have been demonstrated morphologically by scanning electron microscopy (36). In fact, highly organized networks of SR interconnected with mitochondria were revealed in adult skeletal muscle fibers (36). Moreover, ryanodine receptors (RyR), as sensors of depolarization, could play a central role in generating local domains of high [Ca2+]c that would be sensed by the neighboring mitochondria, in a manner similar to IP3 receptors in HeLa cells (Fig. 1 (20)). This is supported by the effects of caffeine, known to release Ca2+ from the SR by RyR activation bypassing depolarization. As Caf at low concentrations induced a [Ca2+]m peak followed by oscillations, in a way comparable to KCl response, RyRs seem to be oscillation triggers (Fig. 4, E and F). This has been already proposed for dyspedic myotubes (37) or cardiac cells (25). At the single cell level, Caf induced rapid perimembrane and mitochondrial [Ca2+] spikes, with a frequency increasing with the Caf concentration to give a sustained [Ca2+]m elevation at 20 mM (25). In our study, RyR localization could explain the restriction of the [Ca2+]m oscillations to KCl stimulation as opposed to the CCh effect. At low concentrations, Caf would act on RyR localized on KCl-sensitive Ca2+ stores and would initiate a global oscillatory behavior in the neighboring mitochondria, whereas at high concentrations, Caf would open all RyR and thus give rise to a single [Ca2+]m peak. The presence of discrete Ca2+ stores in C2C12 myotubes has previously been suggested by Henning et al. (23). These authors proposed separate noninteracting stores for KCl and CCh and for KCl and ATP.
Because of the potential existence of discrete SR Ca2+ stores, alterations in their filling state might influence mitochondrial Ca2+ responses. To address this issue, desensitization and cross-desensitization studies were performed. Repeated applications of KCl led to a down-regulation of the second [Ca2+]m transient, whereas dual nicotinic stimulation up-regulated the intensity of the second transient (Fig. 7) indicating different pathways of cellular activation with respect to mitochondria. The state of cell polarization was not responsible for this desensitization. A 5-min wash between two KCl pulses appeared to be sufficient for cell repolarization, in contrast to a 2-min wash, as shown by the CCh-induced [Ca2+]m desensitization (Fig. 7, see also Ref. 23). Furthermore, the voltage-operated Ca2+ channel was probably not desensitized because 45Ca2+ influx caused by a second KCl pulse was the same as the first one. The KCl-induced [Ca2+]m desensitization may rather be due to an incomplete Ca2+ store refilling or to desensitization of the mitochondrial Ca2+ uniporter, as suggested in insulinoma cells (38). However, partial aequorin consumption cannot be ruled out. mtAeq could be consumed in specific regions of mitochondria, and a redistribution of mtAeq by movements of mitochondria would require some time. But another phenomenon participates in [Ca2+]m desensitization, as a partial recovery was observed when the washing time between two pulses of KCl was increased to at least 10 min (data not shown). The same might apply to ATP- and CPA-induced [Ca2+]m desensitization.
In contrast to the other stimuli, repeated stimulations with CCh yielded a [Ca2+]m potentiation (Fig. 7). Discrepancy between Ca2+ homeostasis after CCh and KCl depolarization has been observed in rat skeletal muscle cells (39). These authors showed that CCh induced a drop followed by a large overshoot of sarcoplasmic reticulum [Ca2+] ([Ca2+]SR), while KCl only elicited a [Ca2+]SR drop followed by a return to almost basal level. This over-accumulation of Ca2+ in the SR during cholinergic stimulation could be related to Ca2+ uptake by SR portions strategically located near the nicotinic channels (24). A similar situation could occur in C2C12 cells and could be the reason for the CCh-induced [Ca2+]m potentiation observed in our work. The CCh-sensitive stores could contain more Ca2+ after the first pulse, thus producing a higher second [Ca2+]m peak. Alternatively, ATP may be locally depleted by SERCA-mediated Ca2+ uptake into the SR after a first CCh pulse leading to decreased SERCA activity during the second CCh pulse. As SR Ca2+ uptake should then be reduced, Ca2+ may accumulate in mitochondria resulting in a higher [Ca2+]m. This mechanism could also be the reason for the cross-potentiation between CCh and KCl, where the oscillatory second phase induced by KCl was increased after a previous stimulation with CCh (Fig. 8B). In a manner similar to double KCl stimulation, the CCh-induced response was desensitized after a previous KCl pulse, again suggesting incomplete SR refilling. In summary, it seems that discrete Ca2+ stores exist but some interactions between KCl- and CCh-sensitive stores occur. In addition, those mobilized by ATP appear to be separate, as no cross-reactivity between KCl and ATP was observed.
In conclusion, the present data point to a complex spatiotemporal
Ca2+ pattern implicating cellular organelles. On the one
hand, portions of the SR sensitive to KCl would contact mitochondria,
establishing a cross-talk between these two organelles, therefore
permitting repetitive Ca2+ exchanges and subsequent
[Ca2+]m oscillations. On the other hand, distinct
Ca2+ stores probably exist in C2C12 myotubes, explaining
the discrepancy between the effects of KCl and CCh on mitochondrial
Ca2+ signaling. The aequorin technology is helpful to
estimate Ca2+ redistribution into intracellular
compartments, and it has already proven its usefulness in detecting
defective Ca2+ signaling. For example, in cells of patients
with myoclonic epilepsy with ragged-red fibers, histamine stimulation
caused a higher [Ca2+]m peak, as compared with
controls (40). As mitochondria are likely to be involved in dystrophic
muscle (41), a similar approach with aequorin might therefore shed
light on a better understanding of the Ca2+ dysregulation
in Duchenne muscular dystrophy.
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ACKNOWLEDGEMENTS |
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We thank G. Chaffard, P. Lhote, and M. Renard for excellent technical assistance; R. Pitarelli (Swiss Federal Institute of Technology, Lausanne, Switzerland) for mathematical analysis of oscillations; and T. Buetler for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by the Association Française Contre les Myopathies, by the Swiss Foundation of Research on Muscular Diseases, and by Swiss National Science Foundation Grants 31.56877.99 (to U. R.) and 32.49755.96 (to C. B. W.).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: Pharmacology Group, School of Pharmacy, University of Lausanne/BEP, CH-1015 Lausanne, Switzerland. Tel.: 41 21 692 45 31; Fax: 41 21 692 45 15; E-mail: Urs.Ruegg@ict.unil.ch.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M006209200
2 The 45Ca2+ influx assay was as previously described (42). C. Challet, P. Maechler, C. B. Wollheim, and U. T. Rüegg, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: [Ca2+]c, cytosolic Ca2+ concentration; [Ca2+]m, mitochondrial Ca2+ concentration; Caf, caffeine; CCh, carbachol; CPA, cyclopiazonic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; IP3, inositol 1,4,5-trisphosphate; mtAeq, aequorin targeted to mitochondria; SERCA, sarcoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; RyR, ryanodine receptor; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.
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