Article |
Address correspondence to P. Gailly, Département de Physiologie, Université Catholique de Louvain (UCL 5540), Av. Hippocrate 55, 1200 Brussels, Belgium. Tel.: 32-2-764-55-42. Fax: 32-2-764-55-80. E-mail: gailly{at}fymu.ucl.ac.be
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Abstract |
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Key Words: calcium channels; dystrophy; sarcoplasmic reticulum; capacitative entry; TRP channel
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Introduction |
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Results |
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The results given in the previous section were obtained on depolarized cells (fibers maintained 35 min in potassium aspartate solution, as described previously [Franco-Obregon and Lansman, 1994]). This was necessary to correctly impose a holding potential (cell-attached configuration). Therefore, to mimic as much as possible the patch-clamp experiments, the effect of thapsigargin on Ca stores was studied in fibers depolarized in potassium aspartate solution (also containing 10 mM EGTA; see Materials and methods). Depolarization in this solution induced a [Ca2+]i transient that was taken as an index of the amount of releasable Ca (Fig. 3). After 5 min in this solution in the absence or presence of 1 µM thapsigargin, fibers were repolarized for 5 min in a Krebs solution (to prepare for the next depolarization) containing 50 µM EGTA to avoid any refilling of the stores during this period of time. On return in potassium aspartate solution and in the absence of thapsigargin, this produced a peak of [Ca2+]i 200 nM (from the resting level just before stimulation to the peak of [Ca2+]i, n =10). No difference could be observed between C57 and mdx fibers (n = 5 for each). Fibers treated with 1 µM thapsigargin showed an increase of resting [Ca2+]i from their basal level (60 ± 9 nM) to 111 ± 24 nM (n = 10). Upon depolarization, the peak of [Ca2+]i was significantly reduced by a factor of 6 (n = 10). This effect was observed in both C57 and mdx fibers. In conclusion, thapsigargin-treated fibers had lost 85% of their depolarization-releasable content of Ca. This result contrasts with the milder effect of thapsigargin observed by Kurebayashi and Ogawa (2001), and is probably due to the fact that thapsigargin-induced depletion is obtained in depolarized fibers.
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Expression and localization of mammalian TRPC isoforms in C57 and mdx fibers
The mRNA expression of the seven known mammalian TRPC isoforms was examined in adult skeletal muscle fibers using RT-PCR technique. Fig. 4 A shows that TRPC1, 2, 3, 4, and 6 mRNAs are detected in wild-type and mdx adult skeletal muscle fibers, whereas TRPC5 and TRPC7 isoforms were not. As a control, these two isoforms were found in another tissue (mouse brain).
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Repression of TRPC in transfected mdx fibers
To examine the role of endogenous TRPC in the Ca currents observed, we tried to specifically repress the expression of these proteins and to study the functional repercussions. As the occurrence of Ca channels was higher in mdx than in C57 fibers, we chose to repress the expression in this type of fibers. We transiently transfected mdx fibers with antisense and sense oligonucleotides whose sequence was chosen in the most conserved region of the mRNA sequences of the different TRPCs. 48 h after transfection, the efficiency, assessed by the percentage of fluorescent fibers, was 60%.
Mdx fibers treated with sense or antisense oligonucleotides were submitted to fractionation, SDS-PAGE, and Western blot analysis. TRPC1 and 4 isoforms were significantly repressed by the treatment with antisense oligonucleotides (residual expression of 40 and 50%, respectively), but TRPC6 expression was not modified (Fig. 5, A and B). Note that only 60% of the studied fibers were successfully transfected.
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As the repression of the TRPC protein expression led to an important decrease of the occurrence of Ca activity, we investigated whether the repression was specific and did not affect the expression of other proteins. To do so, we studied sodium currents in the same population of transfected mdx fibers. Sodium currents were elicited by 50-ms depolarizing pulses from a holding potential of -70 to -30 mV. Single openings generally occurred at the beginning of the test pulse. Average unitary currents showed the characteristics of sodium macroscopic currents with rapid onset and inactivation (Fig. 6). In both sense- and antisense-transfected mdx fibers, peak current amplitudes of average currents, recorded at 30 mV, were identical. Single-channel conductance was calculated as the slope of linear regression of single-channel current-voltage curves between-60 and 0 mV (Fig. 6 B). It was close to 18 pS in the mdx fibers transfected with sense and antisense oligonucleotides. This value of conductance is similar to the one obtained previously by others in FDB fibers (Desaphy et al., 2001).
