Decreased expression of ryanodine receptors alters calcium-induced calcium release mechanism in mdx duodenal myocytes.

It is generally believed that alterations of calcium homeostasis play a key role in skeletal muscle atrophy and degeneration observed in Duchenne's muscular dystrophy and mdx mice. Mechanical activity is also impaired in gastrointestinal muscles, but the cellular and molecular mechanisms of this pathological state have not yet been investigated. We showed, in mdx duodenal myocytes, that both caffeine- and depolarization-induced calcium responses were inhibited, whereas acetylcholine- and thapsigargin-induced calcium responses were not significantly affected compared with control mice. Calcium-induced calcium release efficiency was impaired in mdx duodenal myocytes depending only on inhibition of ryanodine receptor expression. Duodenal myocytes expressed both type 2 and type 3 ryanodine receptors and were unable to produce calcium sparks. In control and mdx duodenal myocytes, both caffeine- and depolarization-induced calcium responses were dose-dependently and specifically inhibited with the anti-type 2 ryanodine receptor antibody. A strong inhibition of type 2 ryanodine receptor in mdx duodenal myocytes was observed on the mRNA as well as on the protein level. Taken together, our results suggest that inhibition of type 2 ryanodine receptor expression in mdx duodenal myocytes may account for the decreased calcium release from the sarcoplasmic reticulum and reduced mechanical activity.

It is generally believed that alterations of calcium homeostasis play a key role in skeletal muscle atrophy and degeneration observed in Duchenne's muscular dystrophy and mdx mice. Mechanical activity is also impaired in gastrointestinal muscles, but the cellular and molecular mechanisms of this pathological state have not yet been investigated. We showed, in mdx duodenal myocytes, that both caffeine-and depolarization-induced calcium responses were inhibited, whereas acetylcholine-and thapsigargin-induced calcium responses were not significantly affected compared with control mice. Calcium-induced calcium release efficiency was impaired in mdx duodenal myocytes depending only on inhibition of ryanodine receptor expression. Duodenal myocytes expressed both type 2 and type 3 ryanodine receptors and were unable to produce calcium sparks. In control and mdx duodenal myocytes, both caffeineand depolarization-induced calcium responses were dose-dependently and specifically inhibited with the anti-type 2 ryanodine receptor antibody. A strong inhibition of type 2 ryanodine receptor in mdx duodenal myocytes was observed on the mRNA as well as on the protein level. Taken together, our results suggest that inhibition of type 2 ryanodine receptor expression in mdx duodenal myocytes may account for the decreased calcium release from the sarcoplasmic reticulum and reduced mechanical activity.
Dystrophin is a cytoskeletal structural protein present in skeletal, cardiac, and smooth muscles (1). Although it is well established that the lack of dystrophin expression is the primary genetic defect in Duchenne's muscular dystrophy, functionality of smooth muscles in patients with Duchenne's muscular dystrophy and in mdx mice has received little attention. However, different degrees of disorders have been observed in mdx smooth muscles of the digestive track (impaired nitrergic relaxation and increase of spontaneous tone, Refs. 2 and 3), and different clinical manifestations, including gastric dilatation and intestinal pseudo-obstructions, have been reported in patients with Duchenne's muscular dystrophy (4,5). The role of dystrophin in smooth muscle contraction is still largely unknown.
In skeletal and cardiac muscles, it has been suggested that an elevation in [Ca 2ϩ ] i under resting conditions may activate Ca 2ϩ -dependent proteases inducing muscle damage (1). In fact, some groups have found a difference in [Ca 2ϩ ] i between normal and dystrophic skeletal muscles from patients and mdx mice (6,7). Other groups have not been able to confirm these data (8), although an elevated subsarcolemmal Ca 2ϩ concentration has been reported by studying activation of Ca 2ϩ -activated K ϩ channels (9). An increased Ca 2ϩ influx through cationic channels has been detected in mdx skeletal fibers, suggesting that a dysregulation of channel activity may be involved in this neuromuscular disorder (10). Controversial data also have been reported for the peak Ca 2ϩ responses upon stimulation. Some groups have found larger Ca 2ϩ rises in mdx mice (7), others have found them to be similar to controls (11), and some have even reported reductions (12).
