Mitochondrial Calcium Oscillations in C2C12 Myotubes*

Corinne ChalletDagger , Pierre Maechler§, Claes B. Wollheim§, and Urs T. RueggDagger

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



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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: alpha -Bgt, alpha -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).


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha -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,


[<UP>Ca<SUP>2+</SUP></UP>]<SUB>m</SUB>(M)=<RAD><RCD>L/L<SUB><UP>max</UP></SUB><UP> · 10<SUP>−9</SUP>,</UP></RCD></RAD> (Eq. 1)
where L are the recorded photons/s and Lmax the remaining photons which correspond to the total light output during the whole experiment minus the photons emitted up to the measured point. Total light output was obtained by exposing cells to 10 mM CaCl2, after permeabilization with 100 µM digitonin, to consume all aequorin.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   Effect of KCl on [Ca2+]m and its modulation by agents acting on mitochondria. C2C12 myotubes stably expressing mtAeq were exposed to a depolarizing solution (55 mM KCl) for 5 min. In A, [Ca2+]m is indicated by a black line and [Ca2+]c by a gray line. The [Ca2+]m transient caused by a short KCl pulse (20 s) is shown in the inset of A. In B and C, agents acting on mitochondria were added 5 min before and during KCl stimulation: the protonophore FCCP (1 µM; black line, B) and the mitochondrial Na+/Ca2+ exchange inhibitor CGP (10 µM; black line, C). The gray lines in B and C show the effect of KCl (55 mM) alone. The traces are representative of at least three independent experiments performed at least in duplicate.

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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Fig. 3.   Modulation of KCl-induced [Ca2+]m increase with agents not acting on mitochondria. C2C12 myotubes stably expressing mtAeq were depolarized with 55 mM KCl. To modulate the subsequent [Ca2+]m increase, agents were added 5 min (or 20 min in B) before and during KCl stimulation. EGTA (3 mM; black line, A), nifedipine, an inhibitor of the voltage-operated Ca2+ channel (Nif, 1 µM; black line, B), and cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase (CPA, 100 µM; black line, C), were tested. The effect of KCl alone is shown with gray lines in A-C. The traces are representative of at least three independent experiments performed at least in duplicate.

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 alpha -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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Fig. 4.   Other [Ca2+]m raising agents in C2C12 myotubes stably expressing mtAeq. C2C12 myotubes stably expressing mtAeq were stimulated for 5 min with the nicotinic agonist carbachol (CCh 100 µM; A), with the purinergic agonist ATP (1 mM; B), with cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase (CPA 100 µM; C), or with caffeine, a RyR activator (Caf 5 mM; E). The subsequent [Ca2+]m transients were recorded. Mean values of the [Ca2+]m peaks evoked by these agents and 55 mM KCl are represented in D and F. In F, the caffeine concentration dependence is shown, and the presence or absence of oscillations is indicated by + or -. Values significantly different from basal [Ca2+]m are indicated with **(0.001 < p < 0.01) or *** (p < 0.001; Student's t test after analysis of variance test; n >=  3, performed at least in duplicate).



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Fig. 5.   Characterization of the carbachol-induced [Ca2+]m peak. The cholinergic agonist carbachol (CCh, 100 µM) induced a [Ca2+]m peak in C2C12 myotubes stably expressing mtAeq (gray lines in A-C). This response was attenuated by a 5-min pretreatment with the nicotinic antagonist alpha -bungarotoxin (alpha -Bgt, 100 nM; black line, A) or with EGTA (3 mM; black line, C) but not with the muscarinic antagonist atropine (Atrop, 10 µM; black line, B). Mean values of the [Ca2+]m peak evoked by CCh with or without one of the above agents are represented in the insets. Values significantly different from the CCh response are indicated with * (0.05 < p < 0.01) or *** (p < 0.001; Student's t test; n >=  3, performed at least in duplicate).

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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Fig. 6.   Modulation of the ATP-induced [Ca2+]m increase. Under purinergic stimulation with ATP (1 mM), C2C12 myotubes stably expressing mtAeq displayed a biphasic [Ca2+]m increase (gray lines, A-C). Modulating agents were added 5 min before and during the 5-min ATP stimulation. EGTA (3 mM) affected the ATP response in two ways as shown in A and B (black lines). Half of the preparations tested showed behavior A, and the other part showed behavior B. The effect of cyclopiazonic acid (CPA, 100 µM) is shown in C (black line). The traces are representative of at least three independent experiments performed at least in duplicate.

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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Fig. 7.   Second pulses of [Ca2+]m raising agents causing desensitization of [Ca2+]m except for carbachol. C2C12 myotubes stably expressing mtAeq were stimulated twice with the same [Ca2+]m raising agent, and both pulses were separated by a 5-min wash with PSS (except in E where the wash lasted 2 min). KCl (55 mM; A), ATP (1 mM; B), cyclopiazonic acid (CPA, 100 µM; C), and carbachol (CCh, 100 µM; D and E) were tested. Mean values of the [Ca2+]m peaks evoked by these agents are represented in F. Second peaks significantly different from the first peaks are indicated with ** (0.001 < p < 0.01) or *** (p < 0.001; Student's t test; n >=  3, performed at least in duplicate).

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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Fig. 8.   Cross-reactivity between KCl and other [Ca2+]m raising agents. C2C12 myotubes stably expressing mtAeq were challenged with KCl and another [Ca2+]m raising agent, and both pulses were separated by a 5-min wash with PSS. KCl stimulation (55 mM) was followed by the addition of carbachol (CCh, 100 µM; A), ATP (1 mM; C), or cyclopiazonic acid (CPA, 100 µM; E). In B, D, and F, KCl (55 mM) was used as the second stimulus after a pulse of CCh (100 µM; B), of ATP (1 mM; D), or of CPA (100 µM; F). The traces are representative of at least three independent experiments performed at least in duplicate.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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