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Discussion |
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The importance of an abnormal influx of Ca has been noticed in skeletal myotubes from DMD patients (Imbert et al., 1996) and in myotubes and adult fibers from mdx mice (Turner et al., 1988; Haws and Lansman, 1991; Tutdibi et al., 1999; Mallouk et al., 2000). This influx seems to implicate various spontaneously active and/or mechanosensitive cationic channels (Imbert et al., 1996; Vandebrouck et al., 2001), whereas the voltage-dependent Ca channels do not seem to be involved (Imbert et al., 2001). In myotubes, Hopf et al. (1996a) described a Ca leak channel whose activity was increased in mdx and which was activated by store depletion. The identity of this channel was unknown; it might be similar to the voltage-independent Ca channels found in adult fibers (Franco and Lansman, 1990a, and this paper) although its Po was 30 times lower than in adult fibers. In this paper, we show that the voltage-independent Ca channels that are abnormally activated in adult mdx skeletal muscle fibers are sensitive to Ca stores depletion by thapsigargin or caffeine. Indeed, store depletion increases the Po of these channels by keeping them opened for a longer time (increased
0, same
c). The Ca channel studied here is similarly permeable to Ba2+, Ca2+, and Mn2+, as is the store-dependent channel from endothelial cells (Luckhoff and Clapham, 1994).
The identification of the channel involved in store-dependent Ca entry has been largely investigated in nonexcitable cells. Candidate molecules for this pathway probably belong to the TRPC protein family (for review see Clapham et al., 2001). This has been shown by heterologous expression of TRPC proteins. Using this methodology, TRPC15 have been shown to be possibly activated by store depletion (Groschner et al., 1998; Philipp et al., 1998; Vannier et al., 1999; Warnat et al., 1999; Liu et al., 2000). However, other authors showed that heterologous expression of TRPC36 also gave channels whose activation was independent of store depletion (Zitt et al., 1997; Hofmann et al., 1999; Schaefer et al., 2000; Wu et al., 2000). Therefore, different groups tried to evaluate the functional role of TRPC proteins by repressing their expression by transfection with antisense nucleotide sequences. This strategy has been used to clearly demonstrate that TRPC4 is part of the native Ca-release activated Ca channel in adrenal cells (Philipp et al., 2000), that TRPC1 and/or TRPC3 are involved in the thapsigargin-induced entry of Ca in HEK-293 cells (Wu et al., 2000), and that TRPC2 protein is involved in store-operated influx of Ca in fibroblasts (Gailly and Colson-Van Schoor, 2001). As the voltage-independent Ca channels found in C57 and mdx adult fibers were store dependent, we hypothesized that they might be constituted of TRPC proteins. By using the most conserved coding sequence in the different TRPC genes, we repressed their expression by transfection with antisense oligonucleotides. Among the seven known TRPC isoforms, five were endogenously expressed at the mRNA level, and three were present at the plasma membrane (TRPC1, 4, and 6). After 48-h repression with antisense oligonucleotides, we observed a 5060% decrease in the amount of TRPC expressed. As transfection efficiency was also 60%, it seems reasonable to think that these proteins were almost absent from the transfected fibers. In contrast, TRPC6 expression was not diminished in transfected cells. This might be due to the ternary configuration of the mRNA or to a slow turnover of the protein. In parallel with these observations, we found that the channel occurrence was reduced by a factor of 10 in transfected fibers (selected by their fluorescein tag). The behavior of the channel was unchanged (Po,
0,
c, activation by stores depletion) but its presence became very rare. Therefore, we conclude that the abnormal influx of Ca observed in adult mdx fibers occurs through store-dependent Ca channels constituted, at least partially, of TRPC proteins. The remaining occurrence might be due to an incomplete repression of the TRPC1 and 4 isoforms, or to the presence of TRPC6 protein that was not repressed. Further study will be necessary to precisely identify which TRPC isoform(s) is (are) involved in this process, TRPC1 and/or TRPC4. However, it is interesting to note that the TRPC1 channel is known to have a dystrophin homology domain (Lockwich et al., 2000). Therefore, the lack of dystrophin and the consequent disorganization of the cytoskeleton observed in dystrophic fibers might induce a disregulation of this protein, leading to an increased influx of Ca.
The mechanisms coupling depletion of the stores to subsequent entry of Ca remain unknown. Almost all cells presenting this kind of Ca entry release Ca from the ER through IP3 receptors. Furthermore, recent studies strongly suggest the involvement of the IP3 receptor in capacitative Ca entry (Kiselyov et al., 1998; Boulay et al., 1999; Putney, 1999; Ma et al., 2000). Skeletal muscle sarcoplasmic reticulum releases Ca essentially through ryanodine receptors, which might therefore be involved in the coupling process (Kiselyov et al., 2001). However, skeletal muscle also contains low amounts of IP3 receptors (Valdivia et al., 1992), and Liberona et al. (1998) observed that dystrophic muscle cell lines (human and mouse) have a two- to threefold increase in basal levels of IP3 and an increased number of IP3 receptors (Jaimovich et al., 2000). This might also be involved in the abnormal regulation of these Ca channels.