A key aspect of the Ca 2ϩ signaling pathway is represented by its spatial and temporal complexity. Localized changes in [Ca 2ϩ ] i are pivotal events in triggering important cellular responses such as contraction, secretion, gene expression, and metabolic activation. In smooth muscle cells, Ca 2ϩ release channels of the sarcoplasmic reticulum (SR) 1 modulate the [Ca 2ϩ ] i in response to activation of voltage-gated Ca 2ϩ channels (13) and membrane receptors (14,15).
In this study, we tested the hypothesis that, in visceral smooth muscle lacking dystrophin, the Ca 2ϩ responses evoked by the Ca 2ϩ -induced Ca 2ϩ release (CICR) mechanism could be affected by the mutation. We addressed this issue by using patch clamp technique coupled to confocal microscopy with Fluo-4 to analyze Ca 2ϩ signals, binding experiments, and Western blotting to evaluate the expression of ryanodine receptors (RYRs) in duodenal myocytes from wild-type and mdx mice. We show for the first time that the RYR2 expression is impaired in mdx duodenal myocytes and that this alteration may account for the reduced Ca 2ϩ responses evoked by caffeine and activation of voltage-gated Ca 2ϩ channels.

EXPERIMENTAL PROCEDURES
Cell Preparation-The investigation conformed with the European Community and French guiding principles in the care and use of animals. Authorization to perform animal experiments (A-33-063-003) was obtained from the Préfecture de la Gironde (France).
Wild-type control (C57BL/10) and mdx (C57BL/10 mdx) mice aged 5-8 months were killed by cervical dislocation. Isolated myocytes were obtained from the longitudinal layer of the duodenum by enzymatic dispersion as described previously (16). Cells were seeded on glass slides in M199 culture medium containing 10% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, 20 units/ml penicillin, and 20 g/ml strep-* This work was supported by a grant from the Association Française contre les Myopathies (France). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
tomycin. Cells were kept in an incubator gassed with 95% air and 5% CO 2 at 37°C and used within 8 h.
Reverse Transcription-Polymerase Chain Reaction-Total RNA was extracted from freshly isolated mouse duodenal smooth muscle cells using an RNeasy minikit (Qiagen, Hilden, Germany) following the instructions of the supplier. The reverse transcription (RT) reaction was performed using a Sensiscript RT kit (Qiagen). Total RNA was incubated with oligo(dT) primers (Promega, Lyon, France) at 65°C for 5 min. After a cooling time of 15 min at 25°C, RT mixture was added, and the total mixture was incubated for 60 min at 37°C. The resulting cDNA was stored at Ϫ20°C. The PCR was performed with 1 g of cDNA, 1.25 units of HotStart Taq DNA polymerase (Qiagen), a 1 M concentration of each primer, and a 200 M concentration of each deoxynucleotide triphosphate in a final volume of 50 l. The PCR conditions were 95°C for 15 min, then 25-35 cycles at 94°C for 1 min, 60°C (RYR1 and RYR2) or 56°C (RYR3) for 1 min, and 72°C for 1 min. At the end of PCR, samples were kept at 72°C for 10 min for final extension and then stored at 4°C. Reverse transcription and PCR were performed with a thermal cycler (Eppendorf, Le Pecq, France). Amplification products were separated by electrophoresis (2% agarose gel) and visualized by ethidium bromide staining. The minimum detection of RYR amplification products was obtained with 15 ng of cDNA. Gels were photographed with EDAS 120 and analyzed with KDS1D 2.0 software (Kodak Digital Science, Paris, France). The relative amount of the amplification products was determined and normalized to that of the glyceraldehyde-3-phosphate dehydrogenase fragment. The identity of the PCR products was verified by DNA sequencing.