In this paper, we have thus identified the molecular nature of the channels involved in the abnormal influx of Ca observed in adult dystrophic muscle fibers. The originality of our paper stands in the fact that TRPCs are involved in capacitative Ca entry in excitable cells and, as shown for the first time, that a disregulation of their activity is involved in a neuromuscular disorder. Hopefully, this might give a new target in the search of drug therapies of DMD.
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Materials and methods |
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Single fibers were mechanically dissociated by passing the muscle repeatedly through fire-polished Pasteur pipettes. Dissociated fibers were plated onto tissue culture dishes coated with ECM basement membrane (Harbor Bio-Products) and allowed to adhere to the bottom of the dish for 2 h.
Measurements of cytosolic [Ca2+]
Muscle fibers were loaded for 1 h at RT with the membrane-permeant Ca indicator Fura-PE3/AM (Calbiochem) 1µM and Pluronic F-127 0.004%. Fura-PE3/AM was preferred over Fura-2/AM because it is stable during long lasting experiments, with little or no compartmentation (Hopf et al., 1996b). Fibers were illuminated through an inverted microscope (40x objective; Nikon) alternatively at 340 and 380 nm (20 Hz), and the fluorescent light emitted at 510 nm was measured. The ratio R340/380 of the fluorescence intensity emitted at the two excitation wavelengths was calculated, and cytosolic concentration of calcium ([Ca2+]i) was determined by using a cuvette calibration (Hopf et al., 1996b).
PCR amplification
Total RNA was extracted from C57 and mdx tibialis anterior muscles using the Tripure RNA extraction reagent (Roche). First-strand cDNA was synthesized from 2 µg of total RNA in a final volume of 20 µl using oligo dTPrimer (Thermoscript RT-PCR System; Life Technologies). PCR reaction was conducted using 2-µl aliquots of the RT reaction and specific primers for TRPC17 mouse sequences. These primers were chosen in a region known to cross a sizeable intron (Zhu et al., 1996), thus eliminating contribution from genomic DNA amplification. Primers used for amplification of TRPC1, 3, 4, 5, and 6 fragments were those designed by Garcia and Schilling (1997). For TRPC7 amplification, we used primers described by Okada et al. (1999), and for TRPC2, the primers we designed in a previous study (Gailly and Colson-Van Schoor, 2001). PCR was performed with Expand High Fidelity Taq polymerase (Roche) for 35 cycles (Idaho rapid light cycler), consisting of denaturation at 94°C for 30 s, annealing at 53 to 62°C (optimized for each TRPC fragment amplified) for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 6 min. The expected sizes of the different PCR products were 387, 345, 317, 415, 340, 327, and 701 bp for TRPC17, respectively.
Transient transfection of adult skeletal muscle fibers with TRPC antisense oligonucleotides
C57 and mdx fibers were plated onto dishes 212 h before transfection. They were transiently transfected with degenerated 5' fluorescein-labeled TRPC antisense or sense oligonucleotides (Genset). The antisense sequence was chosen in a region conserved in all TRPC isoforms, as follows: Fluo 5'-aagtarswvadsyanaryttbghhckwgcaaayttccaytc-3'. The sense sequence was the reverse of the antisense sequence and was used as a control. Transient transfection was performed with 50-nM antisense or sense oligonucleotides and oligofectAMINETM reagent (Life Technologies) in Ham's F12/DME without serum. Fibers were incubated for 4 h at 30°C in the presence of oligonucleotides before adding Ham's F12/DME with 6% FBS, and the culture was maintained at 30°C for 48 h. Transfection efficiency was determined by the fluorescence of cells. Electrophysiological recordings of transfected (fluorescent) cells and Western blot analyses were performed 48 h after transfection.
Western blotting
Dissociated fibers were harvested by centrifugation and resuspended in lysis buffer (20 mM Na2HPO4, pH 7.2, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5% Triton X-100) containing protease inhibitor cocktail (Roche). Fibers were incubated for 1 h on ice and centrifuged at 10,000 g for 15 min at 4°C, generating Triton-soluble (supernatant) and Triton-insoluble (pellet) proteins. In some experiments, dissociated mdx fibers were directly lysed and boiled for 5 min in 2x SDS-loading sample buffer containing 1% (vol/vol) 2-mercaptoethanol (Laemmli, 1970).