Fluorescence and Patch Clamp Measurements-Measurements of [Ca 2ϩ ] i were performed, in part, with an Indo-1 setup as described elsewhere (16). Briefly cells were loaded either with 1 M Indo-1 acetoxymethyl ester for 30 min or with 50 M Indo-1 added to the pipette solution and entering the cells after establishment of the whole-cell recording mode. [Ca 2ϩ ] i was estimated from the 405/480 nm fluorescence ratio using a calibration determined within cells (16). Briefly fluorescence ratios were calculated for each cell with a pipette solution containing 10 mM EGTA (R min ) or after application of a 200-mV hyperpolarizing step causing membrane breakdown (R max ). R min and R max values from control (n ϭ 94) and mdx myocytes (n ϭ 95) were 0.36 Ϯ 0.05, 4.27 Ϯ 0.03, 0.38 Ϯ 0.05, and 4.44 Ϯ 0.09, respectively. An intracellular value for the quantity K ϭ K d ␤ was determined according to Almers and Neher (21) in control and mdx duodenal myocytes. ␤ is defined as F 480 (at R min )/F 480 (at R max ). K values from control (n ϭ 94) and mdx myocytes (n ϭ 95) were 979 Ϯ 116 and 1357 Ϯ 376 nM, respectively. These parameters were used to calculate the [Ca 2ϩ ] i values according to Grynkiewicz's formula (22). All measurements were made at 25 Ϯ 1°C.
For other experiments, Fluo-4 (50 M) was dialyzed into the cell through the patch clamp pipette. Images were acquired using the line scan mode of a confocal Bio-Rad MRC1024 microscope connected to a Nikon Diaphot microscope. Excitation light was delivered by a 25milliwatt argon ion laser (Ion Laser Technology, Salt Lake City, UT) through a Nikon Plan Apo ϫ60, 1.4 numerical aperture objective lens. Fluo-4 was excited at 488 nm, and emitted fluorescence was filtered and measured at 522 Ϯ 35 nm. At the setting used to detect Fluo-4 fluorescence, the resolution of the microscope was near 0.4 ϫ 0.4 ϫ 1.5 m (x, y, and z axis). Scanned lines were plotted vertically, and each line was added to the right of the preceding line to form the line scan image. Fluorescence signals are expressed as pixel per pixel fluorescence ratios (F/F 0 ) where F is the fluorescence during a response and F 0 is the rest level fluorescence of the same pixel. Image processing and analysis were performed by using Lasersharp 2000 (Bio-Rad) and IDL softwares (Research Systems, Inc., Boulder, CO).
Voltage clamp was made with a standard patch clamp technique using a List EPC-7 patch clamp amplifier (Darmstadt-Eberstadt, Germany). Patch pipettes had resistances of 3-4 megaohms. Anti-ryanodine receptor antibodies were added to the pipette solution to allow dialysis of the cell after a breakthrough in whole-cell recording mode for at least 5 min, a time longer than that expected for diffusion of substances in solution (23).
Flash Photolysis-Caged Ca 2ϩ , 1-(4,5-dimethoxy-2-nitrophenyl) EDTA (1 mM, in the presence of 0.25 mM CaCl 2 ) was introduced into the cell via the patch clamp pipette with 5 min allowed for equilibration. Photolysis was produced by a 1-ms pulse from a xenon flash lamp (Hi-Tech Scientific, Salisbury, UK) focused to a ϳ2-mm-diameter spot around the cell. Light was band pass-filtered with a UG11 glass between 300 and 350 mm. Flash intensity could be adjusted by varying the capacitor-charging voltage between 0 and 380 V, which corresponded to a change in the energy input into the flash lamp from 0 to 240 J. On flash photolysis, Ca 2ϩ was released within 2-4 ms, and the small percentage of conversion of the caged compound (ϳ20%) allows the application of a second pulse without altering the Ca 2ϩ responses (13,24).
RYR Immunostaining-Myocytes were washed with phosphate-buffered saline, fixed with 4% (v/v) formaldehyde and 0.5% glutaraldehyde for 10 min at room temperature, and permeabilized in phosphatebuffered saline containing 3% fetal calf serum and 1 mg/ml saponin for 20 min. Cells were incubated with phosphate-buffered saline, saponin (1 mg/ml), and either specific anti-RYR2 or anti-RYR3 antibody (1 g/ml) overnight at 4°C (25). Then cells were washed (4 ϫ 5 min) and incubated with the appropriate secondary antibody conjugated to fluorescein isothiocyanate for 45 min at room temperature. After washing in phosphate-buffered saline, cells were mounted in Vectashield (Ab-Cys, Paris, France). Images of the stained cells were obtained with a confocal microscope (Bio-Rad MRC1024), and fluorescence was estimated by gray level analysis using Laserpix software (Bio-Rad) in 0.5-m confocal sections. On each cell, fluorescence was acquired from a z-series analysis (20 Ϯ 5 sections) using Lasersharp software (Bio-Rad) and expressed by volume unit. Cells were compared by keeping acquisition parameters (gray scale, exposure time, iris aperture, gain, laser power, etc.) constant. Nonspecific fluorescence (NSF) was determined when specific anti-RYR subtype antibody was preincubated with its antigen peptide for 1 h before application of the immunostaining protocol. When the cell fluorescence obtained with the anti-RYR subtype antibody was higher than NSF, the cell was considered to be immunopositive, and specific fluorescence (F Ϫ NSF) was estimated.
[ 3 H]Ryanodine Binding Assay-Microsomal membranes from the longitudinal layer of mouse duodenum were prepared by homogenization with a Kontes Potter-Elvehjem pestle in a solution containing 20 mM Tris-HCl, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7. The homogenate was centrifuged at 1200 rpm for 10 min at 4°C. Microsomal membranes were obtained as a pellet by centrifugation of the supernatant at 40,000 rpm for 90 min at 4°C. Microsomal membranes were then resuspended in the buffer and stored at Ϫ80°C. Protein concentration was determined according to Bradford (18).
[ 3 H]Ryanodine binding was carried out as described previously (26). For saturation experiments, the incubation medium contained 1 M KCl, 25 mM HEPES (pH 7.4 at 37°C), and 0.1 mM CaCl 2 . After a 3-h incubation at 37°C, aliquots were filtered through Whatmann GF/C glass fiber filters and washed three times with 8 ml of ice-cold 0.1 M Tris (pH 7.4 at 4°C). The filters were placed into scintillation vials containing 4 ml of liquid scintillation mixture, and the retained radioactivity was measured in a Packard 1500 Tri-Carb counter. The specific binding was defined as the difference between binding in the absence (total binding) and in the presence (nonspecific binding) of 10 M ruthenium red. Nonspecific binding accounted for less than 5% of total binding at 2 nM [ 3 H]ryanodine.
Solutions-The physiological solution contained 130 mM NaCl, 5.6 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 11 mM glucose, and 10 mM HEPES, pH 7.4 with NaOH. The basic pipette solution contained 130 mM CsCl, 10 mM HEPES, pH 7.3 with CsOH. Acetylcholine and active compounds were applied to the recorded cell by pressure ejection for the period indicated on the records.
Data Analysis-Data are expressed as means Ϯ S.E.; n represents the number of cells or experiments. Significance was tested by means of paired Student's t test when cells were their own control; otherwise an unpaired t test was used. p values Ͻ0.05 were considered significant.

Ca 2ϩ Responses in Duodenal
Myocytes from mdx Mice-In control duodenal myocytes from C57BL/10 mice, dystrophin was present and located at the periphery of the cell sections, whereas dystrophin was absent in mdx mice (not shown). In freshly isolated single myocytes from control and mdx mice, the resting [Ca 2ϩ ] i levels were estimated to be 61 Ϯ 5 nM (n ϭ 94) and 59 Ϯ 5 nM (n ϭ 95), respectively, and were not significantly different (p Ͼ 0.05). Applications of caffeine (10 mM) or acetylcholine (ACh, 10 M) activated transient increases in [Ca 2ϩ ] i that have been shown to depend essentially on Ca 2ϩ release from the SR (16). With time intervals of 3 min between successive applications of the stimulating substances, similar Ca 2ϩ responses were obtained in the same cell, indicating complete refilling of the internal Ca 2ϩ store within 3 min (16). As shown in Fig. 1, the caffeine-induced Ca 2ϩ responses were decreased by about 50% in mdx duodenal myocytes, whereas the AChinduced Ca 2ϩ responses were not significantly reduced when compared with control mice. As caffeine is known as a pharmacological activator of RYRs, we tested the effects of membrane depolarizations on Ca 2ϩ -induced Ca 2ϩ release. In control mice, maximal Ca 2ϩ inward currents in response to depolarizing steps from Ϫ70 to 0 mV triggered maximal transient Ca 2ϩ responses ( Fig. 2A). In mdx mice, the Ca 2ϩ responses were reduced by about 45%, whereas the Ca 2ϩ currents were similar (Fig. 2, A and B). Quantitative results indicated that Ca 2ϩ current densities evoked by a depolarizing step from Ϫ70 to 0 mV were similar in control (13.4 Ϯ 1.4 pA/picofarad, n