Proteins were separated by 8% SDS-PAGE and transferred onto PVDF membranes (Amersham). After incubation of these membranes in Tris-buffered saline containing 0.05% Tween 20 and 5% skimmed milk (wt/vol), rabbit anti-TRPC1, 3, 4, and 6 (1/600) pAbs (Alomone Labs) or anti-TRPC2 (1/100) pAb (a gift of Dr. H. Florman [University of Massachusetts Medical School, Worcester, MA]; Jungnickel et al., 2001) were added for 2 h at RT, and bound antibodies were detected by peroxidase-conjugated antirabbit IgG serum (1/10,000 for 1 h at RT). The immunoreactive bands were detected using a chemiluminescence ECL+ Western blotting detection kit (Amersham Biosciences) and quantified by densitometry after correction for gel loading assayed by Coomassie blue staining and/or actin content (detected using an anti-ß actin antibody; Sigma-Aldrich).
Immunocytochemistry
The following protocol was used: fixation in 4% PFA for 20 min; permeabilization in 0.2% Triton/PBS for 15 min; preincubation in 10% normal goat serum/PBS for 1 h; incubation in 100-fold diluted TRPC1, 2, 3, 4, or 6 antiserum for 1 h; and incubation in 200-fold diluted FITC-labeled antirabbit goat antiserum for 1 h. Immunostained cells were observed under an inverted microscope (Axiovert T-100; Carl Zeiss MicroImaging, Inc.; excitation, 488 nm; emission, 505 nm). Images were acquired with a CCD camera (model 1300-v; Princeton Instruments) and analyzed with MetaFluor® software.
Electrophysiological methods
Single-channel Ca activity was recorded from cell-attached patches using the technique described by Hamill and co-workers (1981). Patch electrodes were pulled on a DMZ-Universal puller (Zeitz-Instruments) in three stages from borosilicate glass capillaries (1.5 mm diameter; Harvard Apparatus, Inc.) to a tip diameter of 12 µm. Patch electrodes had a resistance of 25 M. Cells were viewed under phase-contrast with an inverted microscope (Axiovert S-100; Carl Zeiss MicroImaging, Inc.).
Single channel activity was recorded at a constant holding potential of -60 mV and at RT using a patch-clamp amplifier (EPC-9; HEKA). Current records were filtered with a Bessel filter at 3 kHz and digitized at 10 kHz. Data were analyzed using Pulse-Fit, Pulse-Tools, and Origin 6.1 software. The intrapipette solution contained the following (mM): 110 CaCl2, 10 Hepes, and 0.01 4.4'-diisothyocyanate stilbene-2.2' disulfonic acid (DIDS). The bathing solution was an isotonic potassium aspartate solution containing the following (mM): 150 potassium aspartate, 5 MgCl2, 10 EGTA, and 10 Hepes, pH 7.4. The osmolarity of each solution was adjusted to 320330 mosmol.l-1 by adding glucose. The potassium aspartate bathing solution was used to render null the resting membrane potential so that the patch potential would be equal to the applied voltage command. DIDS was used to block possible chloride conductances.
Thapsigargin (Sigma-Aldrich) was dissolved in DMSO and diluted 1:2,000 into the bath to a final concentration of 1 µM. The solvent alone had no effect on channel activity. Caffeine (Merck Eurolab) was used at a final concentration of 10 mM.
Sodium currents were recorded at RT in cell-attached configuration. The intrapipette solution contained the following (mM): 150 NaCl, 1 MgCl2, 1 CaCl2, and 10 Hepes, pH 7.3. The bath solution contained Cs+ ions as the main cations in order to inhibit potassium currents and to depolarize the fiber: 145 CsCl, 5 EGTA, 1 MgCl2, 10 Hepes, and 5 glucose, pH 7.3. After 510 min of incubation in this solution, the intrinsic resting membrane potential of the FDB muscle fibers was consistently close to 0 mV. Capacitance currents were almost totally cancelled by the compensation circuit of the amplifier. Sodium currents were elicited by 50-ms depolarizing pulses from a holding potential of -70 to -30 mV applied every 0.5 s.
Statistics
Data are presented as mean ± SD. x2 test, ANOVA, or t tests were used to determine statistical significance.
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Footnotes |
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* Abbreviations used in this paper: DMD, Duchenne muscular dystrophy; FDB, flexor digitorum brevis; mdx, murine X-linked dystrophy; Po, open probability;
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Acknowledgments |
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This work was supported by the Association française contre les myopathies, the Association belge contre les maladies neuro-musculaires, and by a grant from the General Direction of Scientific Research of the French Community of Belgium (ARC 00/05-260).
Submitted: 20 March 2002
Revised: 29 July 2002
Accepted: 2 August 2002
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References |
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