ϭ 13) and mdx mice (14.1 Ϯ 1.1 pA/picofarad, n ϭ 14). This current is due to activation of two different types of calcium channels: a typical L-type calcium channel and a second type resistant to dihydropyridines but inhibited by mapacalcine (27). It is noteworthy that, in the presence of a mixture of 1 M oxodipine and 5 M mapacalcine for 5 min to block sarcolemmal Ca 2ϩ channels, both inward current and increase in [Ca 2ϩ ] i were suppressed during test depolarizations in control and mdx mice (not shown). As illustrated by the current-voltage relationships in Fig. 2B, the threshold potential and the potential corresponding to peak current were not different in control and mdx mice. The ⌬[Ca 2ϩ ] i -voltage relationship revealed that, in mdx mice, the peak Ca 2ϩ responses were significantly reduced in the voltage range from Ϫ10 to ϩ20 mV (Fig. 2B). To establish whether mdx mutation may reduce the loading of the intracellular Ca 2ϩ store, we studied the effect of thapsigargin (a SR Ca 2ϩ ATPase inhibitor) to deplete the SR. In myocytes from control mice, application of 1 M thapsigargin (in Ca 2ϩ -free 0.5 mM EGTA-containing solution for 30 s) evoked a sustained increase in [Ca 2ϩ ] i of 112 Ϯ 14 nM (n ϭ 11). In mdx mice, the thapsigargin-induced Ca 2ϩ response was not significantly affected (110 Ϯ 21 nM, n ϭ 13). Taken together, these results indicate that, in mdx mice, the decrease of Ca 2ϩ responses evoked by caffeine and depolarizing steps did not appear to be due to an inhibition of voltage-dependent Ca 2ϩ channels or Ca 2ϩ loading of the SR.
Confocal Ca 2ϩ Signals Evoked by Activation of Voltage-gated Ca 2ϩ Channels and Flash Photolysis of Caged Ca 2ϩ -Spontaneous Ca 2ϩ sparks were not detected in control and mdx duodenal myocytes (n ϭ 155). Various experimental conditions, such as applications of low concentrations of caffeine or Bay K 8644 (an L-type Ca 2ϩ channel agonist) or low membrane depolarizations, have been reported to trigger and increase the frequency of Ca 2ϩ sparks in vascular myocytes (17). Applications of 5 nM Bay K 8644 (n ϭ 79), low depolarizations (from Ϫ70 to Ϫ50 mV or from Ϫ50 to Ϫ20 mV, n ϭ 41), or 1 mM caffeine (n ϭ 35) were ineffective in inducing generation of Ca 2ϩ sparks in control and mdx mice. In contrast, depolarizing steps applied from Ϫ70 mV elicited propagated Ca 2ϩ waves. As shown in Fig. 3, A and B, Ca 2ϩ responses evoked by depolarizing steps from Ϫ70 to Ϫ30 mV were not statistically different in control and mdx mice, whereas Ca 2ϩ responses evoked by higher depolarizing steps (from Ϫ70 to 0 mV) were reduced by about 40% in mdx mice compared with control mice (Fig. 3,  A-C). These results show that inhibition of Ca 2ϩ responses in mdx myocytes can also be detected in line scan images.
RYRs can be directly activated by an increase in [Ca 2ϩ ] i in the vicinity of the receptors as demonstrated previously in vascular myocytes (13). Flash photolysis of DM-nitrophen (caged Ca 2ϩ ) instantaneously elevated (within 2 ms) the Ca 2ϩ concentration and evoked Ca 2ϩ transients in the entire line scan image (Fig. 4A). Plotting the peak of the Ca 2ϩ transients as a function of flash intensity revealed that the Ca 2ϩ -induced increase in [Ca 2ϩ ] i in mdx mice was significantly reduced compared with control mice, particularly for high UV flash intensities (Fig. 4B). The Ca 2ϩ sensitivity of RYRs can be estimated by plotting the ratio between the peak Ca 2ϩ transients and the maximal Ca 2ϩ transient at different UV flash intensities for control and mdx mice. The points appeared to be superimposed suggesting no changes in the Ca 2ϩ sensitivity of RYRs in mdx mice (Fig. 4C).
Applications of 10 mM caffeine evoked propagating Ca 2ϩ waves in duodenal myocytes from control and mdx mice. The peak amplitude of these responses was reduced by about 40% in mdx compared with control mice (Fig. 8) in good agreement with the results obtained from experiments using whole-cell Indo-1 fluorescence.
[ 3 , n ϭ 4). These results support the idea that the density of RYRs was decreased in mdx duodenal myocytes.

H]Ryanodine Binding in Duodenal Microsomes-The
RYR Subtypes Expressed in Duodenal Myocytes and Effects of Anti-RYR Subtype Antibodies-Expression of RYR subtypes was detected in duodenal myocytes from control and mdx mice. To compare expression levels obtained from independent experiments, we normalized the data by using glyceraldehyde-3phosphate dehydrogenase as an internal standard. Only RYR2 and RYR3 mRNAs were detected in freshly isolated myocytes from control and mdx mice (Fig. 6A), whereas RYR1 mRNA was never observed (not shown). Similar levels of mRNA were detected for RYR3 in control and mdx mice. In contrast, the expression of RYR2 was strongly inhibited in mdx duodenal myocytes (Fig. 6A). These PCR experiments were confirmed by Western blots. The amount of RYR2 protein was substantially decreased in duodenum of mdx mice, whereas the expression levels of RYR3 and ␤-actin (not shown) were not affected by the absence of dystrophin (Fig. 6B). Immunodetection of RYRs in cell confocal sections with the specific anti-RYR2 and anti-RYR3 antibodies revealed that both RYR2 and RYR3 were distributed in the whole sections (Fig. 7A). Compiled data showed the selective inhibition of specific RYR2 fluorescence in mdx mice with no significant modification of the specific RYR3 fluorescence (Fig. 7B). These results indicate that mice duodenal myocytes expressed both RYR2 and RYR3 and that expression of RYR2 but not RYR3 was reduced in mdx mice.
To confirm that the decrease in RYR2 expression could be responsible for the reduced Ca 2ϩ responses in mdx mice, we tested the effects of both anti-RYR2 and anti-RYR3 antibodies on duodenal Ca 2ϩ responses. Concentration-dependent inhibitory effects and specificity of these antibodies have been reported previously in other smooth muscle cells (25,28). In both control and mdx mice, intracellular applications of 10 g/ml anti-RYR3 antibody for 7 min had no significant effect on the caffeine-induced Ca 2ϩ responses (Fig. 8). In contrast, the anti-RYR2 antibody inhibited in a concentration-dependent manner the Ca 2ϩ responses in control mice (Fig. 8A) as well as in mdx mice (Fig. 8B). The specificity of the anti-RYR2 antibody was confirmed by the absence of effect of the antibody when it was preincubated with its peptide epitope before intracellular application (Fig. 8). We also found that the Ca 2ϩ responses evoked by depolarizing steps (from Ϫ70 to 0 mV) in both control and mdx duodenal myocytes were selectively inhibited by the anti-RYR2 antibody (n ϭ 7), whereas they were unaffected by the anti-RYR3 antibody (n ϭ 5).

DISCUSSION
In Duchenne's muscular dystrophy patients and mdx mice, it is generally accepted that the missing link between the absence of dystrophin and muscle hypotonia is due to an alteration in Ca 2ϩ homeostasis, but no firm conclusions have yet been reached. In the gastrointestinal track, impaired nitrergic relaxation and increase of spontaneous tone have been reported in mdx mice (2, 3). We have readdressed this issue by meas- uring [Ca 2ϩ ] i in localized areas with confocal microscopy, voltage-gated Ca 2ϩ currents and global Ca 2ϩ release from the SR, and ryanodine receptor expression in duodenal myocytes from control and mdx mice.
Under resting conditions, no significant difference in bulk cytosolic [Ca 2ϩ ] i between mdx and control mice was observed in duodenal myocytes as well as in line scan images using two different Ca 2ϩ dyes in agreement with previous data obtained in vas deferens (29). In addition, no variation in SR Ca 2ϩ loading was detected as illustrated by the absence of significant reduction in ACh-induced Ca 2ϩ release at a concentration of ACh (10 M) that had been shown to completely deplete the SR (16). Capacitive Ca 2ϩ entry was not affected in mdx duodenal myocytes as revealed by similar thapsigargin-induced Ca 2ϩ responses obtained in control and mdx mice. In contrast, both caffeine-and depolarization-induced Ca 2ϩ responses were significantly inhibited in mdx compared with control duodenal myocytes. The reduction of depolarization-induced Ca 2ϩ responses was not dependent on a reduced voltage-gated Ca 2ϩ current as the current densities in mdx and control mice were similar. These results were therefore consistent with the possibility that Ca 2ϩ influx was less able to activate the CICR mechanism and/or with a reduction in RYR expression in mdx duodenal myocytes. Ca 2ϩ responses evoked by flash photolysis of caged Ca 2ϩ were reduced in mdx duodenal myocytes, but the Ca 2ϩ sensitivity of these responses, illustrated by the normalized curves of Ca 2ϩ release versus flash photolysis intensity, was not different in mdx compared with control mice suggesting that the gain of function of the CICR was not altered. In contrast, RYR expression was reduced in mdx mice as the maximal binding capacity of [ 3 H]ryanodine to duodenal membranes was strongly decreased, suggesting that the reduction of RYRs might account for the diminished Ca 2ϩ responses to caffeine and voltage-gated Ca 2ϩ currents. However, three RYR subtypes are generally expressed in smooth muscles, and their roles as functional Ca 2ϩ release channels have been questioned (17,28).
Duodenal myocytes expressed both RYR2 and RYR3 isoforms but not RYR1. This is in contrast with vascular myocytes that express all three RYR subtypes (17) and non-pregnant myometrial cells that express only RYR3 (28). At physiological extracellular Ca 2ϩ concentration, RYR3 is insensitive to caffeine in non-pregnant myometrial cells. In vascular myocytes, both RYR1 and RYR2 are required for triggering Ca 2ϩ sparks and Ca 2ϩ waves induced by activation of L-type Ca 2ϩ current (17). Our results show that both RYR2 and RYR3 are unable to induce Ca 2ϩ sparks in control duodenal myocytes, supporting our model that co-expression of RYR1 and RYR2 is needed to trigger elementary Ca 2ϩ signals in smooth muscle. In duodenal myocytes, Ca 2ϩ responses are dependent on the expression of RYR2 as the anti-RYR2-specific antibody inhibited in a concentration-dependent manner the caffeine-and the depolarization-induced Ca 2ϩ waves. In contrast, the anti-RYR3-specific antibody was ineffective, indicating that, at physiological extracellular Ca 2ϩ concentration, RYR3 did not participate in the CICR mechanism in duodenal myocytes in agreement with previous data obtained in vascular myocytes (17). In both control and mdx mice, caffeine-and depolarization-induced Ca 2ϩ waves revealed the same sensitivity to the anti-RYR2-specific antibody and the same absence of effects of the anti-RYR3-specific antibody, suggesting that inhibition of RYR2 expression was responsible for the reduced Ca 2ϩ responses observed in mdx duodenal myocytes. These results were supported by the specific inhibition of RYR2 expression in duodenum of mdx mice as revealed by both RT-PCR and Western blot experiments. This is the first data showing that inhibition of RYR2 expression may be involved in dystrophic smooth muscle. However, a mutation of the RYR1 gene has been reported in human congenital myopathies, resulting in reduced level of SR Ca 2ϩ release (30).
The absence of dystrophin generates modifications of ion channels that may lead to alterations of Ca 2ϩ flux. Changes of Ca 2ϩ channel activity or appearance of novel forms of cationic channels have been reported in dystrophic muscles (10,31). It has been speculated that the lack of dystrophin in skeletal muscle may induce localized structural disorders leading to disrupted excitation-contraction coupling in relation to chronic elevation of [Ca 2ϩ ] i , which may stop Ca 2ϩ release from the SR in localized areas and contribute to muscle weakness (1). However, the contractile responses of mechanically skinned muscle fibers are practically similar in both control and mdx mice (32), suggesting that the fundamental mechanisms of muscle contractility are not impaired in the absence of dystrophin. In contrast, our results in dystrophic duodenum show that the selective inhibition of RYR2 expression is responsible for the decreased CICR efficiency, which may account for a reduced mechanical activity in smooth muscle. Further experiments are necessary that are beyond the scope of this work to establish the sequence of events from the lack of dystrophin to alterations of protein expression, particularly of RYR isoforms, and to correlate inhibition of RYR expression with disorders of CICR and contraction in other muscles.